TY - JOUR A1 - Harrison, Odile B. A1 - Claus, Heike A1 - Jiang, Ying A1 - Bennett, Julia S. A1 - Bratcher, Holly B. A1 - Jolley, Keith A. A1 - Corton, Craig A1 - Care, Rory A1 - Poolman, Jan T. A1 - Zollinger, Wendell D. A1 - Frasch, Carl E. A1 - Stephens, David S. A1 - Feavers, Ian A1 - Frosch, Matthias A1 - Parkhill, Julian A1 - Vogel, Ulrich A1 - Quail, Michael A. A1 - Bentley, Stephen D. A1 - Maiden, Martin C. J. T1 - Description and Nomenclature of Neisseria meningitidis Capsule Locus JF - Emerging Infectious Diseases N2 - Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference. KW - genetics KW - nuclear magnetic resonance KW - structural determination KW - meningococcal polysaccharides KW - chemical properties KW - serogroup-Y KW - group-B KW - antigen KW - biosynthesis KW - elucidation Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131703 VL - 19 IS - 4 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 - Endres, Leo M. A1 - Jungblut, Marvin A1 - Divyapicigil, Mustafa A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Christodoulides, Myron A1 - Kim, Brandon J. A1 - Schubert-Unkmeir, Alexandra T1 - Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection JF - Fluids and Barriers of the CNS N2 - Background Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction. Methods We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection. Results Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection. Conclusions Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting. KW - brain endothelial cells KW - bacterial meningitis KW - meningeal blood-csf barrier KW - induced pluripotent stem cells KW - neisseria meningitidis KW - leptomeningeal cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300208 VL - 19 IS - 1 ER - TY - THES A1 - Endres, Leo Maximilian T1 - Development of multicellular \(in\) \(vitro\) models of the meningeal blood-CSF barrier to study \(Neisseria\) \(meningitidis\) infection T1 - Entwicklung multizellulärer \(in\) \(vitro\) Modelle der meningealen Blut-Liquor Schranke zur Untersuchung der \(Neisseria\) \(meningitidis\) Infektion N2 - Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies. Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis. N2 - Neisseria meningitidis (der Meningokokkus) ist einer der Hauptursachen bakterieller Meningitis, einer lebensbedrohlichen Entzündung der Hirnhäute. Entscheidend für das für das Voranschreiten der Krankheit ist die Fähigkeit des Erregers, die meningeale Blut-Liquor-Schranke (mBCSFB), bestehend aus spezialisierten Hirnendothelzellen (BECs) und leptomeningealen Zellen (LMCs), zu überwinden und in den submeningealen Raum einzudringen. Da es sich bei N. meningitidis um ein rein humanes Pathogen handelt, beschränkt sich die Erforschung dieser speziellen Interaktion primär auf die Verwendung von in vitro Modellen. Bisher wurden hierfür hauptsächlich periphere oder immortalisierte BECs verwendet, welchen jedoch wichtige Barriere-Eigenschaften fehlen. Um die Interaktion von N. meningitidis mit der mBCSFB in einem physiologisch relevanteren Umfeld zu untersuchen, wurden in dieser Arbeit neuartige BEC-LMC Kokulturmodelle entwickelt. Dabei wurden sowohl BEC-ähnliche Zellen, die aus induzierten pluripotenten Stammzellen generiert wurden (iBECs), als auch hCMEC/D3 Zellen verwendet und zusammen mit LMCs aus Tumorbiopsien kultiviert. Mittels Transmissions-Elektronenmikroskopie und Immunfluoreszenzfärbung konnten die unterschiedlichen Zellschichten und deren Expression charakteristischer zellulärer Marker dargestellt werden. Durchgängige Expression von wichtigen Bestandteilen Barriere-formender Zellverbindungen, sogenannter Tight und Adherens Junctions, wurde in der iBEC-Schicht beobachtet. Die Integrität der zellulären Barriere wurde mittels transendothelialer elektrischer Resistenz (TEER) und Permeabilität gegenüber Natrium-Fluorescein (NaF) bestimmt. Erhöhte TEER-Werte und verringerte NaF-Permeabilität, gemessen über einen Zeitraum von sieben Tagen, zeigten eine durch die Kokultur mit LMCs ausgelöste Steigerung der Dichtigkeit und Stabilität der iBEC-Barriere. Infektionsexperimente mit N. meningitidis zeigten in allen Modellen vergleichbare bakterielle Adhäsion und Invasion der BEC-Schicht. Bakterielle Transmigration durch die gesamten Zellbarriere war im iBEC-LMC Modell kurz nach Infektion nur in geringem Maße detektierbar, nahm jedoch innerhalb von 24 Stunden deutlich zu. Interessanterweise wurde bis zu 24 Stunden nach Infektion noch eine hohe Integrität der Barriere gemessen, welche allerdings im weiteren Verlauf verloren ging. Neben signifikantem TEER-Verlust wurde eine verringerte Expression der Tight und Adherens Junction Proteine ZO-1, claudin-5, und VE-cadherin mittels qPCR festgestellt. qPCR und siRNA Knockdown Experimente deuteten darauf hin, dass dies möglicherweise, aber nicht ausschließlich, auf den Transkriptionsfaktor Snail1 zurückzuführen war. Zusätzlich zu den beobachteten Effekten auf die zelluläre Transkription von Tight Junction Genen, zeigten Immunfluoreszenzfärbungen eine verringerte Expression von Occludin an den Zell-Zell-Verbindungen, was auf eine post-translationale Modulation schließen lässt. Zusammen deuten die Ergebnisse dieser Infektionsstudien auf eine mögliche Kombination aus trans- und parazellulärer bakterieller Transmigration der mBCSFB hin. Zuletzt wurden in dieser Arbeit noch die Immunaktivierung von BECs nach N. meningitidis Infektion in den neuen BEC-LMC Kokulturmodellen untersucht. Hierbei wurde eine erhöhte Expression von Zytokinen, insbesondere Interleukin-8, beobachtet. Insgesamt konnten in dieser Arbeit neue, fortschrittlicher in vitro Modelle der mBCSFB entwickelt werden, welche die humane Physiologie besser widerspiegeln und daher für Infektionsstudien mit Meningitis-verursachenden Erregern wie N. meningitidis von besonderem Nutzen sind. KW - Bakterielle Hirnhautentzündung KW - Blut-Liquor-Schranke KW - Induzierte pluripotente Stammzelle KW - Neisseria meningitidis KW - In-vitro-Kultur KW - Brain endothelial cells KW - Leptomeningeal cells KW - Hirnendothelzellen KW - Leptomeningealzellen Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-346216 ER - TY - JOUR A1 - Strobel, Katharina A1 - Sickenberger, Christina A1 - Schoen, Christoph A1 - Kneitz, Hermann A1 - Kolb-Mäurer, Annette A1 - Goebeler, Matthias T1 - Diagnosis and therapy of Mycobacterium marinum: a single-center 21-year retrospective analysis JF - Journal der Deutschen Dermatologischen Gesellschaft N2 - Background and Objectives In Europe, infections with Mycobacterium (M.) marinum are rare. We conducted a retrospective single-center study to assess the clinical spectrum of M. marinum infection and its diagnosis, treatment and outcome under real-world conditions. Patients and Methods Eighteen patients presenting with M. marinum infections between 1998 and 2018 were identified in the data warehouse of the University Hospital Würzburg and considered for detailed analysis. Results Twelve patients reported aquatic exposure. In 16/18 cases the upper extremities were affected. No invasive infections were detected. Mean time to diagnosis was 15 weeks. Histology revealed granulomatous inflammation in 14 patients while mycobacterial cultures were positive for M. marinum in 16 cases. Most patients received antibiotic monotherapy (14/18) while combination therapy was administered in four cases. Treatment (with a median duration of 10 weeks) was successful in 13 patients. Five patients were lost to follow-up. Conclusions Our retrospective analysis of M. marinum infections at a German tertiary referral center revealed a considerable diagnostic delay and the relevance of microbiological culture, PCR and histology for diagnosis. Monotherapy with clarithromycin (rather than doxycycline) appeared as a reasonable treatment option while immunosuppressed or -compromised patients and those with extended disease received combination therapy. KW - Mycobacterium marinum KW - diagnosis KW - therapy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318428 VL - 20 IS - 9 SP - 1211 EP - 1218 ER - TY - JOUR A1 - Rohde, Jörn A1 - Himmel, Wolfgang A1 - Hofinger, Clemens A1 - Lâm, Thiên-Trí A1 - Schrader, Hanna A1 - Wallstabe, Julia A1 - Kurzai, Oliver A1 - Gágyor, Ildikó T1 - Diagnostic accuracy and feasibility of a rapid SARS-CoV-2 antigen test in general practice - a prospective multicenter validation and implementation study JF - BMC Primary Care N2 - Background PCR testing is considered the gold standard for SARS-CoV-2 diagnosis but its results are earliest available hours to days after testing. Rapid antigen tests represent a diagnostic tool enabling testing at the point of care. Rapid antigen tests have mostly been validated by the manufacturer or in controlled laboratory settings only. External validation at the point of care, particularly in general practice where the test is frequently used, is needed. Furthermore, it is unclear how well point of care tests are accepted by the practice staff. Methods In this prospective multicenter validation study in primary care, general practitioners included adult individuals presenting with symptoms suggesting COVID-19. Each patient was tested by the general practitioner, first with a nasopharyngeal swab for the point of care test (Roche SARS-CoV-2 Rapid Antigen Test) and then with a second swab for PCR testing. Using the RT-PCR result as a reference, we calculated specificity, sensitivity, positive predictive value and negative predictive value, with their 95% confidence intervals. General practitioners and medical assistants completed a survey to assess feasibility and usefulness of the point of care tests. Results In 40 practices in Würzburg, Germany, 1518 patients were recruited between 12/2020 and 06/2021. The point of care test achieved a sensitivity of 78.3% and a specificity of 99.5% compared to RT-PCR. With a prevalence of 9.5%, the positive predictive value was 93.9% and the negative predictive value was 97.8%. General practitioners rated the point of care test as a helpful tool to support diagnostics in patients with signs and symptoms suggestive for infection, particularly in situations where decision on further care is needed at short notice. Conclusion The point of care test used in this study showed a sensitivity below the manufacturer’s specification (Sensitivity 96.25%) in the practice but high values for specificity and high positive predictive value and negative predictive value. Although widely accepted in the practice, measures for further patient management require a sensitive interpretation of the point of care test results. KW - COVID-19 testing KW - feasibility study KW - attitude of health personnel KW - sensitivity and specificity KW - general practice Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299659 VL - 23 IS - 1 ER - TY - JOUR A1 - Springer, Jan A1 - Held, Jürgen A1 - Mengoli, Carlo A1 - Schlegel, Paul Gerhardt A1 - Gamon, Florian A1 - Träger, Johannes A1 - Kurzai, Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen A1 - Eyrich, Matthias T1 - Diagnostic performance of (1→3)-β-D-glucan alone and in combination with aspergillus PCR and galactomannan in serum of pediatric patients after allogeneic hematopoietic stem cell transplantation JF - Journal of Fungi N2 - Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation (alloSCT). Sera were tested prospectively twice weekly for Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-β-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% (95% confidential interval (CI): 28–99%) and 55% (95% CI: 32–77%) for BDG, 40% (95% CI: 5–85%) and 100% (95% CI: 83–100%) for GM, and 60% (95% CI: 15–95%) and 95% (95% CI: 75–100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56–94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68–99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay. KW - beta-D-glucan KW - galactomannan KW - real-time PCR KW - Aspergillus KW - pediatric Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234179 SN - 2309-608X VL - 7 IS - 3 ER - TY - JOUR A1 - Schubert-Unkmeir, Alexandra A1 - Schneider-Schaulies, Sibylle A1 - Gulbins, Erich A1 - Hebling, Sabrina A1 - Simonis, Alexander T1 - Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells N2 - The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. Author Summary Neisseria meningitidis, an obligate human pathogen, is a causative agent of septicemia and meningitis worldwide. Meningococcal infection manifests in a variety of forms, including meningitis, meningococcemia with meningitis or meningococcemia without obvious meningitis. The interaction of N. meningitidis with human cells lining the blood vessels of the blood-cerebrospinal fluid barrier is a prerequisite for the development of meningitis. As a major pathogenicity factor, the meningococcal outer membrane protein Opc enhances bacterial entry into brain endothelial cells, however, mechanisms underlying trapping of receptors and signaling molecules following this interaction remained elusive. We now show that Opc-expressing meningococci activate acid sphingomyelinase (ASM) in brain endothelial cells, which hydrolyses sphingomyelin to cause ceramide release and formation of extended ceramide-enriched membrane platforms wherein ErbB2, an important receptor involved in bacterial uptake, clusters. Mechanistically, ASM activation relied on binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of PC-PLC. Meningococcal isolates of the ST-11 clonal complex, which are reported to be more likely to cause severe sepsis, but rarely meningitis, barely invaded brain endothelial cells and revealed a highly restricted ability to induce ASM and ceramide release. Thus, our results unravel a differential activation of the ASM/ceramide system by the species N. meningitidis determining its invasiveness into brain endothelial cells. KW - small interfering RNAs KW - Neisseria meningitidis KW - bacterial pathogens KW - endothelial cells KW - meningococcal disease KW - flow cytometry KW - cell staining KW - Escherichia coli infections Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113031 ER - TY - JOUR A1 - Weiß, Martin A1 - Gründahl, Marthe A1 - Deckert, Jürgen A1 - Eichner, Felizitas A. A1 - Kohls, Mirjam A1 - Störk, Stefan A1 - Heuschmann, Peter U. A1 - Hein, Grit T1 - Differential network interactions between psychosocial factors, mental health, and health-related quality of life in women and men JF - Scientific Reports N2 - Psychosocial factors affect mental health and health-related quality of life (HRQL) in a complex manner, yet gender differences in these interactions remain poorly understood. We investigated whether psychosocial factors such as social support and personal and work-related concerns impact mental health and HRQL differentially in women and men during the first year of the COVID-19 pandemic. Between June and October 2020, the first part of a COVID-19-specific program was conducted within the “Characteristics and Course of Heart Failure Stages A-B and Determinants of Progression (STAAB)” cohort study, a representative age- and gender-stratified sample of the general population of Würzburg, Germany. Using psychometric networks, we first established the complex relations between personal social support, personal and work-related concerns, and their interactions with anxiety, depression, and HRQL. Second, we tested for gender differences by comparing expected influence, edge weight differences, and stability of the networks. The network comparison revealed a significant difference in the overall network structure. The male (N = 1370) but not the female network (N = 1520) showed a positive link between work-related concern and anxiety. In both networks, anxiety was the most central variable. These findings provide further evidence that the complex interplay of psychosocial factors with mental health and HRQL decisively depends on gender. Our results are relevant for the development of gender-specific interventions to increase resilience in times of pandemic crisis. KW - anxiety KW - depression KW - human behaviour KW - quality of life Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357858 VL - 13 ER - TY - JOUR A1 - Sturm, Laura A1 - Geißel, Bernadette A1 - Martin, Ronny A1 - Wagener, Johannes T1 - Differentially Regulated Transcription Factors and ABC Transporters in a Mitochondrial Dynamics Mutant Can Alter Azole Susceptibility of Aspergillus fumigatus JF - Frontiers in Microbiology N2 - Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold. KW - mitochondrial dynamics KW - azole resistance KW - efflux pumps KW - transcription factors Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204874 SN - 1664-302X VL - 11 ER - TY - JOUR A1 - Muenstermann, Marcel A1 - Strobel, Lea A1 - Klos, Andreas A1 - Wetsel, Rick A. A1 - Woodruff, Trent M. A1 - Köhl, Jörg A1 - Johswich, Kay O. T1 - Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections JF - Virulence N2 - The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar\(^{-/-}\) mice, whereas C5ar1\(^{-/-}\) and C5ar2\(^{-/-}\) mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1\(^{-/-}\) mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8\(^{Δ71−73}\)) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a “super-agonist” peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis. KW - inflammation KW - C3a KW - C5a KW - C3aR KW - C5aR1 KW - C5aR2 KW - meningococcal disease KW - sepsis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200496 VL - 10 IS - 1 ER - TY - JOUR A1 - Stijnis, Kees A1 - Dijkmans, Anneke C. A1 - Bart, Aldert A1 - Brosens, Lodewijk A. A. A1 - Muntau, Birgit A1 - Schoen, Christoph A1 - Barth, Thomas F. A1 - van Gulik, Thomas A1 - van Gool, Tom A1 - Grobusch, Martin P. A1 - Tappe, Dennis T1 - Echinococcus vogeli in Immigrant from Suriname to the Netherlands JF - Emerging Infectious Diseases KW - alveolar echinococcosis KW - monoclonal antibody KW - multilocularis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143953 VL - 21 IS - 3 ER - TY - GEN A1 - Tort, Jose F. A1 - Mitreva, Makedonka A1 - Brehm, Klaus R. A1 - Rinaldi, Gabriel T1 - Editorial: Novel Frontiers in Helminth Genomics T2 - Frontiers in Genetics N2 - No abstract available. KW - flatworm KW - nematodes KW - genomics KW - helminths KW - neglected diseases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-210209 SN - 1664-8021 VL - 11 IS - 791 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 - Brehm, Klaus A1 - Komguep Nono, Justin A1 - Lutz, Manfred B. T1 - EmTIP, a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important N2 - Background Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown. Principal Findings We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro. Conclusions We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host. KW - T Cells KW - primary cells KW - parasitic diseases KW - vesicles KW - larvae KW - immunoprecipitation KW - host-pathogen interactions KW - immune response Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111407 ER - TY - JOUR A1 - Koch, J. A1 - Hellenbrand, W. A1 - Schink, S. A1 - Wichmann, O. A1 - Carganico, A. A1 - Drewes, J. A1 - Kruspe, M. A1 - Suckau, M. A1 - Claus, H. A1 - Marcus, U. T1 - Evaluation of a temporary vaccination recommendation in response to an outbreak of invasive meningococcal serogroup C disease in men who have sex with men in Berlin, 2013-2014 JF - Eurosurveillance N2 - Meningococcal serogroup C (MenC) vaccination of men who have sex with men (MSM) was temporarily recommended to control an outbreak of invasive MenC disease among MSM in Berlin in 2012–2013. Vaccination was offered to HIV-infected MSM free of charge; others had to request reimbursement or pay out of pocket. We aimed to assess (i) awareness and acceptance of this recommendation through an online survey of MSM, (ii) implementation through a survey of primary care physicians and analysis of vaccine prescriptions, and (iii) impact through analysis of notified cases. Among online survey respondents, 60% were aware of the recommendation. Of these, 39% had obtained vaccination (70% of HIV-infected, 13% of HIV-negative/non-tested MSM). Awareness of recommendation and vaccination were positively associated with HIV infection, primary care physicians’ awareness of respondents’ sexual orientation, and exposure to multiple information sources. Most (26/30) physicians informed clients about the recommendation. Physicians considered concerns regarding reimbursement, vaccine safety and lack of perceived disease risk as primary barriers. After the recommendation, no further outbreak-related cases occurred. To reach and motivate target groups, communication of a new outbreak-related vaccination recommendation should address potential concerns through as many information channels as possible and direct reimbursement of costs should be enabled. KW - Meningococcal serogroup C Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165070 VL - 21 IS - 5 ER - TY - JOUR A1 - Straub, Anton A1 - Vollmer, Andreas A1 - Lâm, Thiên-Trí A1 - Brands, Roman C. A1 - Stapf, Maximilian A1 - Scherf-Clavel, Oliver A1 - Bittrich, Max A1 - Fuchs, Andreas A1 - Kübler, Alexander C. A1 - Hartmann, Stefan T1 - Evaluation of advanced platelet-rich fibrin (PRF) as a bio-carrier for ampicillin/sulbactam JF - Clinical Oral Investigations N2 - Objectives Mechanisms of wound healing are often impaired in patients with osteonecrosis of the jaw (ONJ). According to the guidelines for the treatment of this disease, early surgical intervention is indicated. However, surgery often faces complications such as wound healing disorders. The application of platelet-rich fibrin (PRF) after necrosectomy between bone and mucosa may constitute a promising approach to improve surgical results. An aspect that was not investigated until now is that PRF acts as a “bio-carrier” for antibiotics previously applied intravenously. Materials and methods We investigated the antimicrobial properties of PRF in 24 patients presenting ONJ undergoing systemic antibiosis with ampicillin/sulbactam. We measured the concentration of ampicillin/sulbactam in plasma and PRF and performed agar diffusion tests. Ampicillin/sulbactam was applied intravenously to the patient 10 minutes for blood sampling for PRF. No further incorporation of patients’ blood or PRF product with antibiotic drugs was obtained. Four healthy patients served as controls. Results Our results revealed that PRF is highly enriched with ampicillin/sulbactam that is released to the environment. The antibiotic concentration in PRF was comparable to the plasma concentration of ampicillin/sulbactam. The inhibition zone (IZ) of PRF was comparable to the standard ampicillin/sulbactam discs used in sensitivity testing. Conclusions The results of our study demonstrated that PRF is a reliable bio-carrier for systemic applied antibiotics and exhibits a large antimicrobial effect. Clinical relevance We describe a clinically useful feature of PRF as a bio-carrier for antibiotics. Especially when applied to poorly perfused tissues and bone such as in ONJ, the local release of antibiotics can reduce wound healing disorders like infections. KW - osteonecrosis of the jaw KW - osteoradionecrosis KW - antiresorptive drug-related osteonecrosis of the jaw KW - ARONJ KW - oral microbiome KW - agar diffusion test Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324515 VL - 26 IS - 12 ER - TY - JOUR A1 - Herz, Michaela A1 - Brehm, Klaus T1 - Evidence for densovirus integrations into tapeworm genomes JF - Parasites & Vectors N2 - Background Tapeworms lack a canonical piRNA-pathway, raising the question of how they can silence existing mobile genetic elements (MGE). Investigation towards the underlying mechanisms requires information on tapeworm transposons which is, however, presently scarce. Methods The presence of densovirus-related sequences in tapeworm genomes was studied by bioinformatic approaches. Available RNA-Seq datasets were mapped against the Echinococcus multilocularis genome to calculate expression levels of densovirus-related genes. Transcription of densovirus loci was further analyzed by sequencing and RT-qPCR. Results We herein provide evidence for the presence of densovirus-related elements in a variety of tapeworm genomes. In the high-quality genome of E. multilocularis we identified more than 20 individual densovirus integration loci which contain the information for non-structural and structural virus proteins. The majority of densovirus loci are present as head-to-tail concatemers in isolated repeat containing regions of the genome. In some cases, unique densovirus loci have integrated close to histone gene clusters. We show that some of the densovirus loci of E. multilocularis are actively transcribed, whereas the majority are transcriptionally silent. RT-qPCR data further indicate that densovirus expression mainly occurs in the E. multilocularis stem cell population, which probably forms the germline of this organism. Sequences similar to the non-structural densovirus genes present in E. multilocularis were also identified in the genomes of E. canadensis, E. granulosus, Hydatigera taeniaeformis, Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana, Taenia asiatica, Taenia multiceps, Taenia saginata and Taenia solium. Conclusions Our data indicate that densovirus integration has occurred in many tapeworm species. This is the first report on widespread integration of DNA viruses into cestode genomes. Since only few densovirus integration sites were transcriptionally active in E. multilocularis, our data are relevant for future studies into gene silencing mechanisms in tapeworms. Furthermore, they indicate that densovirus-based vectors might be suitable tools for genetic manipulation of cestodes. KW - Echinococcus KW - Echinococcosis KW - Densovirus KW - Parvovirus KW - Mobile genetic element KW - Gene silencing KW - Stem cell KW - Epigenetic Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202478 VL - 12 ER - TY - JOUR A1 - Taha, Muhamed-Kheir A1 - Claus, Heike A1 - Lappann, Martin A1 - Veyrier, Frédéric J. A1 - Otto, Andreas A1 - Becher, Dörte A1 - Deghmane, Ala-Eddine A1 - Frosch, Matthias A1 - Hellenbrand, Wiebke A1 - Hong, Eva A1 - du Châtelet, Isabelle Parent A1 - Prior, Karola A1 - Harmsen, Dag A1 - Vogel, Ulrich T1 - Evolutionary Events Associated with an Outbreak of Meningococcal Disease in Men Who Have Sex with Men JF - PLoS ONE N2 - Meningococci spread via respiratory droplets, whereas the closely related gonococci are transmitted sexually. Several outbreaks of invasive meningococcal disease have been reported in Europe and the United States among men who have sex with men (MSM). We recently identified an outbreak of serogroup C meningococcal disease among MSM in Germany and France. In this study, genomic and proteomic techniques were used to analyze the outbreak isolates. In addition, genetically identical urethritis isolates were recovered from France and Germany and included in the analysis. Genome sequencing revealed that the isolates from the outbreak among MSM and from urethritis cases belonged to a clade within clonal complex 11. Proteome analysis showed they expressed nitrite reductase, enabling anaerobic growth as previously described for gonococci. Invasive isolates from MSM, but not urethritis isolates, further expressed functional human factor H binding protein associated with enhanced survival in a newly developed transgenic mouse model expressing human factor H, a complement regulatory protein. In conclusion, our data suggest that urethritis and outbreak isolates followed a joint adaptation route including adaption to the urogenital tract. KW - nitrites KW - genome sequencing KW - men who have sex with men KW - meningococcal disease KW - Neisseria meningitidis KW - Neisseria gonorrhoeae KW - mammalian genomics KW - mouse models Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179870 VL - 11 IS - 5 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 - Nono, Justin Komguep A1 - Pletinckx, Katrien A1 - Lutz, Manfred B. A1 - Brehm, Klaus T1 - Excretory/Secretory-Products of Echinococcus multilocularis Larvae Induce Apoptosis and Tolerogenic Properties in Dendritic Cells In Vitro JF - PLoS Neglected Tropical Diseases N2 - Background: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E. multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. Methodology/Principal Findings: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naive T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. Conclusions: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis. KW - granulosus KW - hydatid disease KW - metacestode vesicles KW - antigen-B KW - alveoar echinococcosis KW - TGF-BETA KW - regulatory T cells KW - gene expression KW - Brugia Malayi KW - TNF-alpha Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134280 VL - 6 IS - 2 ER - TY - JOUR A1 - Götz, Ralph A1 - Panzer, Sabine A1 - Trinks, Nora A1 - Eilts, Janna A1 - Wagener, Johannes A1 - Turrà, David A1 - Di Pietro, Antonio A1 - Sauer, Markus A1 - Terpitz, Ulrich T1 - Expansion Microscopy for Cell Biology Analysis in Fungi JF - Frontiers in Microbiology N2 - Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes. KW - Expansion microscopy KW - fluorescence microscopy KW - fungi KW - sporidia KW - hyphae Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202569 SN - 1664-302X VL - 11 ER - TY - JOUR A1 - Aldejohann, Alexander Maximilian A1 - Wiese-Posselt, Miriam A1 - Gastmeier, Petra A1 - Kurzai, Oliver T1 - Expert recommendations for prevention and management of Candida auris transmission JF - Mycoses N2 - Candida auris was first described as a yeast pathogen in 2009. Since then, the species has emerged worldwide. In contrast to most other Candida spp., C. auris frequently exhibits multi-drug resistance and is readily transmitted in hospital settings. While most detections so far are from colonised patients, C. auris does cause superficial and life-threatening invasive infections. During management of the first documented C. auris transmission in a German hospital, experts from the National Reference Centers for Invasive Fungal Infections (NRZMyk) and the National Reference Center for Surveillance of Nosocomial Infections screened available literature and integrated available knowledge on infection prevention and C. auris epidemiology and biology to enable optimal containment. Relevant recommendations developed during this process are summarised in this guidance document, intended to assist in management of C. auris transmission and potential outbreak situations. Rapid and effective measures to contain C. auris spread require a multi-disciplinary approach that includes clinical specialists of the affected unit, nursing staff, hospital hygiene, diagnostic microbiology, cleaning staff, hospital management and experts in diagnostic mycology / fungal infections. Action should be initiated in a step-wise process and relevant interventions differ between management of singular C. auris colonised / infected patients and detection of potential C. auris transmission or nosocomial outbreaks. KW - Candida auris KW - nosocomial transmission KW - infection prevention KW - expert recommendation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318570 VL - 65 IS - 6 SP - 590 EP - 598 ER - TY - JOUR A1 - Walther, Grit A1 - Zimmermann, Anna A1 - Theuersbacher, Johanna A1 - Kaerger, Kerstin A1 - Lilienfeld-Toal, Marie von A1 - Roth, Mathias A1 - Kampik, Daniel A1 - Geerling, Gerd A1 - Kurzai, Oliver T1 - Eye infections caused by filamentous fungi: spectrum and antifungal susceptibility of the prevailing agents in Germany JF - Journal of Fungi N2 - Fungal eye infections can lead to loss of vision and blindness. The disease is most prevalent in the tropics, although case numbers in moderate climates are increasing as well. This study aimed to determine the dominating filamentous fungi causing eye infections in Germany and their antifungal susceptibility profiles in order to improve treatment, including cases with unidentified pathogenic fungi. As such, we studied all filamentous fungi isolated from the eye or associated materials that were sent to the NRZMyk between 2014 and 2020. All strains were molecularly identified and antifungal susceptibility testing according to the EUCAST protocol was performed for common species. In total, 242 strains of 66 species were received. Fusarium was the dominating genus, followed by Aspergillus, Purpureocillium, Alternaria, and Scedosporium. The most prevalent species in eye samples were Fusarium petroliphilum, F. keratoplasticum, and F. solani of the Fusarium solani species complex. The spectrum of species comprises less susceptible taxa for amphotericin B, natamycin, and azoles, including voriconazole. Natamycin is effective for most species but not for Aspergillus flavus or Purpureocillium spp. Some strains of F. solani show MICs higher than 16 mg/L. Our data underline the importance of species identification for correct treatment. KW - eye infection KW - fungal infection KW - keratitis KW - antifungal susceptibility KW - natamycin KW - Fusarium KW - Purpureocillium KW - Aspergillus KW - Alternaria KW - Scedosporium Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241810 SN - 2309-608X VL - 7 IS - 7 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - Faecal carriage of CTX-M extended-spectrum beta-lactamase-producing Enterobacteriaceae among street children dwelling in Mwanza city, Tanzania JF - PLoS ONE N2 - Background Data on ESBL carriage of healthy people including children are scarce especially in developing countries. We analyzed the prevalence and genotypes of ESBL-producing Enterobacteriaceae (EPE) in Tanzanian street children with rare contact to healthcare facilities but significant interactions with the environment, animals and other people. Methodology/ Principle findings Between April and July 2015, stool samples of 107 street children, who live in urban Mwanza were analyzed for EPE. Intestinal carriage of EPE was found in 34 (31.8%, 95% CI; 22.7–40.3) children. Of the 36 isolates from 34 children, 30 (83.3%) were Escherichia coli (E. coli) and six Klebsiella pneumoniae (K. pneumoniae). Out of 36 isolates, 36 (100%), 35 (97%), 25 (69%) and 16 (44%) were resistant to tetracycline, trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin, respectively. Beta-lactamase genes and the multilocus sequence types of E. coli and K. pneumoniae were characterized. ESBL gene bla\(_{CTX-M-15}\) was detected in 75% (27/36) of ESBL isolates. Sequence types (STs) 131, 10, 448 and 617 were the most prevalent in E. coli. Use of local herbs (OR: 3.5, 95% CI: 1.51–8.08, P = 0.003) and spending day and night on streets (OR: 3.6, 95% CI: 1.44–8.97, P = 0.005) were independent predictors of ESBL carriage. Conclusions/ Significance We observed a high prevalence of bla\(_{CTX-M-15}\) in EPE collected from street children in Tanzania. Detection of E. coli STs 131, 10, 38 and 648, which have been observed worldwide in animals and people, highlights the need for multidisciplinary approaches to understand the epidemiology and drivers of antimicrobial resistance in low-income countries. KW - Tanzania KW - children KW - Enterobacteriaceae KW - ESBL Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170331 VL - 12 IS - 9 ER - TY - JOUR A1 - Soundararajan, Manonmani A1 - Marincola, Gabriella A1 - Liong, Olivia A1 - Marciniak, Tessa A1 - Wencker, Freya D. R. A1 - Hofmann, Franka A1 - Schollenbruch, Hannah A1 - Kobusch, Iris A1 - Linnemann, Sabrina A1 - Wolf, Silver A. A1 - Helal, Mustafa A1 - Semmler, Torsten A1 - Walther, Birgit A1 - Schoen, Christoph A1 - Nyasinga, Justin A1 - Revathi, Gunturu A1 - Boelhauve, Marc A1 - Ziebuhr, Wilma T1 - Farming practice influences antimicrobial resistance burden of non-aureus staphylococci in pig husbandries JF - Microorganisms N2 - Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms. KW - non-aureus staphylococci KW - NAS KW - alternative pig farming KW - antimicrobial resistance KW - one-health approach KW - intervention strategies KW - livestock-associated staphylococci KW - organic farming KW - pig farming methods Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312750 SN - 2076-2607 VL - 11 IS - 1 ER - TY - JOUR A1 - Weiss, Esther A1 - Ziegler, Sabrina A1 - Fliesser, Mirjam A1 - Schmitt, Anna-Lena A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Morton, Charles-Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - First Insights in NK—DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus JF - Frontiers in Cellular and Infection Microbiology N2 - Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal. KW - natural killer cells KW - dendritic cells KW - NK-DC cross-talk KW - Aspergillus fumigatus KW - soluble factors KW - innate immunity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233565 VL - 8 ER - TY - THES A1 - Schielke, Stephanie T1 - Functional and molecular characterization of FarR – a transcriptional regulator of the MarR family in Neisseria meningitidis T1 - Funktionelle und molekulare Charakterisierung von FarR, einem Transkriptionsregulator der MarR Familie in Neisseria meningitidis N2 - Neisseria meningitidis is a facultatively pathogenic human commensal and strictly adapted to its niche within the human host, the nasopharynx. Not much is known about the regulatory processes required for adaptation to this environment. Therefore the role of the transcriptional regulator NMB1843, one of the two predicted regulators of the MarR family in the meningococcal genome, was investigated. As this gene displayed a high sequence homology to FarR, the Fatty acid resistance Regulator in N. gonorrhoeae, we designated the meningococcal protein FarR (NmFarR). Homology modeling of this protein revealed a dimeric structure with the characteristic winged helix-turn-helix DNA binding motif of the MarR family. NmFarR is highly conserved among meningococcal strains and expression of farR during exponential growth is controlled post-transcriptionally, being highest in the late exponential phase. By means of electrophoretic mobility shift assays (EMSAs) the direct and specific binding of FarR to the farAB promoter region was shown, comparable to its homologue in gonococci. As FarR is involved in fatty acid resistance in N. gonorrhoeae, susceptibility assays with the medium chain lauric acid (C12:0), the long chain saturated palmitic acid (C16:0) and the long chain unsaturated linoleic acid (C18:2) were performed, testing a wide variety of strains of both species. In contrast to the unusually susceptible gonococci, a high intrinsic fatty acid resistance was detected in almost all meningococcal isolates. The molecular basis for this intrinsic resistance in N. meningitidis was elucidated, showing that both a functional FarAB efflux pump system as well as an intact lipopolysaccharide (LPS) are responsible for palmitic acid resistance. However, even despite circumvention of the intrinsic resistance, FarR could not be connected with fatty acid resistance in meningococci. Instead, FarR was shown to directly and specifically repress expression of the Neisseria adhesin A (nadA), a promising vaccine candidate absent in N. gonorrhoeae. Microarray analyses verified these results and disclosed no further similarly regulated genes, rendering the FarR regulon the smallest regulon in meningococci reported until now. The exact FarR binding site within the nadA promoter region was identified as a 16 bp palindromic repeat and its influence on nadA transcription was proved by reporter gene fusion assays. This repression was also shown to be relevant for infection as farR deficient mutant strains displayed an increased attachment to epithelial cells. Furthermore, farR transcription was attested to be repressed upon contact with active complement components within human serum. Concluding, it is shown that FarR adopted a role in meningococcal host niche adaptation, holding the balance between immune evasion by repressing the highly antigenic nadA and host cell attachment via this same adhesin. N2 - Neisseria meningitidis ist ein fakultativ pathogener menschlicher Kommensale und eng an die Bedingungen seiner spezifischen Nische, den Nasopharynx, angepasst. Über die regulatorischen Mechanismen, die für diese Anpassung vonnöten sind, ist nicht viel bekannt. Daher wurde die Rolle des Transkriptionsregulators NMB1843 untersucht, eines der beiden prognostizierten Regulatoren der MarR Familie im Meningokokken-Genom. Aufgrund einer hohen Sequenzhomologie dieses Gens zu FarR, dem Fatty acid resistance Regulator in N. gonorrhoeae, nannten wir das Meningokokken-Protein ebenfalls FarR (NmFarR). Homologie-Modellierung dieses Proteins ergab eine dimere Struktur mit dem charakteristischen winged helix-turn-helix DNA-Bindemotiv der MarR Familie. Es wurde gezeigt, dass NmFarR in Meningokokken-Stämmen hochkonserviert ist. Die Expression von farR wird während des exponentiellen Wachstums posttranskriptional kontrolliert und erreicht ihren Höchststand in der spätexponentiellen Phase. Wie bei seinem Homolog in Gonokokken konnte die direkte und spezifische Bindung von FarR an die farAB Promotorregion nachgewiesen werden. Da FarR in N. gonorrhoeae an der Fettsäureresistenz beteiligt ist, wurde die Suszeptibilität einer großen Auswahl von Stämmen beider Spezies gegenüber drei unterschiedlichen Fettsäuren getestet: Laurinsäure (C12:0), Palmitinsäure (C16:0) und Linolsäure (C18:2). Im Gegensatz zu den ungewöhnlich sensitiven Gonokokken konnte eine hohe inhärente Fettsäureresistenz in fast allen Meningokokken-Isolaten beobachtet werden. Nach Analyse der molekularen Grundlage dieser Resistenz konnte gezeigt werden, dass sowohl eine funktionale FarAB Efflux-Pumpe als auch ein intaktes Lipopolysaccharid (LPS) für die Palmitinsäureresistenz verantwortlich sind. Trotz Umgehung der inhärenten Resistenz konnte keine Verbindung von FarR mit Fettsäureresistenz in Meningokokken hergestellt werden. Stattdessen reprimiert FarR direkt und spezifisch die Expression des Neisseria Adhäsins A (nadA), eines vielversprechenden Impfstoffbestandteils. Microarrays bestätigten diese Ergebnisse, zeigten aber keine weiteren ähnlich regulierten Gene auf. Somit ist das FarR-Regulon das bisher kleinste Regulon in Meningokokken. Die genaue FarR-Bindestelle innerhalb des nadA Promotors wurde als ein 16 bp Palindrom identifiziert und dessen Einfluss auf die Transkription von nadA mittels Reportergenanalysen gezeigt. Auch in Infektionsversuchen wurde die Relevanz dieser Repression deutlich, da ein farR-deletierter Stamm eine höhere Adhärenz an Epithelzellen aufwies. Die Transkription von farR sank nach Kontakt mit aktiven Komplementbestandteilen aus humanem Serum. Zusammenfassend wurde gezeigt, dass FarR eine Rolle in der Nischenadaptation von Meningokokken zukommt, indem er zwischen Immunevasion durch Repression des hoch-immunogenen nadA und Wirtszelladhäsion durch eben dieses Adhäsin vermittelt. KW - Transkription KW - Neisseria gonorrhoeae KW - Neisseria meningitidis KW - Adhäsine KW - FarR KW - Transkriptionsregulation KW - NadA KW - Neisseria meningitdis KW - transcriptional regulation KW - FarR KW - Neisseria gonorrhoeae KW - niche adaptation Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48550 ER - TY - THES A1 - Pawlik, Marie-Christin T1 - Gene expression in the human pathogen Neisseria meningitidis: Adaptation to serum exposure and zinc limitation T1 - Genexpression im humanen Pathogen Neisseria meningitidis: Adaptation an Serumexposition und Zinkmangel N2 - Neisseria meningitidis is a facultative human pathogen that occasionally shows strong resistance against serum complement exposure. Previously described factors that mediate meningococcal serum resistance are for example the capsule, LPS sialylation, and expression of the factor H binding protein. I aimed for identification of novel serum resistance factors, thereby following two approaches, i) the analysis of the impact of global regulators of gene expression on serum resistance; and ii) a comparative analysis of closely related strains differing in serum resistance. (i) Of six meningococcal global regulators of gene expression studied, only mutation of the zinc uptake regulator Zur reduced complement deposition on meningococci. Little was known about meningococcal Zur and regulatory processes in response to zinc. I therefore elucidated the yet unidentified meningococcal Zur regulon comparing the transcriptional response of the N. meningitidis strain MC58 under zinc-rich and zinc-deficient conditions using a common reference design of microarray analysis. The meningococcal Zur regulon comprises 17 genes, of which 15 genes were repressed and two genes were activated at high zinc condition. Amongst the Zur-repressed genes were genes involved in zinc uptake, tRNA modification, and ribosomal assembly. A 23 bp meningococcal consensus Zur binding motif (Zur box) with a conserved central palindrome was established (TGTTATDNHATAACA) and detected in the promoter region of all regulated transcriptional units (genes/operons). In vitro binding of meningococcal Zur to the Zur box of three selected genes was shown for the first time using EMSAs. Binding of meningococcal Zur to DNA depended specifically on zinc, and mutations in the palindromic sequence constrained Zur binding to the DNA motif. ii) Three closely related strains of ST-41/44 cc from invasive disease and carriage which differed in their resistance to serum complement exposure were analysed to identify novel mediators of serum resistance. I compared the strains’ gene content by microarray analysis which revealed six genes being present in both carrier isolates, but absent in the invasive isolate. Four of them are part of two Islands of horizontally transferred DNA, i.e. IHT-B and –C. The working group furthermore applied a comprehensive screening assay, a transcriptome and a proteome analysis leading to identification of three target proteins. I contributed to establish the role of these three proteins in serum resistance: The adhesin Opc mediates serum resistance by binding of vitronectin, a negative regulator of the complement system; the hypothetical protein NMB0865 slightly contributes to serum resistance by a yet unknown mechanism; and NspA, recently identified to bind the negative complement regulator factor H, led to considerable reduced complement-mediated killing. N2 - Neisseria meningitidis ist ein fakultatives Humanpathogen, welches mitunter sehr resistant gegenüber Serumkomplement-Exposition ist. Bereits beschriebene Faktoren, welche die Serumresistenz von Meningokokken fördern, sind beispielsweise die Kapsel, LPS-Sialylierung und Expression des fH-bindenden Proteins. Das Ziel dieser Arbeit war die Identifikation neuartiger Serumresistenzfaktoren, wobei ich zwei Ansätzen verfolgte: i) Die Analyse des Einflusses von globalen Regulatoren der Genexpression auf die Serumresistenz; und ii) eine vergleichenden Analyse von eng verwandten Stämmen, die sich hinsichtlich ihrer Serumresistanz unterschieden. i) Von sechs untersuchten globalen Regulatoren der Genexpression, war die Komplementdeposition auf Meningokokken nur nach Mutation des Regulators der Zinkaufnahme, Zur, reduziert. Über Zur selbst und die regulatorischen Prozesse in Reaktion auf Zink war in Meningokokken wenig bekannt. Ich habe daher das bisher nicht bestimmte Zur-Regulon von Meningokokken aufgeklärt, wofür ich mittels Mikroarrays die transkriptionelle Antwort des N. meningitidis-Stammes MC58 unter Zink-Überfluss und Zink-Mangel zu vergleichen. Das Zur-Regulon von Meningokokken umfasst 17 Gene, von denen unter Zinküberfluss 15 reprimiert und zwei aktiviert wurden. Unter den Zur-reprimierten Genen fanden sich Gene, die in die Aufnahme von Zink, die Modikation von tRNAs und den Zusammenbau des Ribosoms involviert sind. Ein 23 bp langes Binde-Konsensusmotiv für Meningokokken-Zur (Zur-Box) mit einem konservierten zentralen Palindrom wurde ermittelt (TGTTATDNHATAACA) und in der Promotorregion aller regulierten Transkriptionseinheiten (Gene/Operons) detektiert. In vitro-Bindung des N. meningitidis Zur an die Zur-Box dreier ausgewählter Genen konnte mittels EMSAs erstmals gezeigt werden. Die Bindung von Zur an DNA war spezifisch abhängig von Zink, und Mutationen in der palindromischen Sequenz hemmten die Zur-Bindung an das DNA-Motiv. ii) Drei eng verwandte Stämme des ST-41/44-Komplexes aus invasiver Erkrankung und Trägertum, die sich in ihrer Resistenz gegenüber Serumkomplement-Exposition unterschieden, wurden analysiert um neuartige Mediatoren der Serumresistenz zu identifizieren. Der Gengehalt der Stämme wurde mittels Mikroarray-Analyse verglichen. Dies offenbarte sechs Gene, die in den beiden Trägerstämmen vorhanden, aber in dem invasiven Isolat abwesend waren. Vier dieser Gene liegen innerhalb zweier Inseln horizontal transferierter DNA, d.h. IHT-B und –C. Weiterhin führte die Arbeitsgruppe eine Transkriptom- und Proteom-Analyse der drei Stämme sowie einen umfangsreichen Screening-Assay durch. Diese Ansätze führten zur Identifikation dreier Kandidaten-Proteine für die weitere Analyse. Ich wirkte daran mit, die Rolle dieser Proteine für die Serumresistenz von Meningokokken zu ermitteln: Das Adhäsin Opc vermittelt Serumresistenz durch Bindung von Vitronektin, einem negativen Regulator des Komplementsystems; das hypothetische Protein NMB0865 trägt über einen bisher unbekannten Mechanismus geringfügig zur Serumresistanz bei; und NspA, für welches vor Kurzem erkannt wurde, dass es den negativen Komplementregulator Faktor H bindet, führte zu beträchtlich reduzierter Abtötung durch Komplement. KW - Komplement KW - Neisseria meningitidis KW - Genregulation KW - Zinkmangel KW - Serumresistenz KW - Meningokokken KW - globale Regulatoren KW - Serum KW - Bakterien KW - Zink KW - serum resistance KW - meningococci KW - global regulators KW - zinc limitation Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78758 ER - TY - THES A1 - Herz, Michaela T1 - Genome wide expression profiling of Echinococcus multilocularis T1 - Genomweite Expressionsanalysen von Echinococcus multilocularis N2 - Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis. N2 - Alveoläre Echinokokkose wird durch das Metazestodenstadium des kleinen Fuchsbandwurms Echinococcus multilocularis verursacht und medizinisch als eine schwere Zoonose mit begrenzten Behandlungsmöglichkeiten betrachtet. Um ein besseres Verständnis für die Biologie der Zestoden zu erlangen, wurde das Genom von E. multilocularis, zusammen mit denen anderer Zestoden, bereits sequenziert. Bisher wurden nur wenige Studien zum Transkriptom von E. multilocularis durchgeführt und eine umfassende Analyse der Transkriptionsprofile über verschiedene Stadien des Lebenszyklus hinweg fehlt bislang. Diese Arbeit stellt die bisher umfassendste Untersuchung des Transkriptoms von E. multilocularis dar. Unterschiede in der Genexpression in verschiedenen Stadien des Lebenszyklus und unter experimentellen Bedingungen wurden qualitativ und quantitativ untersucht. Dazu wurden Daten aus RNA-Sequenzierungen in drei biologischen Replikaten verwendet. Die untersuchten Datensätze beruhen auf Proben von Metazestoden, die unter aeroben und anaeroben Bedingungen kultiviert, sowie von Metazestoden, die direkt aus Gerbilen isoliert wurden. Weitere Proben umfassen Stammzellkulturen zu drei verschiedenen Entwicklungszeitpunkten sowie nicht-aktivierte und aktivierte Protoskolizes, das Larvenstadium das sich zu Adulten entwickeln kann. Zusätzlich wurden zwei Datensätze von Metazestoden unter experimentellen Bedingungen, die zur Identifizierung stammzellspezifischer (keimzellspezifischer) Gene geeignet sind, sowie ein Datensatz von einem siRNA-Experiment untersucht. Die Analyse dieser Datensätze führte zu Genexpressionsprofilen für alle annotierten Gene, unter anderem für Gene, die im Tegument des Metazestoden exprimiert werden und eine Rolle spielen bei Wirt-Parasit-Interaktionen und der Modulierung der Immunantwort des Wirts. Genexpressionsprofile liefern zudem Informationen über Gene, die für das infiltrative Wachstum des Parasiten in der Leber verantwortlich sein könnten. Des Weiteren wurden keimzellspezifische Gene identifiziert. Keimzellen sind die einzigen proliferierenden Zellen in E. multilocularis und daher von essentieller Bedeutung für die Entwicklung und das Wachstum des Parasiten. Durch eine Kombination von Keimzelldepletierungs- und Keimzellanreicherungsverfahren wurden Gene mit keimzellspezifischer Expression identifiziert. Wie erwartet, sind viele dieser Gene in der Translation, der Zellzyklusregulation oder DNA-Replikation und –Reparatur involviert. Darüber hinaus wurden keimzellspezifisch exprimierte Transkriptionsfaktoren detektiert, von denen viele in der Festlegung des Zellschicksals eine Rolle spielen. Als Beispiel eines keimzellspezifischen Genes wurde das Gen, das für die reverse Transkriptase (TERT) kodiert, genauer untersucht. Die Expression von E. multilocularis tert in Keimzellen wurde experimentell bestätigt. Zellkulturexperimente weisen darauf hin, dass TERT für die Proliferation und die Entwicklung essentiell ist. TERT ist daher ein potentiell interessantes Wirkstofftarget für die chemotherapeutische Behandlung der alveolären Echinokokkose. Zu den keimzellspezifischen Genen in E. multilocularis gehören auch Gene densoviralen Ursprungs. Es wurden mehr als 20 Densovirusloci mit Informationen für nicht-strukturelle und strukturelle Densovirusproteine im E. multilocularis-Genom identifiziert. Densovirale Elemente wurden auch in vielen anderen Zestodengenomen detektiert. Die genomische Integration dieser Elemente deutet darauf hin, dass densovirus-basierte Vektoren zur genetischen Manipulation von Zestoden geeignet sein könnten. Interessanterweise sind nur drei von mehr als 20 Densovirusloci im E. multilocularis-Genom exprimiert. Da es in Zestoden keinen kanonischen piRNA-Signalweg gibt, stellt sich die Frage nach möglichen Genabschaltungsmechanismen. Die Analyse der RNA-Sequenzierdaten ergab Hinweise auf natürliche Antisense-Transkripte als einen möglichen Genregulationsmechanismus in E. multilocularis. Vorläufige Experimente und bisherige Studien deuten weiterhin darauf hin, dass DNA-Methylierung ein Mechanismus der Genregulation und -abschaltung in Zestoden sein könnte. Die Transkriptionsdaten enthalten auch Informationen zu Genen, die in differenzierten Zellen exprimiert werden, wie zum Beispiel das Serotonintransportergen, das in Nervenzellen exprimiert wird. Zellkulturversuche weisen darauf hin, dass Serotonin und Serotonintransport eine wichtige Rolle bei der Proliferation, der Entwicklung und dem überleben von E. multilocularis spielen. Insgesamt bietet diese Arbeit einen umfassenden Transkriptionsdatenatlas über die Stadien des Lebenszyklus von E. multilocularis. Die Identifizierung von keimzellspezifischen Genen und Genen, die für die Interaktion zwischen Wirt und Parasit wichtig sind, wird die zukünftige Forschung erheblich erleichtern. Ein globaler Überblick über die Genexpressionsprofile wird zudem hilfreich sein bei der Entdeckung geeigneter Wirkstofftargets und bei der Entwicklung neuer Chemotherapeutika gegen die alveoläre Echinokokkose. KW - Fuchsbandwurm KW - Serotonin KW - Telomerase KW - Stammzelle KW - Transkriptomanalyse KW - foxtapeworm KW - transcriptome data analysis KW - germinative cell Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203802 ER - TY - JOUR A1 - Nyawale, Helmut A. A1 - Moremi, Nyambura A1 - Mohamed, Mohamed A1 - Njwalila, Johnson A1 - Silago, Vitus A1 - Krone, Manuel A1 - Konje, Eveline T. A1 - Mirambo, Mariam M. A1 - Mshana, Stephen E. T1 - High seroprevalence of SARS-CoV-2 in Mwanza, northwestern Tanzania: a population-based survey JF - International Journal of Environmental Research and Public Health N2 - The transmission of the SARS-CoV-2 virus, which causes COVID-19, has been documented worldwide. However, the evidence of the extent to which transmission has occurred in different countries is still to be established. Understanding the magnitude and distribution of SARS-CoV-2 through seroprevalence studies is important in designing control and preventive strategies in communities. This study investigated the seropositivity of the SARS-CoV-2 virus antibodies in the communities of three different districts in the Mwanza region, Tanzania. A household cross-sectional survey was conducted in September 2021 using the modified African Centre for Disease and Prevention (ACDC) survey protocol. A blood sample was obtained from one member of each of the selected households who consented to take part in the survey. Immunochromatographic rapid test kits were used to detect IgM and IgG SARS-CoV-2 antibodies, followed by descriptive data analysis. Overall, 805 participants were enrolled in the study with a median age of 35 (interquartile range (IQR):27–47) years. The overall SARS-CoV-2 seropositivity was 50.4% (95%CI: 46.9–53.8%). The IgG and IgM seropositivity of the SARS-CoV-2 antibodies was 49.3% and 7.2%, respectively, with 6.1% being both IgG and IgM seropositive. A history of runny nose (aOR: 1.84, 95%CI: 1.03–3.5, p = 0.036), loss of taste (aOR: 1.84, 95%CI: 1.12–4.48, p = 0.023), and living in Ukerewe (aOR: 3.55, 95%CI: 1.68–7.47, p = 0.001) and Magu (aOR: 2.89, 95%CI: 1.34–6.25, p= 0.007) were all independently associated with SARS-CoV-2 IgM seropositivity. Out of the studied factors, living in the Ukerewe district was independently associated with IgG seropositivity (aOR 1.29, CI 1.08–1.54, p = 0.004). Twenty months after the first case of COVID-19 in Tanzania, about half of the studied population in Mwanza was seropositive for SARS-CoV-2. KW - SARS-CoV-2 KW - COVID-19 KW - seroprevalence KW - antibodies KW - Mwanza KW - Tanzania Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288134 SN - 1660-4601 VL - 19 IS - 18 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Scholtes, Katja A1 - Kolibay, Felix A1 - Schorscher, Nora A1 - Ertl, Georg A1 - Ernestus, Ralf-Ingo A1 - Vogel, Ulrich A1 - Franke, Axel A1 - Kowalzik, Barbara T1 - Hospital preparedness for mass critical care during SARS-CoV-2 pandemic JF - Critical Care N2 - Mass critical care caused by the severe acute respiratory syndrome corona virus 2 pandemic poses an extreme challenge to hospitals. The primary goal of hospital disaster preparedness and response is to maintain conventional or contingency care for as long as possible. Crisis care must be delayed as long as possible by appropriate measures. Increasing the intensive care unit (ICU) capacities is essential. In order to adjust surge capacity, the reduction of planned, elective patient care is an adequate response. However, this involves numerous problems that must be solved with a sense of proportion. This paper summarises preparedness and response measures recommended to acute care hospitals. KW - Mass critical care KW - Disaster response KW - SARS-CoV-2 KW - Hospital emergency plan Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230201 VL - 24 ER - TY - JOUR A1 - Brehm, Klaus A1 - Hemer, Sarah A1 - Konrad, Christian A1 - Spiliotis, Markus A1 - Koziol, Uriel A1 - Schaack, Dominik A1 - Förster, Sabine A1 - Gelmedin, Verena A1 - Stadelmann, Britta A1 - Dandekar, Thomas A1 - Hemphill, Andrew T1 - Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development N2 - Background The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. Results Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. Conclusions Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs. KW - Cestode KW - Tapeworm KW - Echinococcus KW - Echinococcosis KW - Insulin KW - Receptor kinase KW - Kinase inhibitor KW - Host-parasite interaction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110357 ER - TY - JOUR A1 - Doran, Kelly S. A1 - Fulde, Marcus A1 - Gratz, Nina A1 - Kim, Brandon J. A1 - Nau, Roland A1 - Prasadarao, Nemani A1 - Schubert-Unkmeir, Alexandra A1 - Tuomanen, Elaine I. A1 - Valentin-Weigand, Peter T1 - Host-pathogen interactions in bacterial meningitis JF - Acta Neuropathologica N2 - Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. Pathogenesis is based on complex host-pathogen interactions, some of which are specific for certain bacteria, whereas others are shared among different pathogens. In this review, we summarize the recent progress made in understanding the molecular and cellular events involved in these interactions. We focus on selected major pathogens, Streptococcus pneumonia, S. agalactiae (Group B Streptococcus), Neisseria meningitidis, and Escherichia coli K1, and also include a neglected zoonotic pathogen, Streptococcus suis. These neuroinvasive pathogens represent common themes of host-pathogen interactions, such as colonization and invasion of mucosal barriers, survival in the blood stream, entry into the central nervous system by translocation of the blood-brain and blood-cerebrospinal fluid barrier, and induction of meningeal inflammation, affecting pia mater, the arachnoid and subarachnoid spaces. KW - microvascular endothelial cells KW - outer membrane protein KW - Neuroinfectiology KW - Bacterial meningitis KW - Pneumococci KW - Meningococci KW - Group B Streptococcus KW - Streptococcus suis KW - Escherichia coli K1 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191034 VL - 131 IS - 2 ER - TY - THES A1 - Nono, Justin T1 - Immunomodulation through Excretory/Secretory Products of the parasitic Helminth Echinococcus multilocularis T1 - Immunmodulation durch Exkretorisch/Sekretorischen Produkten der parasitären Helminthen Echinococcus multilocularis N2 - Die Alveoläre Echinokokkose (AE) ist eine lebensbedrohliche Zoonose, die durch das Metazestoden-Larvenstadium des Fuchsbandwurms Echinococcus multilocularis ausgelöst wird. Nach Eintritt des Parasiten in den Zwischenwirt wird zunächst eine potentiell anti-parasitische, Th1-dominierte Immunantwort ausgelöst, welche anschließend in der chronischen Phase graduell durch eine permissive, Th2-dominierte Antwort ersetzt wird. Als Ergebnis einer zugrunde liegenden Immunmodulation durch den Parasiten können Echinococcus-Larven für Jahre bis Jahrzehnte im Wirt persistieren und verhalten sich ähnlich einem perfekt transplantierten Organ. Über die molekulare Basis der Immunmodulation durch den Parasiten ist derzeit wenig bekannt. In dieser Arbeit wurden geeignete Kultursysteme für verschiedene E. multilocularis Larvenstadien verwendet, um den Einfluss exkretorisch/sekretorischer Metaboliten (E/S-Produkte) auf Wirts-Immuneffektor-Zellen zu studieren. E/S-Produkte kultivierter Larven, die die frühe (Primärzellen) und chronische (Metazestode) Phase der Infektion repräsentieren induzierten Apoptose und tolerogene Eigenschaften in Dendritischen Zellen (DC) des Wirts, während solche von Kontroll-Larven (Protoskolizes) keine derartigen Effekte zeigten. Dies zeigt, dass die frühen infektiösen Stadien von E. multilocularis in DC ein tolerierendes Milieu erzeugen, welches sehr wahrscheinlich die initiale Etablierung des Parasiten in einer Phase begünstigt, in der er höchst sensitiv gegenüber Wirtsangriffen ist. Interessanterweise förderten E/S-Produkte des Metazestoden in vitro die Konversion von CD4+ T-Zellen in Foxp3+, regulatorische T-Zellen (Treg) während E/S-Produkte von Primärzellen oder Protoskolizes dies nicht vermochten. Da Foxp3+ Tregs generell als immunosuppressorisch bekannt sind, deuten diese Daten an, dass der Metazestode aktiv eine Induktion von Tregs herbeiführt, um eine permissive Immunsuppression während einer Infektion zu erreichen. Eine substantielle Zunahme von Anzahl und Frequenz Foxp3+ Tregs konnte zudem in Peritoneal-Exsudaten von Mäuuen nach intraperitonealer Injektion von Parasitengewebe gemessen werden, was anzeigt, dass eine Expansion von Foxp3+ Tregs auch während der in vivo Infektion von Bedeutung ist. Interessanterweise konnte in dieser Arbeit ein Activin-Orthologes des Parasiten, EmACT, identifiziert werden, weleches vom Metazestoden sekretiert wird und ähnlich wie humanes Activin in der Lage ist, eine TGF-β-abhängige Expansion von Tregs in vitro zu induzieren. Dies zeigt an, dass E. multilocularis evolutionsgeschichtlich konservierte Zytokine nutzt, um aktiv die Wirts-Immunantwort zu beeinflussen. Zusammenfassend deuten die gewonnenen Daten auf eine wichtige Rolle Foxp3+ Tregs, welche u.a. durch EmACT induziert werden, im immunologischen geschehen der AE hin. Ein weiterer Parasiten-Faktor, EmTIP, mit signifikanten Homologien zum T-cell Immunomodulatory Protein (TIP) des Menschen wurde in dieser Arbeit näher charakterisiert. EmTIP konnte in der E/S-Fraktion von Primärzellen nachgewiesen werden und induzierte die Freisetzung von IFN-γ in CD4+ T-Helferzellen. Durch Zugabe von anti-EmTIP-Antikörpern konnte zudem die Entwicklung des Parasiten zum Metazestoden in vitro gehemmt werden. EmTIP dürfte daher einerseits bei der frühen Parasiten-Entwicklung im Zwischenwirt eine Rolle spielen und könnte im Zuge dessen auch die Ausprägung der frühen, Th-1-dominierten Immunantwort während der AE begünstigen. Zusammenfassend wurden in dieser Arbeit zwei E. multilocularis E/S-Faktoren identifiziert, EmACT und EmTIP, die ein hohes immunmodulatorisches Potential besitzen. Die hier vorgestellten Daten liefern neue, fundamentale Einsichten in die molekularen Mechanismen der Parasiten-induzierten Immunmodulation bei der AE und sind hoch relevant für die Entwicklung anti-parasitischer Immuntherapien. N2 - Alveolar echinococcosis (AE) is a severe and life-threatening disease caused by the metacestode larva of the fox-tapeworm Echinococcus multilocularis. Parasite entry into the host evokes an early and potentially parasiticidal Th1 immune response that is gradually replaced by a permissive Th2 response. An immunoregulatory environment has also been reported in the host as the disease progresses. As a result of immunomodulation, E. multilocularis larvae persist in the host for decades without being expelled, and thus almost act like a perfect transplant. Very little is currently known on the molecular basis of the host immunomodulation by E. multilocularis. In this work, in vitro cultivation systems were used to assess the influence of metabolites released by the parasite larvae (E/S products) on host immune effector cells. E/S products of cultivated larvae that respresent the early (primary cells) and chronic (metacestode vesicles) phase of AE induced apoptosis and tolerogenic properties (poor responsiveness to LPS stimulation) in host dendritic cells (DC) whereas those of control larvae (protoscoleces) failed to do so. These findings show that the early infective stage of E. multilocularis induces tolerogenicity in host DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. Interestingly, metacestode E/S products promoted the conversion of naïve CD4+ T-cells into Foxp3+ regulatory T-cells in vitro, whereas primary cell and protoscolex E/S products failed to do it. Since Foxp3+ regulatory T-cells are generally known to mediate immunosuppression, the present finding indicates that Foxp3+ regulatory T-cells, expanded by E/S products of the metacestode larva, could play a role in the parasite-driven immunomodulation of the host observed during AE. Furthermore, a substantial increase in number and frequency of suppressive Foxp3+ regulatory T-cells could be observed within peritoneal exudates of mice following intraperitoneal injection of E. multilocularis metacestodes, indicating that Foxp3+ regulatory T-cells could also play an important role in E. multilocularis-driven immunomodulation in vivo. Interestingly, a parasite activin ortholog, EmACT, secreted by metacestodes, was shown to expand host regulatory T-cells in a TGF-β-dependent manner, similarly to mammalian activin A. This observation indicated that E. multilocularis utilizes evolutionarily conserved TGF-β superfamily ligands, like EmACT, to expand host regulatory T-cells. Taken together, the present findings suggest EmACT, a parasite activin secreted by the metacestode and capable of expanding host regulatory T-cells, as an important player in the host immunomodulation by E. multilocularis larvae. Another parasite factor EmTIP, homologous to mammalian T-cell immunomodulatory protein (TIP) was characterized in this work. EmTIP could be detected in the secretions of the parasite primary cells and localized to the intercellular space within the parasite larvae. EmTIP blockade inhibited the proliferation of E. multilocularis primary cells and the formation of metacestode vesicles indicating a major role for parasite development. Furthermore, EmTIP evoked a strong release of IFN-γ by CD4+ T-cells hence suggesting that the secretion of this factor as a result of its role in parasite development could “secondarily” induce a potentially protective Th1 response. In conclusion, this work identified two molecules, EmACT and EmTIP, with high immunomodulatory potential that are released by E. multilocularis larvae. The data presented do provide insights into the mechanisms of parasite-driven host immunomodulation during AE that are highly relevant for the development of anti-parasitic immune therapies. KW - Immunmodulation KW - Fuchsbandwurm KW - Regulatorischer T-Lymphozyt KW - Dendritische Zelle KW - Immunomodulation KW - Helminths KW - Tapeworm KW - Echinococcus KW - Regulatory T-cell KW - Dendritic cell KW - Würmer Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85449 ER - TY - JOUR A1 - Brehony, Carina A1 - Trotter, Caronline L. A1 - Ramsay, Mary E. A1 - Chandra, Manosree A1 - Jolley, Keith A. A1 - van der Ende, Arie A1 - Carion, Françoise A1 - Berthelsen, Lene A1 - Hoffmann, Steen A1 - Harðardóttir, Hjördís A1 - Vazques, Julio A. A1 - Murphy, Karen A1 - Toropainen, Maija A1 - Caniça, Manuela A1 - Ferreira, Eugenia A1 - Diggle, Mathew A1 - Edwards, Giles F. A1 - Taha, Muhamed-Kheir A1 - Stefanelli, Paola A1 - Kriz, Paula A1 - Gray, Steve J. A1 - Fox, Andrew J. A1 - Jacobsson, Susanne A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Tzanakaki, Georgina A1 - Heuberger, Sigrid A1 - Caugant, Dominique A. A1 - Frosch, Matthias A1 - Maiden, Martin C. J. T1 - Implications of Differential Age Distribution of Disease-Associated Meningococcal Lineages for Vaccine Development JF - Clinical and Vaccine Immunology : CVI N2 - New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups. KW - differential age distribution KW - disease-associated KW - meningococcal lineages KW - vaccine development Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120808 VL - 21 IS - 6 ER - TY - JOUR A1 - Stock, Nina Katharina A1 - Petráš, Petr A1 - Melter, Oto A1 - Kapounová, Gabriela A1 - Vopalková, Petra A1 - Kubele, Jan A1 - Vaniš, Václav A1 - Tkadlec, Jan A1 - Bukáčková, Eva A1 - Machová, Ivana A1 - Jindrák, Vlastimil T1 - Importance of Multifaceted Approaches in Infection Control: A Practical Experience from an Outbreak Investigation JF - PLoS ONE N2 - Background This study presents the results of a multidisciplinary, nosocomial MRSA outbreak investigation in an 8-bed medical intensive care unit (ICU). The identification of seven MRSA positive patients in the beginning of 2014 led to the closure of the ward for several weeks. A multidisciplinary, retrospective investigation was initiated in order to identify the reason and the source for the outbreak, describe MRSA transmission in the department and identify limitations in infection control. Methods The investigation comprised an epidemiological description of MRSA cases from 2012 to 2014 and a characterization of MRSA isolates, including phage-, spa- and PFGE-typing. Additionally, MRSA screening was performed from the hospital staff and the environment. To identify the reason for the outbreak, work-related, psychological and behavioral factors were investigated by impartial audits and staff interviews. Results Thirty-one MRSA cases were registered during the study period, and 36 isolates were investigated. Molecular typing determined the outbreak strain (phage type 54/812, PFGE type A4, spa type t003) and identified the probable index case. Nasal carriage in one employee and a high environmental contamination with the outbreak strain was documented. Important gaps in nursing procedures and general management were identified. Elevated stress levels and communication problems preceded the outbreak. Compliance with hand hygiene and isolation procedures was evaluated as appropriate. Conclusion This study demonstrates the complexity of controlling hospital-associated infections. The combined use of different typing methods is beneficial for outbreak investigations. Psychological, behavioral and other work-related factors have an important impact on the spread of nosocomial pathogens. These factors should be addressed and integrated in routine infection control practice. KW - multifaceted approaches KW - infection control KW - Methicillin resistant Staphylococcus aureus KW - MRSA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166891 VL - 11 IS - 6 ER - TY - JOUR A1 - Kohlmorgen, Britta A1 - Elias, Johannes A1 - Schoen, Christoph T1 - Improved performance of the artus Mycobacterium tuberculosis RG PCR kit in a low incidence setting: a retrospective monocentric study JF - Scientific Reports N2 - Tuberculosis (TB) and the spread of Mycobacterium tuberculosis complex (MTBC) strains resistant against rifampin (RIF) and isoniazid (INH) pose a serious threat to global health. However, rapid and reliable MTBC detection along with RIF/INH susceptibility testing are challenging in low prevalence countries due to the higher rate of false positives. Here, we provide the first performance data for the artus MTBC PCR assay in a low prevalence setting. We analyze 1323 respiratory and 311 non-respiratory samples with the artus MTBC PCR assay as well as by mycobacterial culture and microscopy. We propose retesting of specimens in duplicate and consideration of a determined cycle-threshold value cut-off greater than 34, as this significantly increases accuracy, specificity, and negative predictive value without affecting sensitivity. Furthermore, we tested fourteen MTBC positive samples with the GenoType MTBDRplus test and demonstrate that using an identical DNA extraction protocol for both assays does not impair downstream genotypic testing for RIF and INH susceptibility. In conclusion, our procedure optimizes the use of the artus MTB assay with workload efficient methods in a low incidence setting. Combining the modified artus MTB with the GenoType MTBDRplus assays allows rapid and accurate detection of MTBC and RIF/INH resistance. KW - laboratory techniques and procedures KW - Tuberculosis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159248 VL - 7 IS - 14127 ER - TY - JOUR A1 - Walter, T. A1 - Collenburg, L. A1 - Japtok, L. A1 - Kleuser, B. A1 - Schneider-Schaulies, S. A1 - Müller, N. A1 - Becam, J. A1 - Schubert-Unkmeir, A. A1 - Kong, J. N. A1 - Bieberich, E. A1 - Seibel, J. T1 - Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells JF - Chemical Communications N2 - The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells. KW - Ceramide KW - Apoptosis KW - Golgi KW - N-oleoyl serinol KW - Jurkat cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191263 VL - 52 IS - 55 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - THES A1 - Lee, Sae Kyung T1 - Interaction of Helicobacter pylori flagellins with the host innate immune system T1 - Interaktion von Helicobacter pylori Flagellin mit dem angeborenen Immunsystem des Wirtes N2 - Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium. It resides in the gastric mucous layer and epithelial lining of the stomach, often clustering at the junction of epithelial cells. H. pylori colonization usually occurs during childhood, and, when left untreated, generally persists for the host’s lifetime. Persistent H. pylori infection can cause chronic superficial gastritis and gastric duodenal ulcers, which is possibly linked to the development of gastric carcinoma and primary gastric lymphoma, especially of the mucosa-associated lymphoid tissue (MALT) type. It was recently defined as a class 1 carcinogen. The gastric inflammatory response to H. pylori infection is characterized by infiltration of the mucosa by neutrophils, T and B cells, plasma cells and macrophages. This reaction is initially induced by H. pylori attachment, followed by cytokine release by gastric epithelial cells. Epidemiological studies revealed that more than 50% of adults are infected with H. pylori all over the world. However, interestingly, only a subset of individuals develops serious H. pylori-related disease, while most infected individuals show no clinical symptoms. Gastric epithelial cells, like intestinal epithelial cells, express a subset of Toll-like receptors (TLRs) and similar pattern recognition receptors, which are important for the activation of the innate immune system. Bacterial components such as lipopeptides, peptidoglycan, LPS, flagellin, and CpG DNA are the ligands of TLRs. Thus, TLRs in gastric epithelial cells might be able to contribute to innate immune responses to H. pylori infection. However, there is scant knowledge about the mechanisms of innate immune response to acute and chronic H. pylori infection. This study is focused on host cell interaction with H. pylori flagellins, which are major components of the flagellar apparatus, and innate immune responses against them. The flagellins, which are essential for bacterial motility, are important for H. pylori to survive in the stomach mucus during the whole infectious cycle. Flagellins are known to act as the main determinant of many mucosal pathogenic bacteria that mediates proinflammatory signaling, including transcriptional factor NF-B activation via TLR5. In the first part of the study, we investigated the effects of H. pylori flagellins on TLR5 expression, NF-B activation and IL-8 production in various human intestinal and gastric epithelial cell lines by using Western blotting, semi-quantitative RT-PCR and ELISA. IL-8 is a potent neutrophil-activating chemokine expressed by gastric epithelial cells. When we stimulated the cells with the native form of or E. coli-expressed recombinant H. pylori flagellins, FlaA and FlaB, IL-8 was not induced in any case, while S. typhimurium flagellin (FliC) induced it significantly. H. pylori was able to modulate TLR5 protein expression and NF-B activation in epithelial cells regardless of the presence of flagellins. Having established the finding that H. pylori flagellins have unusually low immune-stimulatory properties, we further investigated to find out possible reasons why H. pylori flagellins are distinct from other flagellins of pathogenic bacteria in terms of immune-stimulatory activity. From amino acid sequence comparisons, we found that some regions in the terminal D0D1 protein domains of H. pylori flagellins are different from flagellins of other pathogenic bacteria. D0D1 is the domain which is known to interact with TLR5 in Salmonella FliC. To examine whether the differences endow H. pylori flagellins with low immune-stimulatory properties, we created several mutated H. pylori flagellins (FlaA and FlaB) by site-directed mutagenesis that contain one to four epitopes of Salmonella flagellin D0D1 domain amino acid sequences. The mutant flagellins expressed both in H. pylori and E. coli were used to determine their influence on TLR5-signaling mediators and cytokines, such as MAPkinases, (ERK, p38), NF-B, IL-8, and MIP-3. Salmonella FliC expressed in E. coli induced activation of p38, IB and NF-B leading to IL-8 and MIP-3 production in gastric epithelial cells. However, none of the H. pylori flagellin mutants activated MAP kinases or induced those cytokines. In a co-immunoprecipitation assay none of the recombinant wild type or mutated H. pylori flagellins showed any direct physical interaction with TLR5, while Salmonella FliC significantly co-precipitated with TLR5. Interestingly, we found H. pylori flagellins bind to the surface of gastric epithelial cells like FliC, although they do not bind to or stimulate TLR5. Based on the physical interaction of H. pylori flagellins and FliC with human gastric epithelial cells, we further analyzed transcriptional regulation by H. pylori flagellin in these host cells using microarray analysis. The result showed that H. pylori flagellins modulate host cell gene expression, and many of the identified regulation events overlap with the genes regulated by FliC. These findings imply that H. pylori flagellins do play a role in gene regulation of host cells probably through still unknown factors or receptors, although they do not trigger TLR5-related signaling pathways. The results of our study suggest that, in addition to the low immune-stimulatory activity of H. pylori LPS, the evolutionary reduction in stimulating activity of H. pylori flagellins on the local innate immune responses in the stomach in vivo might be a further strategy of this chronic mucosal pathogen to evade and minimize deleterious host responses, thereby promoting life-long persistence in the host, and possibly contributing to cancerogenesis. N2 - Helicobacter pylori (H. pylori) ist ein gram-negatives, mikroaerophiles, spiralförmiges Bakterium. Es besiedelt die Schleimschicht und die Epitheloberfläche des Magens, wobei es sich besonders an den Kontaktstellen der Epithelzellen anlagert. Die Kolonisation mit H. pylori erfolgt normalerweise während der Kindheit und bleibt, wenn sie nicht behandelt wird, im allgemeinen während der gesamten Lebenszeit des Wirtes bestehen. Die persistierende H. pylori-Infektion kann chronische oberflächliche Gastritis und Zwöffingerdarmgeschwüre verursachen. Die Infektion kann auch zur Entwicklung von Magenkrebs und Lymphomen des Mukosa-assoziierten Lymphgewebes (MALT-Lymphom) führen. H. pylori ist seit 1994 als Typ I-Karzinogen klassifiziert. Die durch eine H. pylori-Infektion induzierte Entzündungsreaktion der Magenschleimhaut ist charakterisiert durch eine Infiltration der Schleimhaut mit neutrophilen Granulozyten, T- und B-Zellen, Plasmazellen und Makrophagen. Diese Reaktion wird ausgelöst durch die Anheftung von H. pylori gefolgt von der Freisetzung von Cytokinen durch die Magenepithelzellen. Epidemiologische Studien haben ergeben, dass weltweit mehr als 50% aller Erwachsenen mit H. pylori infiziert sind. Jedoch entwickelt interessanterweise nur eine Teilgruppe der infizierten Individuen eine ernsthafte H. pylori-assoziierte Krankheit, während die meisten Infizierten keine klinischen Symptome zeigen. Magenepithelzellen exprimieren, genauso wie Darmepithelzellen, eine Reihe von TOLL-like Rezeptoren (TLRs) und ähnliche Musterekennungsrezeptoren, die wichtig für die Aktivierung des angeborenen Immunsystems sind. Bakterielle Komponenten, wie z. B. Lipopeptide, Peptidoglycan, LPS, Flagellin und CpG-DNA sind die Liganden der TLRs. Auf diese Weise könnten die TLRs in den Magenepithelzellen in der Lage sein, zu der angeborenen Immunreaktion auf eine H. pylori-Infektion beizutragen. Jedoch ist bisher nur wenig über die Mechanismen der angeborenen Immunreaktion auf eine akute und chronische H. pylori-Infektion bekannt. Diese Studie befasst sich mit den Zellinteraktionen mit und den Antworten des Wirtsimmunsystems auf H. pylori-Flagelline, stark exprimierte Proteine des Flagellenapparats. Der Flagellenapparat ist essentiell für die Fähigkeit der Bakterien, im Magenschleim (Mukus) beweglich zu sein, und befähigt die Bakterien dazu, während des gesamten Infektionszyklus im Mukus und an der Magenmukosa zu überleben. Flagellin ist für viele pathogene Bakterien im Intestinaltrakt oder auch in der Lunge des Säuger-Wirts ein sehr wichtiger Faktor, der durch Bindung an TLR5 proinflammatorische Signalvorgänge, einschließlich der Aktivierung des Transkriptionsfaktors NF-B, herbeiführt. Im ersten Teil der vorliegenden Studie haben wir die Wirkungen von H. pylori-Flagellinen (FlaA, FlaB) auf TLR5-Expression, NF-B-Aktivierung und IL-8-Produktion in verschiedenen menschlichen Darm- und Magenepithelzelllinien mittels Western Blot, semi-quantitativer RT-PCR und ELISA untersucht. IL-8 ist ein hochwirksames Neutrophilen-aktivierendes Chemokin, welches von den Magenepithelzellen sezerniert wird und als ein Marker für die Zellaktivierung durch H. pylori, seine löslichen Produkte und Kontrollen diente. Nach Stimulation verschiedener Epithelzellen und humaner Makrophagen mit nativen oder in E. coli rekombinant hergestellten H. pylori-Flagellinen FlaA und FlaB wurde in keinem Fall IL-8 gebildet, während S. typhimurium-Flagellin (FliC) IL-8-Bildung und -Sekretion in signifikanter Menge induzierte. H. pylori war in der Lage, TLR5-Protein-Expression und die NF-B-Aktivierung in Epithelzellen zu modulieren, unabhängig von dem Vorhandensein von Flagellinen. Nachdem wir gezeigt hatten, dass H. pylori-Flagelline ungewöhnlich geringe immunstimulierende Eigenschaften besitzen, setzten wir unsere Untersuchungen fort, um mögliche Gründe herauszufinden, warum H. pylori-Flagelline sich von anderen Flagellinen pathogener Bakterien hinsichtlich der das Immunsystem stimulierenden Aktivitäten unterscheiden. Bei Vergleichen von Aminosäuresequenzen fanden wir heraus, dass einige Regionen in den terminalen D0D1-Domänen der H. pylori-Flagelline sich von Flagellinen anderer pathogener Bakterien unterscheiden. D0D1 ist der Funktionsbereich des Flagellins, von dem bekannt ist, dass er bei Salmonellen-FliC mit TLR5 interagiert. Um zu untersuchen, ob diese Unterschiede für die geringe immunstimulierende Wirkung von H. pylori-Flagellinen verantwortlich sind, generierten wir durch eine zielgerichtete Mutagenese mehrere mutierte H. pylori-Flagelline (FlaA und FlaB), die ein bis vier Epitope der Aminosäuresequenzen der D0D1-Domäne des Salmonella-Flagellins enthielten. Die mutierten Flagelline, die sowohl in H. pylori als auch in E. coli exprimiert wurden, wurden genutzt, um ihren Einfluss auf TLR5-Signal-Mediatoren und Cytokine, wie z. B. MAP-Kinasen (ERK, p38), NF-B, IL-8 und MIP-3α herauszufinden. In E. coli exprimiertes Salmonella-FliC bewirkte die Aktivierung von p38, IB, NF-B und ERK und führte zur Produktion von IL-8 und MIP-3α in den Magenepithelzellen. Im Gegensatz dazu aktivierte keine der H. pylori-Flagellinmutanten MAP-Kinasen oder induzierte diese Cytokine. Wir konnten durch Koimmunpräzipitationstechniken zeigen, dass wildtypische oder mutierte H. pylori-Flagelline nicht physisch mit TLR5 interagieren, während Salmonella-FliC spezifisch an TLR5 bindet. Interessanterweise fanden wir heraus, dass H. pylori-Flagelline wie FliC an die Oberfläche verschiedener humaner Epithelzellen binden, obwohl sie nicht TLR5 stimulieren oder an TLR5 binden. Basierend auf der physischen Interaktion von H. pylori-Flagellinen und FliC mit menschlichen Magenepithelzellen haben wir weiterhin die Transkriptionsregulation durch H. pylori-Flagellin in den Wirtszellen mit Hilfe der Microarray-Analyse untersucht. Die Ergebnisse zeigten, dass H. pylori-Flagelline die Gene der Wirtszelle modulieren, und viele der identifizierten Regulationsereignisse überschnitten sich mit den durch FliC regulierten Genen. Diese Ergebnisse implizieren, dass H. pylori-Flagelline doch eine Rolle bei der Genregulierung von Wirtszellen spielen, wahrscheinlich durch noch unbekannte Faktoren und Rezeptoren, obwohl sie keine mit TLR5 in Zusammenhang stehenden Signaltransduktionsketten auslösen. Die Resultate unserer Studien lassen darauf schließen, dass zusätzlich zu der das Immunsystem nur gering stimulierenden Aktivität von H. pylori-LPS die evolutionäre Reduzierung der stimulierenden Aktivität von H. pylori-Flagellinen auf lokale angeborene Immunreaktionen im Magen in vivo eine weitere Strategie dieses chronischen Schleimhautpathogens sein könnte, um schädliche Wirtsreaktionen zu verhindern und zu minimieren und hierdurch seine lebenslange Persistenz, jedoch auch die Krebsentstehung im Wirt zu fördern. KW - Helicobacter pylori KW - TLR5 KW - Helicobacter KW - Flagellin KW - angeborene Immunität KW - TLR5 KW - Helicobacter KW - Flagellin KW - Innate immunity Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-19917 ER - TY - JOUR A1 - Forster, Johannes A1 - Dichtl, Karl A1 - Wagener, Johannes T1 - Lower beta‐1,3‐D‐glucan testing cut‐offs increase sensitivity for non‐albicans Candida species bloodstream infections JF - Mycoses N2 - Purpose Fungal biomarkers support early diagnosis of invasive fungal infections. In this study, we evaluated the impact of a recent update to the manufacturer‐recommended cut‐off for beta‐1,3‐D‐glucan (BDG) testing (Fujifilm Wako BDG assay) on sensitivity and specificity for the detection of candidemia. Additionally, we compared the performance with tests for Candida antigen (Ag by Serion ELISA antigen Candida, Virion\Serion) and anti‐mannan antibodies (Ab by Hemkit Candida IHA, Ravo Diagnostika). Methods Sera of 82 patients with candidemia, which were sampled with a maximum distance of ±14 days from the date of sampling of the corresponding positive blood cultures, were retrospectively analysed for BDG, Ag and Ab. Results of BDG testing were compared with results from sera of 129 patients with candidemia from a different hospital. Results Sensitivity of BDG testing (47%) was higher than for Ag (17%) or Ab (20%). By combining Ag and Ab testing, sensitivity was raised to 32%. Lowering the cut‐off of BDG from 11 pg/ml to the newly recommended cut‐off of 7 pg/ml resulted in a significant increase in sensitivity (47% vs 58%, p = .01 and 63% vs 71% p < .01). At both centres, the increase was significant in NAC but not in C. albicans candidemia. No significant effects on specificity were observed. Conclusion BDG testing outperformed Ag and Ab testing and its combination. Lowering the BDG cut‐off had no significant impact on specificity. The increase in sensitivity can be mainly attributed to a gain in sensitivity for non‐albicans Candida species bloodstream infections. KW - antigen testing KW - BDG KW - beta‐d‐glucan KW - bloodstream infection KW - candidemia KW - mannan Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276515 VL - 65 IS - 5 SP - 500 EP - 507 ER - TY - JOUR A1 - Kurotschka, Peter Konstantin A1 - Tiedemann, Elena A1 - Wolf, Dominik A1 - Thier, Nicola A1 - Forster, Johannes A1 - Liese, Johannes G. A1 - Gagyor, Ildiko T1 - Management of common infections in German primary care: a cross-sectional survey of knowledge and confidence among General Practitioners and outpatient pediatricians JF - Antibiotics N2 - Outpatient antibiotic use is closely related to antimicrobial resistance and in Germany, almost 70% of antibiotic prescriptions in human health are issued by primary care physicians (PCPs). The aim of this study was to explore PCPs, namely General Practitioners' (GPs) and outpatient pediatricians' (PDs) knowledge of guideline recommendations on rational antimicrobial treatment, the determinants of confidence in treatment decisions and the perceived need for training in this topic in a large sample of PCPs from southern Germany. Out of 3753 reachable PCPs, 1311 completed the survey (overall response rate = 34.9%). Knowledge of guideline recommendations and perceived confidence in making treatment decisions were high in both GPs and PDs. The two highest rated influencing factors on prescribing decisions were reported to be guideline recommendations and own clinical experiences, hence patients' demands and expectations were judged as not influencing treatment decisions. The majority of physicians declared to have attended at least one specific training course on antibiotic use, yet almost all the participating PCPs declared to need more training on this topic. More studies are needed to explore how consultation-related and context-specific factors could influence antibiotic prescriptions in general and pediatric primary care in Germany beyond knowledge. Moreover, efforts should be undertaken to explore the training needs of PCPs in Germany, as this would serve the development of evidence-based educational interventions targeted to the improvement of antibiotic prescribing decisions rather than being focused solely on knowledge of guidelines. KW - infectious diseases management KW - general practitioner KW - pediatrician KW - primary care KW - outpatient KW - antibiotic use KW - antimicrobial resistance KW - antimicrobial stewardship KW - survey KW - knowledge Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246272 SN - 2079-6382 VL - 10 IS - 9 ER - TY - JOUR A1 - Schoen, Christoph A1 - Kischkies, Laura A1 - Elias, Johannes A1 - Ampattu, Biju Joseph T1 - Metabolism and virulence in Neisseria meningitidis N2 - A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions. KW - Neisseria meningitidis KW - virulence KW - pathometabolism KW - oxidative stress KW - glutathione KW - γ-glutamyl cycle KW - glutamate dehydrogenase KW - nitrite respiration Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113118 ER - TY - JOUR A1 - Ruf, Dominik A1 - Brantl, Victor A1 - Wagener, Johannes T1 - Mitochondrial Fragmentation in \(Aspergillus\) \(fumigatus\) as Early Marker of Granulocyte Killing Activity JF - Frontiers in Cellular and Infection Microbiology N2 - The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. KW - Aspergillus fumigatus KW - killing KW - assay KW - PMNs KW - granulocytes KW - mitochondria KW - mitochondrial morphology KW - fungicidal activity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227133 VL - 8 IS - 128 ER - TY - THES A1 - Konrad, Christian T1 - Molecular analysis of insulin signaling mechanisms in Echinococcus multilocularis and their role in the host-parasite interaction in the alveolar echinococcosis T1 - Molekulare Analyse der Insulin-Signalmechanismen in Echinococcus multilocularis und ihre Rolle in der Wirt-Parasiten-Interaktion in der Alveolären Echinokokkose N2 - The insulin receptor ortholog EmIR of the fox-tapeworm Echinococcus multilocularis displays significant structural homology to the human insulin receptor (HIR) and has been suggested to be involved in insulin sensing mechanisms of the parasite’s metacestode larval stage. In the present work, the effects of host insulin on Echinococcus metacestode vesicles and the proposed interaction between EmIR and mammalian insulin have been studied using biochemical and cell-biological approaches. Human insulin, exogenously added to in vitro cultivated parasite larvae, (i) significantly stimulated parasite survival and growth, (ii) induced DNA de novo synthesis in Echinococcus, (iii) affected overall protein phosphorylation in the parasite, and (iv) specifically induced the phosphorylation of the parasite’s Erk-like MAP kinase orthologue EmMPK1. These results clearly indicated that Echinococcus metacestode vesicles are able to sense exogenous host insulin which induces a mitogenic response. To investigate whether EmIR mediates these effects, anti-EmIR antibodies were produced and utilized in biochemical assays and immunohistochemical analyses. EmIR was shown to be expressed in the germinal layer of the parasite both on the surface of glycogen storing cells and undifferentiated germinal cells. Upon addition of exogenous insulin to metacestode vesicles, the phosphorylation of EmIR was significantly induced, an effect which was suppressed in the presence of specific inhibitors of insulin receptor-like tyrosine kinases. Furthermore, upon expression of EmIR/HIR receptor chimera containing the extracellular ligand binding domain of EmIR in HEK 293 cells, a specific autophosphorylation of the chimera could be induced through the addition of exogenous insulin. These results indicated the capability of EmIR to sense and to transmit host insulin signals to the Echinococcus signaling machinery. The importance of insulin signaling mechanisms for parasite survival and growth were underscored by in vitro cultivation experiments in which the addition of an inhibitor of insulin receptor tyrosine kinases led to vesicle degradation and death. Based on the above outlined molecular data on the interaction between EmIR and mammalian insulin, the parasite’s insulin receptor orthologue most probably mediates the insulin effects on parasite growth and is, therefore, a potential candidate factor for host-parasite communication via evolutionary conserved pathways. In a final set of experiments, signaling mechanisms that act downstream of EmIR have been analyzed. These studies revealed significant differences between insulin signaling in Echinococcus and the related cestode parasite Taenia solium. These differences could be associated with differences in the organo-tropism of both species. N2 - Der orthologe Insulinrezeptor EmIR des Fuchsbandwurmes Echinococcus multilocularis weist signifikante strukturelle Homologie zum humanen Insulinrezeptor (HIR) auf. Es wurde schon seit geraumer Zeit vermutet, dass EmIR an den Mechanismen beteiligt sein könnte, die es dem Metacestoden Larvenstadium des Parasiten erlauben Insulin zu detektieren. In dieser Arbeit wurden die Effekte von Wirtsinsulin auf Echinococcus Metacestoden-Vesikel und die vermutete Interaktion zwischen EmIR und Insulin von Säugern mittels biochemischer und zellbiologischer experimenteller Ansätze untersucht. Die exogene Zugabe von humanem Insulin zu in vitro kultivierten Parasitenlarven hatte folgende Effekte: (i) das Überleben und das Wachstum des Parasiten wurde signifikant stimuliert; (ii) die DNA de novo Synthese in Echinococcus wurde induziert; (iii) die generelle Proteinphosphorylierung des Parasiten wurde beeinflusst; (iv) die Phosphorylierung der orthologen Erk-like MAP Kinase, EmMPK1, des Parasiten wurde spezifisch induziert. Diese Beobachtungen zeigen deutlich, dass Echinococcus Metacestoden-Vesikel exogenes Insulin des Wirtes detektieren können und dass dieses Insulin einen mitogenischen Effekt auf den Parasiten hat. Um zu untersuchen, ob diese Effekte durch EmIR vermittelt werden, wurden anti-EmIR Antikörper hergestellt und in biochemischen experimentellen Ansätzen und immunohistochemischen Analysen eingesetzt. Es konnte gezeigt werden, dass EmIR in der Germinalschicht des Parasiten expremiert wird, sowohl an der Oberfläche von Glykogen-Speicherzellen als auch von undifferenzierten Germinalzellen. Nach der Zugabe von exogenem Insulin konnte eine signifikante Zunahme der Phosphorylierung von EmIR festgestellt werden. Diese Stimulierung konnte durch die Zugabe eines spezifischen Inhibitors für Insulinrezeptor-ähnliche Tyrosinkinasen unterdrückt werden. Desweiteren konnte mittels der Expression eines chimären EmIR/HIR-Rezeptors, der die extrazelluläre Ligandenbindungsdomäne von EmIR enthielt, in HEK293 Zellen gezeigt werden, dass die Zugabe von exogenem Insulin eine spezifische Autophosphorylierung der Chimäre induziert. Diese Ergebnisse bezeugen die Fähigkeit von EmIR Insulin-abhängige Signale des Wirtes einerseits zu detektieren und andererseits an die Echinococcus Signalwege weiter zu leiten. Die Bedeutung von Insulin-Signalmechanismen für das Überleben und das Wachstum des Parasiten konnte durch in vitro Kultivierungsexperimente aufgezeigt werden. Die Zugabe eines Inhibitors spezifisch für Insulinrezeptor Tyrosinkinasen verursachte die Degradation und den Tod der Metacestoden-Vesikel. Basierend auf den dargelegten molekularen Daten bezüglich der Interaktion zwischen EmIR und Insulin von Säugern erscheint es sehr wahrscheinlich, dass der orthologe Insulinrezeptor des Parasiten die Effekte von Insulin auf das Wachstum des Parasiten vermittelt. Aus diesem Grund ist EmIR ein potentieller Kandidat für die Kommunikation zwischen Wirt und Parasiten mittels evolutionär konservierten Signalwegen. Die Signalmechanismen unterhalb von EmIR wurden in abschließenden Experimenten untersucht. Diese offenbarten deutliche Unterschiede in der Weiterleitung von Insulin induzierten Signalen zwischen Echinococcus und dem verwandten parasitären Zestoden Taenia solium. Diese Unterschiede könnten mit dem unterschiedlichen Organtropismus beider Arten in Verbindung stehen. KW - Fuchsbandwurm KW - Insulin KW - Echinokokkus KW - Insulin KW - Helminth KW - EmERK KW - Echinococcus KW - insulin KW - helminth KW - EmERK Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-22636 ER - TY - THES A1 - Koike, Akito T1 - Molekular und zellbiologischer Ansatz hin zu neuartigen Medikamenten gegen \(Echinococcus\) \(multilocularis\) T1 - Molecular and cell biological approach towards novel drugs against \(Echinococcus\) \(multilocularis\) N2 - Echinococcosis is an important zoonosis. The causative agent of Alveolar Echinococcosis (AE) is Echinococcus multilocularis. The treatment of human AE is limited to surgery and chemotherapy with albendazole (ABZ). However, ABZ works only parasitostatically and it needs to be taken for long periods, although it causes adverse side effects. Thus, development of new, parasiticidal drug with selective toxicity is required. Because undifferentiated stem cells of E. multilocularis play key role in its longevity and regenerative capacity, targeting stem cells is especially important. In vitro screening of protein kinases inhibitors demonstrated that human PIM kinases inhibitors have detrimental effects on E. multilocularis. Through yeast two hybrid assay, the interaction of parasite PIM kinase (EmPIM) and its CDC25 (EmCDC25) was indicated. Through in situ hybridization, expression of EmPIM in the stem cells was observed. Therefore, EmPim is likely to be a positive regulator of cell cycle progression, the same as human Pim1. In addition, 20 compounds against EmPIM were selected through in silico screening and synthesized. One of them has a detrimental effect on E.multilocularis comparable to human pan-PIM inhibitors, but has much weaker toxicity on human cell lines. Furthermore, triclabendazole (TCBZ) and its metabolite TCBZSX, which are approved for another flatworm disease, Fascioliasis were tried on E. multilocularis. With two stem cell markers, damage to stem cells by TCBZSX was shown. In addition, primary cells from treated vesicles never regenerated and the damage to stem cells proved to be irreversible. Our in silico screening method used in EmPIM research has potential to identify compounds which overcome the side effect problem in ABZ-based chemotherapy. On the other hand, it is expected that my research of TCBZ can lead to development of a practical parasiticidal chemotherapy by combining TCBZ, which damages stem cells, and ABZ, which damages differentiated cells. N2 - Die Echinokokkose ist eine der wichtigsten Zoonosen sowohl für die Human- als auch für die Veterinärmedizin. Der Erreger der alveolären Echinokokkose (AE) ist Echinococcus multilocularis. Metazestode Bläschen, das Larvenstadium dieses parasitären Helminthen, können in die Leber eindringen und ungeschlechtlich wie bösartige Tumore wachsen. Dies kann ohne geeignete Behandlung tödlich sein. Die Behandlung von AE beim Menschen beschränkt sich auf Chirurgie und Chemotherapie, aber die Chirurgie ist nur bei einem kleinen Prozentsatz der Patienten anwendbar, die im Frühstadium diagnostiziert werden. Die meisten Patienten können sich nur auf eine Chemotherapie mit Albendazol (ABZ) verlassen. ABZ wirkt jedoch nur parasitostatisch und kann die Krankheit nicht heilen. Daher muss ABZ über einen längeren Zeitraum eingenommen werden, obwohl es mit Nebenwirkungen einhergeht. Daher ist die Entwicklung eines neuen, parasitentötenden und selektiven Medikaments gegen AE erforderlich. Da die undifferenzierte Stammzellpopulation von E. multilocularis eine Schlüsselrolle für seine Langlebigkeit und Regenerationsfähigkeit spielt, ist eine auf Stammzellen abzielende Chemotherapie wichtig. In dieser Arbeit wurde ein In-vitro-Screening verschiedener Hemmstoffe gegen Kinasen und Tubuline durchgeführt. Das Ergebnis des Screenings zeigte, dass Inhibitoren gegen humane pim-Kinasen starke schädliche Auswirkungen auf E. multilocularis haben. Durch ein Hefe-Zwei- Hybrid-System wurde die Interaktion der Parasiten-Pim-Kinase (EmPIM) mit der Zellteilungszyklus 25 (EmCDC25) nachgewiesen, und durch In-situ-Hybridisierung wurde die teilweise Lokalisierung von EmPIM in den Stammzellen beobachtet. Daher ist es wahrscheinlich, dass EmPim ein positiver Regulator der Zellzyklusprogression ist, genau wie menschliches Pim1. ... KW - Bandwürmer KW - Zellzyklus KW - Benzimidazolderivate KW - tapeworm KW - kinase KW - benzimidazole Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288649 ER - TY - THES A1 - Hemer, Sarah T1 - Molecular characterization of evolutionarily conserved signaling systems of Echinococcus multilocularis and their utilization for the development of novel drugs against Echinococosis T1 - Molekulare Charakterisierung evolutionsgeschichtlich konservierter Signalsysteme und deren Nutzung für die Entwicklung neuer Medikamente gegen Echinococcose N2 - Alveolar echinococcosis (AE), a severe and life-threatening disease is caused by the small fox tapeworm Echinococcus multilocularis. Currently, the options of chemotherapeutic treatment are very limited and are based on benzimidazole compounds, which act merely parasitostatic in vivo and often display strong side effects. Therefore, new therapeutic drugs and targets are urgently needed. In the present work the role of two evolutionarily conserved signalling pathways in E. multilocularis, namely the insulin signalling cascade and Abl kinases, has been studied in regard to host-parasite interaction and the possible use in anti-AE chemotherapy. N2 - Die alveoläre Echinokokkose ist eine ernste und lebensgefährliche Erkrankung, die durch den kleinen Fuchsbandwurm ausgelo ̈st wird. Die gegenwärtigen chemotherapeutischen Behandlungsmöglichkeiten beschränken sich auf die Behandlung mit Benzimidazolen, die in vivo nur parasitostatische Wirkung besitzen und häufig sehr starke Nebenwirkungen aufweisen. Aus diesem Grund besteht ein dringendes Bedürfnis nach neuen Medikamenten und Angriffszielen für diese. In der vorliegenden Arbeit wurde die Rolle zweier evolutionsgeschichtlich konservierter Signalsysteme, der Insulin Signalweg und die Abl Kinasen in E. multilocularis in Hinblick auf die Wirt-Parasiten Interaktion und dem mo ̈glichen Nutzen in der AE Chemotherapie untersucht. KW - Fuchsbandwurm KW - Insulin KW - Chemotherapie KW - Echinococcus KW - Insulin KW - Chemotherapie KW - Imatinib KW - Abl KW - Echinococcus KW - Insulin KW - Chemotherapy KW - Abl KW - Imatinib Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74007 ER - TY - THES A1 - Herz, Michaela T1 - Molecular characterization of the serotonin and cAMP-signalling pathways in Echinococcus T1 - Molekulare Charakterisierung der Serotonin- und cAMP-Signalwege in Echinococcus N2 - Alveolar and cystic echinococcosis, caused by Echinococcus multilocularis and Echinococcus granulosus respectively, are severe zoonotic diseases with limited treatment options. The sole curative treatment is the surgical removal of the complete parasite material. Due to late diagnosis, chemotherapeutic treatment often is the only treatment option. Treatment is based on benzimidazoles, which merely act parasitostatic and often display strong side effects. Therefore, new therapeutic drugs are urgently needed. Evolutionarily conserved signalling pathways are known to be involved in hostparasite cross-communication, parasite development and survival. Moreover, they represent potential targets for chemotherapeutic drugs. In this context the roles of the serotonin- and cAMP-signalling pathways in Echinococcus were studied. Genes encoding serotonin receptors, a serotonin transporter and enzymes involved in serotonin biosynthesis could be identified in the E. multilocularis and E. granulosus genomes indicating that these parasites are capable of synthesizing and perceiving serotonin signals. Also the influence of exogenous serotonin on parasite development was studied. Serotonin significantly increased metacestode vesicle formation from primary cells and re-differentiation of protoscoleces. Inhibition of serotonin transport with citalopram significantly reduced metacestode vesicle formation from primary cells and caused death of protoscoleces and metacestodes. Furthermore, it could be shown that serotonin increased phosphorylation of protein kinase A substrates. Taken together, these results show that serotonin and serotonin transport are essential for Echinococcus development and survival. Consequently, components of the serotonin pathway represent potential drug targets. In this work the cAMP-signalling pathway was researched with focus on G-protein coupled receptors and adenylate cyclases. 76 G-protein coupled receptors, including members of all major families were identified in the E. multilocularis genome. Four genes homologous to adenylate cyclase IX were identified in the E. multilocularis genome and three in the E. granulosus genome. While glucagon caused no significant effects, the adenylate cyclase activator forskolin and the adenylate cyclase inhibitor 2’, 5’ didesoxyadenosine influenced metacestode vesicle formation from primary cells, re-differentiation of protoscoleces and survival of metacestodes. It was further shown that forskolin increases phosphorylation of protein kinase A substrates, indicating that forskolin activates the cAMP-pathway also in cestodes. These results indicate that the cAMP signalling pathway plays an important role in Echinococcus development and survival. To complement this work, the influence of different media and additives on E. granulosus protoscoleces was investigated. Anaerobic conditions and the presence of FBS prolonged protoscolex survival while different media influenced protoscolex activation and development. Taken together, this work provided important insights into developmental processes in Echinococcus and potential drug targets for echinococcosis chemotherapy. N2 - Alveoläre und zystische Echinokokkose, hervorgerufen durch Echinococcus multilocularis und Echinococcus granulosus, sind schwere zoonotische Erkrankungen mit eingeschränkten Behandlungsmöglichkeiten. Die einzig kurative Therapie besteht in der chirurgischen Entfernung des gesammten Parasitenmaterials. Aufgrund später Diagnosestellung stellt Chemotherapie oft die einzige Behandlungsmöglichkeit dar. Die derzeitige Therapie basiert auf Benzimidazolen, welche nur parasitostatisch wirken und oft schwere Nebenwirkungen hervorrufen. Neue Medikamente werden daher dringend benötigt. Evolutionär konservierte Signalwege sind bekanntermaßen an Wirt-Parasit Kreuzkommunikation, Parasitenentwicklung und deren Überleben beteiligt. Darüber hinaus stellen sie auch mögliche Angriffspunkte für Chemotherapeutika dar. In diesem Zusammenhang wurden die Rollen des Serotonin- und des cAMP-Signalwegs in Echinococcus untersucht. Gene für Serotoninrezeptoren, einen Serotonintransporter und für Enzyme, die in der Serotoninsynthese involviert sind, konnten in den E. multilocularis und E. granulosus Genomen identifiziert werden, was darauf schließen lässt, dass diese Parasiten in der Lage sind, Serotonin selbst herzustellen und zu sensieren. Des Weiteren wurde der Einfluss von exogenem Serotonin auf die Parasitenentwicklung untersucht. Serotonin förderte die Bildung von Metazestodenvesikeln aus Primärzellen und die Rückdifferenzierung von Protoskolizes signifikant. Die Hemmung des Serotonintransports mit Citalopram reduzierte die Bildung von Metazestodenvesikeln aus Primärzellen signifikant und führte zum Absterben von Protoskolizes undMetazestoden. Des Weiteren konnte gezeigt werden, dass Serotonin die Posphorylierung von Proteinkinase A Substraten erhöht. Zusammengefasst zeigen diese Ergebnisse, dass Serotonin und Serotonintransport essentiell f¨ur die Entwicklung und das Überleben von Echinococcus sind. Folglich stellen Komponenten des Serotoninsignalwegs potentielle Angriffspunkte für Medikamente dar. In dieser Arbeit wurde der cAMP-Signalweg mit Schwerpunkt auf G-Protein gekoppelte Rezeptoren und Adenylatzyklasen untersucht. 76 G-Protein gekoppelte Rezeptoren, inclusive Mitglieder aller Hauptfamilien, wurden im E. multilocularis-Genom identifiziert. Vier Homologe zur Adenylatzyklase IX wurden im E. multilocularis- Genom und drei im E. granulosus-Genom identifiziert. Während Glukagon keine signifikanten Effekte hervorrief, beeinflussten der Adenylatzyklase-Aktivator Forskolin und der Adenylatzyklase-Inhibitor 2’, 5’-Didesoxyadenosin die Bildung von Metazestodenvesikeln aus Primärzellen, die Rückdifferenzierung von Protoskolizes und das Überleben vonMetazestoden. Zudem wurde gezeigt, dass Forskolin die Phosphorylierung von Proteinkinase A-Substraten erhöht. Dies bestätigt, dass Forskolin den cAMP-Signalweg aktiviert. Diese Ergebnisse legen nahe, dass der cAMP-Signalweg eine wichtige Rolle in der Entwicklung und dem Überleben von Echinococcus spielt. Um diese Arbeit zu vervollständigen, wurde der Einfluss von verschiedenen Medien und Zusätzen auf E. granulosus Protoskolizes untersucht. Anaerobe Bedingungen und die Anwesenheit von FBS verlängerten das Überleben von Protoskolizes, während verschiedene Medien die Aktivierung und die Entwicklung von Protoskolizes beeinflussten. Insgesamt gibt diese Arbeit wichtige Einblicke in Entwicklungsprozesse von Echinococcus und zeigt potentielle Angriffspunkte für Medikamente auf. KW - Serotonin KW - Cyclo-AMP KW - Fuchsbandwurm KW - cAMP KW - Echinococcus Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139249 ER - TY - JOUR A1 - Zoran, Tamara A1 - Seelbinder, Bastian A1 - White, Philip Lewis A1 - Price, Jessica Sarah A1 - Kraus, Sabrina A1 - Kurzai, Oliver A1 - Linde, Joerg A1 - Häder, Antje A1 - Loeffler, Claudia A1 - Grigoleit, Goetz Ulrich A1 - Einsele, Hermann A1 - Panagiotou, Gianni A1 - Loeffler, Juergen A1 - Schäuble, Sascha T1 - Molecular profiling reveals characteristic and decisive signatures in patients after allogeneic stem cell transplantation suffering from invasive pulmonary aspergillosis JF - Journal of Fungi N2 - Despite available diagnostic tests and recent advances, diagnosis of pulmonary invasive aspergillosis (IPA) remains challenging. We performed a longitudinal case-control pilot study to identify host-specific, novel, and immune-relevant molecular candidates indicating IPA in patients post allogeneic stem cell transplantation (alloSCT). Supported by differential gene expression analysis of six relevant in vitro studies, we conducted RNA sequencing of three alloSCT patients categorized as probable IPA cases and their matched controls without Aspergillus infection (66 samples in total). We additionally performed immunoassay analysis for all patient samples to gain a multi-omics perspective. Profiling analysis suggested LGALS2, MMP1, IL-8, and caspase-3 as potential host molecular candidates indicating IPA in investigated alloSCT patients. MMP1, IL-8, and caspase-3 were evaluated further in alloSCT patients for their potential to differentiate possible IPA cases and patients suffering from COVID-19-associated pulmonary aspergillosis (CAPA) and appropriate control patients. Possible IPA cases showed differences in IL-8 and caspase-3 serum levels compared with matched controls. Furthermore, we observed significant differences in IL-8 and caspase-3 levels among CAPA patients compared with control patients. With our conceptual work, we demonstrate the potential value of considering the human immune response during Aspergillus infection to identify immune-relevant molecular candidates indicating IPA in alloSCT patients. These human host candidates together with already established fungal biomarkers might improve the accuracy of IPA diagnostic tools. KW - host response KW - invasive pulmonary aspergillosis KW - alloSCT patients KW - galectin-2 KW - caspase-3 KW - matrix metallopeptidase-1 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262105 SN - 2309-608X VL - 8 IS - 2 ER - TY - JOUR A1 - Silwedel, Christine A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Speer, Christian P. A1 - Glaser, Kirsten T1 - More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. Methods Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. Results Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). Conclusions By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp. KW - Ureaplasma urealyticum KW - Ureaplasma parvum KW - Neuroinflammation KW - Meningitis KW - Caspase KW - Apoptosis KW - HBMEC Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200711 VL - 16 ER - TY - JOUR A1 - Schubert-Unkmeir, Alexandra A1 - Konrad, Christian A1 - Slanina, Heiko A1 - Czapek, Florian A1 - Hebling, Sabrina A1 - Frosch, Matthias T1 - Neisseria meningitidis Induces Brain Microvascular Endothelial Cell Detachment from the Matrix and Cleavage of Occludin: A Role for MMP-8 N2 - Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection. KW - Neisseria meningitidis Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68589 ER - TY - JOUR A1 - Silwedel, Christine A1 - Speer, Christian P. A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Buttmann, Mathias A1 - Glaser, Kirsten T1 - Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. Methods: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. Results: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor’s ligands. Conclusions: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism. KW - atypical chemokine receptor 3 KW - human brain microvascular endothelial cells KW - meningitis KW - neuroinflammation KW - Ureaplasma species Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175952 VL - 15 IS - 156 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel T1 - On the importance of targeting parasite stem cells in anti-echinococcosis drug development T1 - De l’importance de cibler les cellules souches du parasite dans la recherche de nouveaux médicaments contre les échinococcoses JF - Parasite N2 - The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite’s stem cell population. N2 - Les échinococcoses alvéolaire et kystique, deux maladies potentiellement mortelles, sont respectivement causées par les larves des vers plats Echinococcus multilocularis et E. granulosus. Dans les deux cas, les hôtes intermédiaires, comme l’homme, s’infectent par l’ingestion des oncosphères, suivie de la multiplication asexuée et la croissance presque illimitée du métacestode dans les organes de l’hôte. À côté de la chirurgie, le traitement des échinococcoses repose sur une chimiothérapie par les benzimidazoles, dont l’action est dirigée contre la bêta-tubuline du parasite. Cependant, comme les bêta-tubulines sont extrêmement similaires chez les cestodes et les humains, les benzimidazoles ne peuvent être utilisés qu’à des posologies parasitostatiques et sont associés à des effets secondaires indésirables. Avec l’objectif principal d’identifier des cibles pour des médicaments alternatifs, le génome nucléaire d’E. multilocularis et d’E. granulosus a été récemment séquencé, et de nombreuses cibles potentielles pour des médicaments sont exprimées par le métacestode. De plus, des études récentes de biologie cellulaire ont montré qu’E. multilocularis dispose de cellules souches multipotentes, appelées cellules germinales, qui sont les seules cellules parasitaires capables de prolifération et à l’origine de toutes les cellules différenciées. Ces cellules germinales représentent donc un type cellulaire crucial pour la prolifération d’E. multilocularis, et très vraisemblablement aussi d’E. granulosus, dans les organes de l’hôte, et vraisemblablement responsables des récurrences parasitaires à l’arrêt de la chimiothérapie. Des études récentes ont aussi indiqué que les cellules germinales pourraient être moins sensibles à la chimiothérapie car elles expriment un isoforme de la bêta-tubuline à affinité limitée vis-à-vis des benzimidazoles. Dans cet article, nous faisons une courte revue des découvertes récentes concernant la génomique d’Echinococcus et la recherche sur les cellules souches. Nous proposons que les recherches futures sur de nouveaux médicaments contre les échinococcoses se focalisent sur la population des cellules souches du parasite. KW - genome KW - chemotherapy KW - benzimidazole KW - stem cells KW - germinative cells KW - beta-tubulin Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118030 SN - 1252-607X VL - 21 ER - TY - JOUR A1 - Hubert, Kerstin A1 - Pawlik, Marie-Christin A1 - Claus, Heike A1 - Jarva, Hanna A1 - Meri, Seppo A1 - Vogel, Ulrich T1 - Opc Expression, LPS Immunotype Switch and Pilin Conversion Contribute to Serum Resistance of Unencapsulated Meningococci JF - PLoS One N2 - Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens. KW - factor H KW - C-reactive protein KW - B neisseria meningitidis KW - outer membrane protein KW - phase variation KW - serogroup B KW - bactericidal activity KW - epithelial cells KW - gene conversion KW - strain MC58 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135421 VL - 7 IS - 9 ER - TY - JOUR A1 - Kim, Brandon J. A1 - Shusta, Eric V. A1 - Doran, Kelly S. T1 - Past and current perspectives in modeling bacteria and blood–brain barrier interactions JF - Frontiers in Microbiology N2 - The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future. KW - bacteria KW - blood–brain barrier KW - meningitis KW - stem cells KW - brain endothelial cell Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201766 VL - 10 IS - 1336 ER - TY - JOUR A1 - Häder, Antje A1 - Schäuble, Sascha A1 - Gehlen, Jan A1 - Thielemann, Nadja A1 - Buerfent, Benedikt C. A1 - Schüller, Vitalia A1 - Hess, Timo A1 - Wolf, Thomas A1 - Schröder, Julia A1 - Weber, Michael A1 - Hünniger, Kerstin A1 - Löffler, Jürgen A1 - Vylkova, Slavena A1 - Panagiotou, Gianni A1 - Schumacher, Johannes A1 - Kurzai, Oliver T1 - Pathogen-specific innate immune response patterns are distinctly affected by genetic diversity JF - Nature Communications N2 - Innate immune responses vary by pathogen and host genetics. We analyze quantitative trait loci (eQTLs) and transcriptomes of monocytes from 215 individuals stimulated by fungal, Gram-negative or Gram-positive bacterial pathogens. We identify conserved monocyte responses to bacterial pathogens and a distinct antifungal response. These include 745 response eQTLs (reQTLs) and corresponding genes with pathogen-specific effects, which we find first in samples of male donors and subsequently confirm for selected reQTLs in females. reQTLs affect predominantly upregulated genes that regulate immune response via e.g., NOD-like, C-type lectin, Toll-like and complement receptor-signaling pathways. Hence, reQTLs provide a functional explanation for individual differences in innate response patterns. Our identified reQTLs are also associated with cancer, autoimmunity, inflammatory and infectious diseases as shown by external genome-wide association studies. Thus, reQTLs help to explain interindividual variation in immune response to infection and provide candidate genes for variants associated with a range of diseases. KW - antimicrobial responses KW - immunogenetics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357441 VL - 14 ER - TY - JOUR A1 - Krüger, Sören A1 - Leskien, Miriam A1 - Schuller, Patricia A1 - Prifert, Christiane A1 - Weißbrich, Benedikt A1 - Vogel, Ulrich A1 - Krone, Manuel T1 - Performance and feasibility of universal PCR admission screening for SARS‐CoV‐2 in a German tertiary care hospital JF - Journal of Medical Virology N2 - Anamnestic screening of symptoms and contact history is applied to identify coronavirus disease 2019 (COVID‐19) patients on admission. However, asymptomatic and presymptomatic patients remain undetected although the viral load may be high. In this retrospective cohort study, all hospitalized patients who received polymerase chain reaction (PCR) admission testing from March 26th until May 24th, 2020 were included. Data on COVID‐19‐specific symptoms and contact history to COVID‐19 cases were retrospectively extracted from patient files and from contact tracing notes. The compliance to the universal testing protocol was high with 90%. Out of 6940 tested patients, 27 new severe acute respiratory syndrome coronavirus‐2 infections (0.4%) were detected. Seven of those COVID‐19 cases (26% of all new cases) were asymptomatic and had no positive contact history, but were identified through a positive PCR test. The number needed to identify an asymptomatic patient was 425 in the first wave of the epidemic, 1218 in the low incidence phase. The specificity of the method was above 99.9%. Universal PCR testing was highly accepted by staff as demonstrated by high compliance. The costs to detect one asymptomatic case in future studies need to be traded off against the costs and damage caused by potential outbreaks of COVID‐19. KW - admission screening KW - COVID‐19 KW - infection control KW - SARS‐CoV‐2 KW - testing strategy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238971 VL - 93 IS - 5 SP - 2890 EP - 2898 ER - TY - JOUR A1 - Elias, Johannes A1 - Findlow, Jamie A1 - Borrow, Ray A1 - Tremmel, Angelika A1 - Frosch, Matthias A1 - Vogel, Ulrich T1 - Persistence of antibodies in laboratory staff immunized with quadrivalent meningococcal polysaccharide vaccine JF - Journal of Occupational Medicine and Toxicology N2 - Background Occupational exposure to live meningococci can potentially cause invasive meningococcal disease in laboratory staff. While, until recently, immunization with quadrivalent polysaccharide vaccine represented one cornerstone of protection, data on long-term persistence of antibodies in adults remain scarce. Methods We analyzed the relationship of antibody levels and time following quadrivalent polysaccharide vaccination (Mencevax® ACWY, GlaxoSmithKline) in a cross-sectional sample of 20 laboratory workers vaccinated at ages between 16.4 to 40.7 years from Germany. Sera were obtained 0.4 to 158.5 (median 35.3) months after vaccination. At the time of sampling, laboratory workers had been regularly exposed to meningococci for periods between 3.2 to 163.8 (median 41.2) months. Serum bactericidal assay (SBA) with rabbit complement and a microsphere-based flow analysis method were used to determine bactericidal titers and concentrations of IgG, respectively, against serogroups A, C, W135, and Y. Decay of antibodies was modeled using linear regression. Protective levels were defined as SBA titers ≥ 8. Results Half-lives of SBA titers against serogroups A, C, W135, and Y were estimated at 27.4, 21.9, 18.8, and 28.0 months, respectively. Average durations of protection were estimated at 183.9, 182.0, 114.6, and 216.4 months, respectively. Inter-individual variation was high; using lower margins of 95% prediction intervals, minimal durations of protection against serogroups A, C, W135 and Y were estimated at 33.5, 24.6, 0.0, and 55.1 months, respectively. The proportion of staff with protective SBA titers against W135 (65.0%) was significantly lower than proportions protected against A (95.0%), C (94.7%), and Y (95.0%). Consistently, geometric mean titer (97.0) and geometric mean concentration of IgG (2.1 μg/ml) was lowest against serogroup W135. SBA titers in a subset of individuals with incomplete protection rose to ≥ 128 (≥ 8 fold) after reimmunization with a quadrivalent glycoconjugate vaccine. Conclusions The average duration of protection following immunization with a quadrivalent polysaccharide vaccine in adults was ≥ 115 months regardless of serogroup. A substantial proportion (approximately 23% according to our decay model) of adult vaccinees may not retain protection against serogroup W135 for five years, the time suggested for reimmunization. KW - Vaccination KW - Meningococcal infection KW - Biohazard KW - Meningococcal polysaccharide caccine Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-95953 UR - http://www.occup-med.com/content/8/1/4 ER - TY - JOUR A1 - Schwerk, Christian A1 - Papandreou, Thalia A1 - Schuhmann, Daniel A1 - Nickol, Laura A1 - Borkowski, Julia A1 - Steinmann, Ulrike A1 - Quednau, Natascha A1 - Stump, Carolin A1 - Weiss, Christel A1 - Berger, Jürgen A1 - Wolburg, Hartwig A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Ishikawa, Hiroshi A1 - Tenenbaum, Tobias A1 - Schroten, Horst T1 - Polar Invasion and Translocation of Neisseria meningitidis and Streptococcus suis in a Novel Human Model of the Blood-Cerebrospinal Fluid Barrier JF - PLoS One N2 - Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens. KW - gene expression KW - plexus epithelial-cells KW - central-nervous-system KW - microvascular endothelial-cells KW - choroid-plexus KW - in vitro KW - brain barrier KW - tight junctions KW - meningococcal disease KW - bacterial meningitis Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131459 VL - 7 IS - 1 ER - TY - JOUR A1 - Dick, Julia A1 - Krauß, Patrizia A1 - Hillenkamp, Jost A1 - Kohlmorgen, Britta A1 - Schoen, Christoph T1 - Postoperative Tropheryma whipplei endophthalmitis – a case report highlighting the additive value of molecular testing JF - JMM Case Reports N2 - Introduction. Tropheryma whipplei is the causative agent of Whipple’s disease. Gastrointestinal and lymphatic tissues are affected in the majority of cases, resulting in diarrhoea, malabsorption and fever. Here, we report a rare case of ocular manifestation in a patient lacking the typical Whipple symptoms. Case presentation. A 74-year-old Caucasian female presented with blurred vision in the right eye over a period of 1–2 months, accompanied by stinging pain and conjunctival hyperaemia for the last 2 days. Upon admission, visual acuity was hand motion in the affected eye. Ophthalmological examination showed typical signs of intraocular inflammation. Diagnostic and therapeutic pars plana vitrectomy including vitreous biopsy and intravitreal instillation of vancomycin and amikacin was performed within hours of initial presentation. Both microscopic analysis and microbial cultures of the vitreous biopsy remained negative for bacteria and fungi. The postoperative antibiotic regime included intravenous administration of ceftriaxone in combination with topical tobramycin and ofloxacin. Due to the empirical therapy the inflammation ceased and the patient was discharged after 5 days with cefpodoxime orally and local antibiotic and steroidal therapy. Meanwhile, the vitreous body had undergone testing by PCR for the eubacterial 16S rRNA gene, which was found to be positive. Analysis of the PCR product revealed a specific sequence of T. whipplei. Conclusion. In our patient, endophthalmitis was the first and only symptom of Morbus Whipple, while most patients with Whipple’s disease suffer from severe gastrointestinal symptoms. 16S rDNA PCR should be considered for any intraocular infection when microscopy and standard culture methods remain negative. KW - intravitreal vancomycin and amikacin KW - intravenous ceftriaxone KW - topic ofloxacin KW - Whipple's disease KW - endophthalmitis KW - Tropheryma whipplei KW - ocular infection KW - vitrectomy KW - oral cefpodoxime KW - oral doxycycline Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158823 VL - 4 ER - TY - JOUR A1 - Prauße, Maria T. E. A1 - Lehnert, Teresa A1 - Timme, Sandra A1 - Hünniger, Kerstin A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Figge, Marc Thilo T1 - Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood JF - Frontiers in Immunology N2 - Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism. KW - immune evasion KW - state-based model KW - innate immune response KW - polymorphonuclear neutrophils KW - whole-blood infection assay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197493 SN - 1664-3224 VL - 9 IS - 560 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Mshana, Stephen E. A1 - Kamugisha, Erasmus A1 - Kataraihya, Johannes B. A1 - Tappe, Dennis A1 - Vogel, Ulrich A1 - Lyamuya, Eligius F. A1 - Claus, Heike T1 - Predominance of methicillin resistant Staphylococcus aureus-ST88 and new ST1797 causing wound infection and abscesses JF - Journal of Infection in Developing Countries N2 - Introduction: Although there has been a worldwide emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA), little is known about the molecular epidemiology of MRSA in Tanzania. Methodology: In this study, we characterized MRSA strains isolated from clinical specimens at the Bugando Medical Centre, Tanzania, between January and December 2008. Of 160 S. aureus isolates from 600 clinical specimens, 24 (15%) were found to be MRSA. Besides molecular screening for the Panton Valentine leukocidin (PVL) genes by PCR, MRSA strains were further characterized by Multi-Locus Sequence Typing (MLST) and spa typing. Results: Despite considerable genetic diversity, the spa types t690 (29.1%) and t7231 (41.6%), as well as the sequence types (ST) 88 (54.2%) and 1797 (29.1%), were dominant among clinical isolates. The PVL genes were detected in 4 isolates; of these, 3 were found in ST 88 and one in ST1820. Resistance to erythromycin, clindamicin, gentamicin, tetracycline and co-trimoxazole was found in 45.8%, 62.5%, 41.6%, 45.8% and 50% of the strains, respectively. Conclusion: We present the first thorough typing of MRSA at a Tanzanian hospital. Despite considerable genetic diversity, ST88 was dominant among clinical isolates at the Bugando Medical Centre. Active and standardized surveillance of nosocomial MRSA infection should be conducted in the future to analyse the infection and transmission rates and implement effective control measures. KW - Tanzania KW - panton-valentine leukocidin KW - field gel-electrophoreresis KW - molecular epidemiology KW - aureus infections KW - MRSA KW - ST88 KW - ST1797 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134746 VL - 6 IS - 8 ER - TY - JOUR A1 - Deogratias, Anna-Pendo A1 - Mushi, Martha F. A1 - Paterno, Laurent A1 - Tappe, Dennis A1 - Seni, Jeremiah A1 - Kabymera, Rogatus A1 - Kidenya, Benson R. A1 - Mshana, Stephen E. T1 - Prevalence and determinants of Campylobacter infection among under five children with acute watery diarrhea in Mwanza, North Tanzania JF - Archives of Public Health N2 - BACKGROUND: Campylobacteriosis, a zoonotic bacterial disease observed world-wide, is becoming the most commonly recognized cause of bacterial gastroenteritis in humans. This study was done to determine the prevalence and determinants of Campylobacter infection among under-fives with acute watery diarrhea in Mwanza City, Tanzania. METHOD: This cross-sectional hospital-based study was conducted at Bugando Medical Centre (BMC) and Sekou Toure Hospital in Mwanza City. All inpatients and outpatients under-fives who met the inclusion criteria from October 2012 to April 2013 were enrolled in the study. Demographic and clinical data were obtained using standardized data collection tools. Stool samples were collected for gram staining and culture for Campylobacter spp. on Preston selective agar media. In addition, blood slides for malaria and HIV tests were done to all patients. RESULTS: A total of 300 children were enrolled with a median age of 12 [interquartile range, 8-19] months. Of these, 169 (56.5%) were from BMC and 131 (43.7%) from Sekou-Toure hospital. One hundred and seventy (56.7%) of the participating children were male. Of 300 under-fives with acute watery diarrhea, 29 patients (9.7%) were found to have Campylobacter infection. A significant higher number of children with Campylobacter infection were found in Sekou Toure hospital compared to BMC [16.0% (21/29) versus 4.7% (8/29), p = 0.002)]. Age above 2 years was independently found to predict campylobacter infection (OR: 2.9, 95% CI 1.1-7.7, p = 0.0037). Of 30 patients with a positive blood slide for Plasmodium falciparum, 20.0% were also positive for Campylobacter infection (OR: 3.9, 95% CI 1.2-10.1, p = 0.021). CONCLUSION: Campylobacter infection shows a comparatively low prevalence in under-fives with acute watery diarrhea in Mwanza city and is independently associated with positive slides for malaria and an age above 2 years. Further studies are needed to type the most prevalent Campylobacter species and to determine their antibiotic susceptibility pattern. KW - acute watery diarrhea KW - campylobacteriosis KW - under five children Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120749 VL - 72 IS - 17 ER - TY - JOUR A1 - Elias, Johannes A1 - Heuschmann, Peter U. A1 - Schmitt, Corinna A1 - Eckhardt, Frithjof A1 - Boehm, Hartmut A1 - Maier, Sebastian A1 - Kolb-Mäurer, Annette A1 - Riedmiller, Hubertus A1 - Müllges, Wolfgang A1 - Weisser, Christoph A1 - Wunder, Christian A1 - Frosch, Matthias A1 - Vogel, Ulrich T1 - Prevalence dependent calibration of a predictive model for nasal carriage of methicillin-resistant Staphylococcus aureus JF - BMC Infectious Diseases N2 - Background Published models predicting nasal colonization with Methicillin-resistant Staphylococcus aureus among hospital admissions predominantly focus on separation of carriers from non-carriers and are frequently evaluated using measures of discrimination. In contrast, accurate estimation of carriage probability, which may inform decisions regarding treatment and infection control, is rarely assessed. Furthermore, no published models adjust for MRSA prevalence. Methods Using logistic regression, a scoring system (values from 0 to 200) predicting nasal carriage of MRSA was created using a derivation cohort of 3091 individuals admitted to a European tertiary referral center between July 2007 and March 2008. The expected positive predictive value of a rapid diagnostic test (GeneOhm, Becton & Dickinson Co.) was modeled using non-linear regression according to score. Models were validated on a second cohort from the same hospital consisting of 2043 patients admitted between August 2008 and January 2012. Our suggested correction score for prevalence was proportional to the log-transformed odds ratio between cohorts. Calibration before and after correction, i.e. accurate classification into arbitrary strata, was assessed with the Hosmer-Lemeshow-Test. Results Treating culture as reference, the rapid diagnostic test had positive predictive values of 64.8% and 54.0% in derivation and internal validation corhorts with prevalences of 2.3% and 1.7%, respectively. In addition to low prevalence, low positive predictive values were due to high proportion (> 66%) of mecA-negative Staphylococcus aureus among false positive results. Age, nursing home residence, admission through the medical emergency department, and ICD-10-GM admission diagnoses starting with “A” or “J” were associated with MRSA carriage and were thus included in the scoring system, which showed good calibration in predicting probability of carriage and the rapid diagnostic test’s expected positive predictive value. Calibration for both probability of carriage and expected positive predictive value in the internal validation cohort was improved by applying the correction score. Conclusions Given a set of patient parameters, the presented models accurately predict a) probability of nasal carriage of MRSA and b) a rapid diagnostic test’s expected positive predictive value. While the former can inform decisions regarding empiric antibiotic treatment and infection control, the latter can influence choice of screening method. KW - Methicillin-resistant staphylococcus aureus KW - Infection control KW - Clinical prediction rule KW - Predictive value of tests KW - False positive reactions KW - Calibration Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96091 UR - http://www.biomedcentral.com/1471-2334/13/111 ER - TY - JOUR A1 - Bauer, Hannah A1 - Concha Mendoza, Gustavo Andrés A1 - Kreienbrock, Lothar A1 - Hartmann, Maria A1 - Frickmann, Hagen A1 - Kann, Simone T1 - Prevalence of common diseases in Indigenous people in Colombia JF - Tropical Medicine and Infectious Disease N2 - The Indigenous tribe called the Wiwa lives retracted in the Sierra Nevada de Santa Marta, Colombia. Little is known about their health status and whether the health care system in place covers their needs. In 2017 and 2018, a permanent physician was in charge for the Wiwa. Diseases and complaints were registered, ranked, and classified with the ICD-10 coding. Datasets from the Indigenous health care provider Dusakawi, collected from local health points and health brigades travelling sporadically into the fields for short visits, were compared. Furthermore, a list of provided medication was evaluated regarding the recorded needs. The most common complaints found were respiratory, infectious and parasitic, and digestive diseases. The top ten diagnoses collected in the health points and in the health brigade datasets were similar, although with a different ranking. The available medication showed a basic coverage only, with a critical lack of treatment for many severe, chronic, and life-threatening diseases. Most of the detected diseases in the Indigenous population are avoidable by an improvement in health care access, an expansion of the provided medication, and an increase in knowledge, hygiene, and life standards. KW - Chagas disease KW - indigenous KW - public health KW - Colombia KW - Sierra Nevada KW - neglected groups Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278953 SN - 2414-6366 VL - 7 IS - 6 ER - TY - JOUR A1 - Duske, Helene A1 - Claus, Heike A1 - Krone, Manuel A1 - Lâm, Thiên-Trí T1 - Prevalence of piperacillin/tazobactam resistance in invasive \(Haemophilus\) \(influenzae\) in Germany JF - JAC-Antimicrobial Resistance N2 - Background Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. Objectives To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. Methods According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller–Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Results Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Conclusions Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations. KW - microbiology KW - immunology KW - generalized anxiety disorder KW - haemophilus influenzae KW - agar KW - Germany KW - piperacillin KW - piperacillin/tazobactam KW - tazobactam KW - Haemophilus influenzae Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350424 SN - 2632-1823 VL - 6 IS - 1 ER - TY - JOUR A1 - Nell, Manuel A1 - Burgkart, Rainer H. A1 - Gradl, Guntmar A1 - von Eisenhart-Rothe, Rüdiger A1 - Schaeffeler, Christoph A1 - Trappe, Dennis A1 - Prazeres da Costa, Clarissa A1 - Gradinger, Reiner A1 - Kirchhoff, Chlodwig T1 - Primary extrahepatic alveolar echinococcosis of the lumbar spine and the psoas muscle JF - Annals of Clinical Microbiology and Antimicrobials N2 - Alveolar echinococcosis (AE) of human being caused by Echinococcus multilocularis is a rare but important zoonosis especially in tempered zones of middle Europe and Northern America with endemic character in many countries. Due to the long incubation period, various clinical manifestations, critical prognosis, and outcome AE presents a serious and severe disease. The primary focus of infection is usually the liver. Although secondary affection of visceral organs is possible extrahepatic AE is highly uncommon. Moreover, the involvement of bone and muscle presents with an even lower incidence. In the literature numerous cases on hepatic AE have been reported. However, extrahepatic AE involving bones and/or muscles was described very rarely. We report a case of an 80-year-old man with primary extrahepatic alveolar Echinococcosis of the lumbar spine and the psoas muscle. The etiology, diagnosis, differential diagnoses, treatment options and outcome of this rare disease are discussed in context with the current literature. KW - Medicine Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141796 VL - 10 IS - 13 ER - TY - JOUR A1 - Vendelova, Emilia A1 - de Lima, Jeferson Camargo A1 - Lorenzatto, Karina Rodrigues A1 - Monteiro, Karina Mariante A1 - Mueller, Thomas A1 - Veepaschit, Jyotishman A1 - Grimm, Clemens A1 - Brehm, Klaus A1 - Hrčková, Gabriela A1 - Lutz, Manfred B. A1 - Ferreira, Henrique B. A1 - Nono, Justin Komguep T1 - Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions JF - PLoS Neglected Tropical Diseases N2 - Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. KW - proteomic analysis KW - excretory-secretory KW - Mesocestoides corti Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166742 VL - 10 IS - 10 ER - TY - JOUR A1 - Wagener, Johannes A1 - Loiko, Veronika T1 - Recent insights into the paradoxical effect of echinocandins JF - Journal of Fungi N2 - Echinocandin antifungals represent one of the most important drug classes for the treatment of invasive fungal infections. The mode of action of the echinocandins relies on inhibition of the β-1,3-glucan synthase, an enzyme essentially required for the synthesis of the major fungal cell wall carbohydrate β-1,3-glucan. Depending on the species, echinocandins may exert fungicidal or fungistatic activity. Apparently independent of this differential activity, a surprising in vitro phenomenon called the “paradoxical effect” can be observed. The paradoxical effect is characterized by the ability of certain fungal isolates to reconstitute growth in the presence of higher echinocandin concentrations, while being fully susceptible at lower concentrations. The nature of the paradoxical effect is not fully understood and has been the focus of multiple studies in the last two decades. Here we concisely review the current literature and propose an updated model for the paradoxical effect, taking into account recent advances in the field. KW - echinocandin KW - caspofungin KW - micafungin KW - anidulafungin KW - paradoxical effect KW - paradoxical growth KW - glucan synthase KW - Fks1 KW - antifungals KW - echinocandins Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197960 SN - 2309-608X VL - 4 IS - 1 ER - TY - JOUR A1 - Weiss, Esther A1 - Schlegel, Jan A1 - Terpitz, Ulrich A1 - Weber, Michael A1 - Linde, Jörg A1 - Schmitt, Anna-Lena A1 - Hünniger, Kerstin A1 - Marischen, Lothar A1 - Gamon, Florian A1 - Bauer, Joachim A1 - Löffler, Claudia A1 - Kurzai, Oliver A1 - Morton, Charles Oliver A1 - Sauer, Markus A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus JF - Frontiers in Immunology N2 - Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility. KW - natural killer cell KW - stem cell transplantation KW - corticosteroids KW - CCL3 KW - CCL4 KW - CCL5 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212581 SN - 1664-3224 VL - 11 ER - TY - JOUR A1 - Hellenbrand, Wiebke A1 - Claus, Heike A1 - Schink, Susanne A1 - Marcus, Ulrich A1 - Wichmann, Ole A1 - Vogel, Ulrich T1 - Risk of Invasive Meningococcal Disease in Men Who Have Sex with Men: Lessons Learned from an Outbreak in Germany, 2012-2013 JF - PLoS ONE N2 - Background We undertook investigations in response to an invasive meningococcal disease (IMD) outbreak in men who have sex with men (MSM) in Berlin 2012–2013 to better understand meningococcal transmission and IMD risk in MSM. Methods We retrospectively searched for further IMD cases in MSM in Germany through local health departments and undertook exploratory interviews. We performed antigen sequence typing, characterized fHbp and aniA genes of strains with the outbreak finetype and reviewed epidemiologically or spatiotemporally linked cases from 2002–2014. Results Among the 148 IMD-cases notified from 01.01.2012–30.09.2013 in 18–59 year-old men we identified 13 MSM in 6 federal states: 11 serogroup C (MenC, all finetype C:P1.5–1,10–8:F3-6), 2 MenB. Interviews with 7 MSM revealed frequent meeting of multiple partners online or via mobile apps and illicit drug use as potential risk factors. MenC incidence was 13-fold higher in MSM than non-MSM. MenC isolates from 9/11 MSM had a novel fHbp allele 766. All C:P1.5–1,10–8:F3-6 strains from MSM versus 16/23 from non-MSM had intact aniA genes (p = 0.04). Although definitive evidence for transmission among MSM in epidemiological or spatiotemporal clusters in 2002–2014 was lacking, clusters were more frequent in men aged 20–49 years. Molecular analysis of C:P1.5–1,10–8:F3-6 strains revealed cases with intact aniA since 2007, mainly associated with fHbp361, fHbp766 and fHbp813, all involving one or more MSM. Conclusions MenC incidence was elevated in MSM during the study period. Multiple casual sexual contacts and illicit drug use were common in affected MSM. In all strains from MSM we detected an intact aniA gene coding for a nitrite reductase, which permits survival in microanaerobic environments and could play a role in meningococcal transmission in MSM through urogenital colonization. Furthermore, meningococcal transmission among MSM may be sustained over large areas and thus require modified spatiotemporal scanning algorithms for timely detection and control. KW - invasive meningococcal disease KW - men who have sex with men KW - Germany Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166842 VL - 11 IS - 8 ER - TY - JOUR A1 - Klotz, Peter A1 - Higgins, Paul G. A1 - Schaubmar, Andreas R. A1 - Failing, Klaus A1 - Leidner, Ursula A1 - Seifert, Harald A1 - Scheufen, Sandra A1 - Semmler, Torsten A1 - Ewers, Christa T1 - Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany JF - Frontiers in Microbiology N2 - Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans. KW - ESKAPE KW - Acinetobacter baumannii KW - antimicrobial susceptibility KW - MLST KW - cattle KW - epidemiology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325927 VL - 10 ER - TY - JOUR A1 - Barth, Thomas F. E. A1 - Herrmann, Tobias S. A1 - Tappe, Dennis A1 - Stark, Lorenz A1 - Grüner, Beate A1 - Buttenschoen, Klaus A1 - Hillenbrand, Andreas A1 - Juchems, Markus A1 - Henne-Bruns, Doris A1 - Kern, Petra A1 - Seitz, Hanns M. A1 - Möller, Peter A1 - Rausch, Robert L. A1 - Kern, Peter A1 - Deplazes, Peter T1 - Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11 JF - PLoS Neglected Tropical Diseases N2 - Background: Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody. Methodology/Principal Findings: We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 mm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11. Conclusions/Significance: Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host. KW - cells KW - multilocularis KW - antigen Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135371 VL - 6 IS - 10 ER - TY - JOUR A1 - Machata, Silke A1 - Sreekantapuram, Sravya A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Dunker, Christine A1 - Schubert, Katja A1 - Krüger, Wibke A1 - Schulze-Richter, Bianca A1 - Speth, Cornelia A1 - Rambach, Günter A1 - Jacobsen, Ilse D. T1 - Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood JF - Frontiers in Immunology N2 - Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts. KW - whole blood ex vivo model KW - host-pathogen interaction KW - neutrophils KW - mice Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222575 SN - 1664-3224 VL - 11 ER - TY - JOUR A1 - Heisig, Martin A1 - Frentzen, Alexa A1 - Bergmann, Birgit A1 - Gentschev, Katharina Ivaylo A1 - Hotz, Christian A1 - Schoen, Christoph A1 - Stritzker, Jochen A1 - Fensterle, Joachim A1 - Rapp, Ulf R. A1 - Goebel, Werner T1 - Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines N2 - Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization. KW - Listeria monocytogenes Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68705 ER - TY - JOUR A1 - Lorson, Thomas A1 - Ruopp, Matthias A1 - Nadernezhad, Ali A1 - Eiber, Julia A1 - Vogel, Ulrich A1 - Jungst, Tomasz A1 - Lühmann, Tessa T1 - Sterilization Methods and Their Influence on Physicochemical Properties and Bioprinting of Alginate as a Bioink Component JF - ACS Omega N2 - Bioprinting has emerged as a valuable threedimensional (3D) biomanufacturing method to fabricate complex hierarchical cell-containing constructs. Spanning from basic research to clinical translation, sterile starting materials are crucial. In this study, we present pharmacopeia compendial sterilization methods for the commonly used bioink component alginate. Autoclaving (sterilization in saturated steam) and sterile filtration followed by lyophilization as well as the pharmacopeia non-compendial method, ultraviolet (UV)-irradiation for disinfection, were assessed. The impact of the sterilization methods and their effects on physicochemical and rheological properties, bioprinting outcome, and sterilization efficiency of alginate were detailed. Only sterile filtration followed by lyophilization as the sterilization method retained alginate's physicochemical properties and bioprinting behavior while resulting in a sterile outcome. This set of methods provides a blueprint for the analysis of sterilization effects on the rheological and physicochemical pattern of bioink components and is easily adjustable for other polymers used in the field of biofabrication in the future. KW - hydrogels Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229460 N1 - Lizenz: https://pubs.acs.org/page/policy/authorchoice_termsofuse.html VL - 5 IS - 12 ER - TY - JOUR A1 - Kim, Brandon J. A1 - McDonagh, Maura A. A1 - Deng, Liwen A1 - Gastfriend, Benjamin D. A1 - Schubert-Unkmeir, Alexandra A1 - Doran, Kelly S. A1 - Shusta, Eric V. T1 - Streptococcus agalactiae disrupts P-glycoprotein function in brain endothelial cells JF - Fluids and Barriers of the CNS N2 - Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood–borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood–brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS. KW - Group B Streptococcus KW - Streptococcus agalactiae KW - Brain endothelial cells KW - P-glycoprotein KW - Efflux transport KW - Meningitis KW - Stem cells KW - P-gp Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201895 VL - 16 ER - TY - THES A1 - Hagmann [geb. Kischkies], Laura Violetta T1 - Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\) T1 - Regulation der stringenten Kontrolle und ihre Auswirkungen auf das ex vivo Überleben des kommensalen Erregers \(Neisseria\) \(meningitidis\) N2 - Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments. N2 - Neisseria meningitidis ist ein kommensal lebendes, fakultativ pathogenes Bakterium, welches unter nicht vollständig verstandenen Umständen lebensbedrohliche Krankheitsbilder bei Menschen verursacht. Aktuelle Studien haben gezeigt, dass die stringente Antwort einen Einfluss auf die bakterielle Virulenz haben kann. Aus diesem Grund untersucht diese Arbeit die Regulation der stringenten Antwort, insbesondere die Rolle von RelA, sowie den Einfluss der stringenten Antwort auf die Ex-vivo-Fitness der Meningokokken. Die Ergebnisse wurden für den Trägerstamm α522 und den hyperinvasiven Stamm MC58 erhoben und miteinander verglichen. Wachstumsexperimente zeigten, dass sich beide Wildtyp-Stämme in ihren Ex-vivo-Phänotypen nicht unterscheiden. Jedoch wurden mittels quantitativer Echtzeit-PCR (qRT-PCR) Unterschiede zwischen beiden Stämmen in der Genexpression von relA gefunden. Zudem war die α522 relA Mutante im Gegensatz zu der MC58 relA Mutante nicht in der Lage, in menschlichem Vollblut zu überleben. Allerdings zeigten in Saliva und Liquor beide Stämme den gleichen Phänotyp. Außerdem war der Trägerstamm auf eine kurze, nicht-codierende AT-reiche Region (ATRrelA) in der Promotorregion von relA angewiesen, um im menschlichen Blut überleben zu können. Darüber hinaus zeigten Zellkulturexperimente mit humanen Epithelzellen, dass die Deletion relA die Invasionsrate in beiden Stämmen signifikant verringerte, obwohl die Adhäsionsrate durch die Deletion unbeeinflusst blieb. Um besser verstehen zu können, weshalb die Deletion von relA unter bestimmten Bedingungen letal ist, wurden mit In-silico- und experimentellen Analysen nach Unterschieden in den Aminosäurebiosynthesewegen beider Stämme gesucht. Es zeigte sich, dass Stamm MC58 in der Lage ist alle 20 Aminosäuren zu synthetisieren, während Stamm α522 eine Auxotrophie für Cystein und Glutamin aufweist. Ferner zeigten die In-vitro-Wachstumsversuche, dass RelA bei Aminosäuremangel essentiell für beide Stämme ist. Darüber hinaus zeigte eine MC58 Mutante mit einer ATRrelA –Kopie in der relA Promotorregion ein im Vergleich zum Wildtyp-Stamm verbessertes Wachstum in mit L-Cystein und/oder L-Glutamin angereichertem Minimalmedium. Gegensätzlich dazu zeigte der Stamm α522 keine Unterschiede im Wachstum zwischen dem Wildtyp und einer ATRrelA Deletions-Mutante. Dies deutet darauf hin, dass ATRrelA an dem komplexen regulatorischen Zusammenspiel der stringenten Antwort und dem L-Cystein- beziehungsweise dem L-Glutamin-Metabolismus beteiligt ist. Es lässt sich vermuten, dass RelA zu der Virulenz von Meningokokken in einer stamm- und umgebungsspezifischen Weise beiträgt. Abschließend hebt diese Arbeit die Rolle von kleinen regulatorischen Elementen für die bakterielle Virulenz hervor und postuliert, dass die Virulenz der Meningokokken auf ihrer Fähigkeit basiert, sich der durch den Wirt gegebenen Umgebung anzupassen. KW - Neisseria meningitidis KW - Stringente Kontrolle KW - Virulenzfaktor KW - Genregulation KW - Transposon KW - Stringent response KW - RelA KW - MITE KW - Stringente Antwort Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144352 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 ER - TY - JOUR A1 - Flemming, S. A1 - Hankir, M. A1 - Ernestus, R.-I. A1 - Seyfried, F. A1 - Germer, C.-T. A1 - Meybohm, P. A1 - Wurmb, T. A1 - Vogel, U. A1 - Wiegering, A. T1 - Surgery in times of COVID-19 — recommendations for hospital and patient management JF - Langenbeck's Archives of Surgery N2 - Background The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), has escalated rapidly to a global pandemic stretching healthcare systems worldwide to their limits. Surgeonshave had to immediately react to this unprecedented clinical challenge by systematically repurposing surgical wards. Purpose To provide a detailed set of guidelines developed in a surgical ward at University Hospital Wuerzburg to safelyaccommodate the exponentially rising cases of SARS-CoV-2 infected patients without compromising the care of emergencysurgery and oncological patients or jeopardizing the well-being of hospital staff. Conclusions The dynamic prioritization of SARS-CoV-2 infected and surgical patient groups is key to preserving life whilemaintaining high surgical standards. Strictly segregating patient groups in emergency rooms, non-intensive care wards andoperating areas prevents viral spread while adequately training and carefully selecting hospital staff allow them to confidentlyand successfully undertake their respective clinical duties. KW - SARS-CoV-2 KW - COVID-19 KW - Surgery Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231766 SN - 1435-2443 VL - 405 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - Surveillance of surgical site infections by Pseudomonas aeruginosa and strain characterization in Tanzanian hospitals does not provide proof for a role of hospital water plumbing systems in transmission JF - Antimicrobial Resistance and Infection Control N2 - Background The role of hospital water systems in the development of Pseudomonas aeruginosa (P. aeruginosa) surgical site infections (SSIs) in low-income countries is barely studied. This study characterized P. aeruginosa isolates from patients and water in order to establish possible epidemiological links. Methods: Between December 2014 and September 2015, rectal and wound swabs, and water samples were collected in the frame of active surveillance for SSIs in the two Tanzanian hospitals. Typing of P. aeruginosa was done by multi-locus sequence typing. Results: Of 930 enrolled patients, 536 were followed up, of whom 78 (14.6%, 95% CI; 11.6–17.5) developed SSIs. P. aeruginosa was found in eight (14%) of 57 investigated wounds. Of the 43 water sampling points, 29 were positive for P. aeruginosa. However, epidemiological links to wound infections were not confirmed. The P. aeruginosa carriage rate on admission was 0.9% (8/930). Of the 363 patients re-screened upon discharge, four (1.1%) possibly acquired P. aeruginosa during hospitalization. Wound infections of the three of the eight P. aeruginosa SSIs were caused by a strain of the same sequence type (ST) as the one from intestinal carriage. Isolates from patients were more resistant to antibiotics than water isolates. Conclusions: The P. aeruginosa SSI rate was low. There was no evidence for transmission from tap water. Not all P. aeruginosa SSI were proven to be endogenous, pointing to other routes of transmission. KW - Tanzania KW - P. aeruginosa KW - surgical site infection KW - water microbiology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158168 VL - 6 IS - 56 ER - TY - JOUR A1 - Schubert, Andreas A1 - Koziol, Uriel A1 - Cailliau, Katia A1 - Vanderstraete, Mathieu A1 - Dissous, Colette A1 - Brehm, Klaus T1 - Targeting Echinococcus multilocularis Stem Cells by Inhibition of the Polo-Like Kinase EmPlk1 N2 - Background Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation. Methodology/Principal findings We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation. Conclusions/Significance We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy. Author Summary The lethal disease AE is characterized by continuous and infiltrative growth of the metacestode larva of the tapeworm E. multilocularis within host organs. This cancer-like progression is exclusively driven by a population of parasite stem cells (germinative cells) that have to be eliminated for an effective cure of the disease. Current treatment options, using benzimidazoles, are parasitostatic only, and thus obviously not effective in germinative cell killing. We herein describe a novel, druggable parasite enzyme, EmPlk1, that specifically regulates germinative cell proliferation. We show that a compound, BI 2536, originally designed to inhibit the human ortholog of EmPlk1, can also inhibit the parasite protein at low doses. Furthermore, low doses of BI 2536 eliminated germinative cells from Echinococcus larvae in vitro and prevented parasite growth and development. We propose that BI 2536 and related compounds are promising drugs to complement current benzimidazole treatment for achieving parasite killing. KW - Vesicles KW - Sequence motif analysis KW - Xenopus oocytes KW - Echinococcus KW - Benzimidazoles KW - Host-pathogen interactions KW - Larvae KW - Cancer treatment Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112806 ER - TY - THES A1 - Gelmedin, Verena Magdalena T1 - Targeting flatworm signaling cascades for the development of novel anthelminthic drugs T1 - Signalkaskaden von Plattwürmern als Angriffspunkte zur Entwicklung neuer Antihelminthika N2 - Echinococcus multilocularis verursacht die Alveoläre Echinokokkose (AE), eine lebendsbedrohliche Krankheit mit limitierten chemotherapeutischen Möglichkeiten. Die jetzige Anti-AE Chemotherapie basiert auf einer einzigen Wirkstoffklasse, den Benzimidazolen. Obwohl Benzimidazole in vitro parasitozid wirken, wirken sie in vivo bei AE-Behandlung lediglich parasitostatisch und rufen schwere Nebenwirkungen hervor. In Fällen operabler Läsionen erfordert die Resektion des Parasitengewebes über einen längeren Zeitraum eine chemotherapeutische Unterstützung. Damit sind die jetzigen Behandlungsmöglichkeiten inadäquat und benötigen Alternativen. In der vorliegenden Arbeit wurden die Signalwege von Plattwürmern analysiert, um potentielle Targets für neue therapeutische Ansätze zu identifizieren. Dabei konzentrierte ich mich unter Anwendung von molekularbiologischer, biochemischer und zellbiologischer Methoden auf Faktoren, die an Entwicklung und Proliferation von E. multilocularis beteiligt sind. Darunter waren die drei MAP kinases des Parasiten EmMPK1, ein Erk1/2-Ortholog, EmMPK2, ein p38-Ortholog und EmMPK3, ein Erk7/8-Ortholog. Des Weiteren identifizierte und charakterisierte ich EmMKK2, ein MEK1/2-Ortholog des Parasiten, welches zusammen mit den bekannten Kinasen EmRaf und EmMPK1 ein Erk1/2-ähnliches MAPK Modul bildet. Ich konnte zudem verschiedene Einflüsse von Wirtswachstumsfaktoren wie EGF (epidermal growth factor) und Insulin auf die Signalmechanismen des Parasiten und das Larvenwachstum zeigen, darunter die Phosphorylierung von Elp, ein Ezrin-Radixin-Moesin ähnliches Protein, die Aktivierung von EmMPK1 und EmMPK3 und eine gesteigerte mitotische Aktivität der Echinokokkenzellen. Zusätzlich wurden verschiedene Substanzen auf ihre letale Wirkung auf den Parasiten untersucht, darunter befanden sich (1.) generelle Inhibitoren von Tyrosinkinasen (PP2, Leflunamid), (2.) gegen die Aktivität von Rezeptor-Tyrosin-Kinasen gerichtete Präparate, (3.) ursprünglich anti-neoplastische Wirkstoffe wie Miltefosin und Perifosin, (4.) Inhibitoren von Serin/ Threonin-Kinasen, die die Erk1/2 MAPK Kaskade blockieren und (5.) Inhibitoren der p38 MAPK. In diesen Untersuchungen hat sich EmMPK2 aus den folgenden Gründen als vielversprechendes Target erwiesen. Aminosäuresequenz-Analysen offenbarten einige Unterschiede zu menschlichen p38 MAP Kinasen, welche sehr wahrscheinlich die beobachtete gesteigerte basale Aktivität des rekombinanten EmMPK2 verursachen, verglichen mit der Aktivität humaner p38 MAPK-α. Zusätzlich suggerieren die prominente Autophosphorylierungsaktivität von rekombinantem EmMPK2 und das Ausbleiben einer Interaktion mit den Echinococcus MKKs einen unterschiedlichen Regulierungsmechanismus im Vergleich zu den humanen Proteinen. Die Aktivität von EmMPK2 konnte sowohl in vitro als auch in kultivierten Metazestodenvesikeln durch die Behandlung mit SB202190 und ML3403, zwei ATP kompetitiven Pyridinylimidazolinhibitoren der p38 MAPK, in Konzentrations-abhängiger Weise inhibiert werden. Zudem verursachten beide Substanzen, insbesondere ML3403 die Inaktivierung von Parasitenvesikeln bei Konzentrationen, die kultivierte Säugerzellen nicht beeinträchtigten. Ebenso verhinderte die Anwesenheit von ML3403 die Generation von neuen Vesikeln während der Kultivierung von Echinococcus Primärzellen. Das Targeting von Mitgliedern des EGF-Signalwegs, insbesondere der Erk1/2-ähnlichen MAPK Kaskade mit Raf- und MEK- Inhibitoren verhinderte die Phosphorylierung von EmMPK1 in in vitro kultivierten Metazestoden. Obwohl das Parasitenwachstum unter diesen Konditionen verhindert wurde, blieb die strukturelle Integrität der Metazestodenvesikeln während der Langzeitkultivierung in Anwesenheit der MAPK Kaskade-Inhibitoren erhalten. Ähnliche Effekte wurden beobachtet nach Behandlung mit den anderen zuvor aufgeführten Inhibitoren. Zusammenfassend lässt sich festhalten, dass verschiedene Targets identifiziert werden konnten, die hoch sensibel auf die Anwesenheit der inhibitorischen Substanzen reagierten, aber nicht zum Absterben des Parasiten führten, mit Ausnahme der Pyridinylimidazolen. Die vorliegenden Daten zeigen, dass EmMPK2 ein Überlebendsignal vermittelnden Faktor darstellt und dessen Inhibierung zur Behandlung der AE benutzt werden könnte. Dabei erwiesen sich p38 MAPK Inhibitoren der Pyridinylimidazolklasse als potentielle neue Substanzklasse gegen Echinokokken. N2 - Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a life-threatening disease with limited options of chemotherapeutic treatment. Anti-AE chemotherapy is currently based on a single class of drugs, the benzimidazoles. Although acting parasitocidic in vitro, benzimidazoles are merely parasitostatic during in vivo treatment of AE and cause severe site effects. In the case of operable lesions, the resection of parasite tissue needs to be supported by a prolonged chemotherapy. Thus, the current treatment options for AE are inadequate and require alternatives. In the present work, the flatworm signaling pathways were analyzed to establish potential targets for novel therapeutic approaches. I focused on factors that are involved in development and proliferation of E. multilocularis using molecular, biochemical and cell biological methods. Among the analysed factors were three MAP kinases of the parasite, EmMPK1, an Erk-1/2 orthologue, EmMPK2, a p38 orthologue and EmMPK3, an Erk7/8 orthologue. Further, I identified and characterized EmMKK2, a MEK1/2 orthologue of the parasite, which, together with the known kinases EmRaf and EmMPK1, forms an Erk1/2-like MAPK module. Moreover, I was able to demonstrate several influences of host growth factors such as EGF (epidermal growth factor) and insulin on worm signaling mechanisms and larval growth, including the phosphorylation of Elp, an ezrin-radixin-moesin like protein, EmMPK1, EmMPK3 and increased mitotic activity of Echinococcus cells. In addition, several substances were examined for their efficacy against the parasite including (i) general tyrosine kinase inhibitors (PP2, leflunamide), (ii) compounds designed to inhibit the activity of receptor tyrosine kinases, (iii) anti-neoplastic agents (miltefosine, perifosine), (iv) serine/threonine kinase inhibitors that have been designed to block the Erk1/2 MAPK cascade and (v) inhibitors of p38 MAPKs. In these studies, EmMPK2 proved to be a promising drug target for the following reasons. Amino acid sequence analysis disclosed several differences to human p38 MAPKs, which is likely to be the reason for the observed enhanced basal activity of recombinant EmMPK2 towards myelin basic protein in comparison to human recombinant p38 MAPK-α. In addition, the prominent auto-phosphorylation activity of the recombinant EmMPK2 protein together with the absence of an interaction with the Echinococcus MKKs suggest a different mechanism of regulation compared to the human enzyme. EmMPK2 activity could be effectively inhibited in vitro and in cultivated metacestode vesicles by treatment with SB202190 and ML3403, two ATP-competitive pyridinyl imidazole inhibitors of p38 MAPKs, in a concentration-dependent manner. Moreover, both compounds, in particular ML3403, caused parasite vesicle inactivation at concentrations which did not affect cultured mammalian cells. Likewise, during the cultivation of Echinococcus primary cells, the presence of ML3403 prevented the generation of new vesicles. Targeting members of the EGF signaling pathway, particulary of the Erk1/2-like MAPK cascade, with Raf and MEK inhibitors prevented the phosphorylation of EmMPK1 in metacestodes cultivated in vitro. However, although parasite growth was prevented under these conditions, the structural integrity of the metacestode vesicles maintained during long-term cultivation in the presence of the MAPK cascade inhibitors. Similar results were obtained when studying the effects of other drugs mentioned above. Taken together, several targets could be identified that reacted with high sensitivity to the presence of inhibitory substances, but did not cause the parasite’s death with one exception, the pyridinyl imidazoles. Based on the presented data, I suggest pyridinyl imidazoles as a novel class of anti-Echinococcus drugs and imply EmMPK2 as survival signal mediating factor, the inhibition of which could be used for the treatment of AE. KW - Fuchsbandwurm KW - Signaltransduktion KW - MAP-Kinase KW - Eingeweidewürmer KW - Proliferation KW - Zelldifferenzierung KW - Inhibitor KW - Entwicklung KW - Heilmittel KW - Fox tapeworm KW - signaltransduction KW - MAP kinase KW - chemotherapy KW - development Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33334 ER - TY - THES A1 - Peters, Simon T1 - The impact of sphingolipids on \(Neisseria\) \(meningitidis\) and their role in meningococcal pathogenicity T1 - Einfluss von Sphingolipiden auf \(Neisseria\) \(meningitidis\) und deren Bedeutung für die Pathogenität N2 - The obligate human pathogen Neisseria meningitidis is a major cause of sepsis and meningitis worldwide. It affects mainly toddlers and infants and is responsible for thousands of deaths each year. In this study, different aspects of the importance of sphingolipids in meningococcal pathogenicity were investigated. In a first step, the acid sphingomyelinase (ASM), which degrades membrane sphingomyelin to ceramide, was studied in the context of meningococcal infection. A requirement for ASM surface activity is its translocation from the lysosomal compartment to the cell surface, a process that is currently poorly understood. This study used various approaches, including classical invasion and adherence assays, flow cytometry, and classical and super resolution immunofluorescence microscopy (dSTORM). The results showed that the live, highly piliated N. meningitidis strain 8013/12 induced calcium-dependent ASM translocation in human brain microvascular endothelial cells (HBMEC). Furthermore, it promoted the formation of ceramide-rich platforms (CRPs). In addition, ASM translocation and CRP formation were observed after treating the cells with pili-enriched fractions derived from the same strain. The importance for N. meningitidis to utilize this pathway was shown by the inhibition of the calcium-dependent ASM translocation, which greatly decreased the number of invasive bacteria. I also investigated the importance of the glycosphingolipids GM1 and Gb3. The results showed that GM1, but not Gb3, plays an important role in the ability of N. meningitidis to invade HBMEC. By combining dSTORM imaging and microbiological approaches, we demonstrated that GM1 accumulated prolifically around bacteria during the infection, and that this interaction seemed essential for meningococcal invasion. Sphingolipids are not only known for their beneficial effect on pathogens. Sphingoid bases, including sphingosine, are known for their antimicrobial activity. In the last part of this study, a novel correlative light and electron microscopy approach was established in the combination with click chemistry to precisely localize azido-functionalized sphingolipids in N. meningitidis. The result showed a distinct concentration-dependent localization in either the outer membrane (low concentration) or accumulated in the cytosol (high concentration). This pattern was confirmed by mass spectrometry on separated membrane fractions. Our data provide a first insight into the underlying mechanism of antimicrobial sphingolipids. N2 - Der obligate Humanpathogen Neisseria meningitidis ist weltweit einer der Hauptursachen für Sepsis und Meningitis. Er befällt vor allem Kleinkinder und Säuglinge und ist jedes Jahr für Tausende von Todesfällen verantwortlich. In dieser Studie wurden verschiedene Aspekte der Bedeutung von Sphingolipiden bei der Pathogenität von Meningokokken untersucht. In einem ersten Schritt wurde die saure Sphingomyelinase (ASM), die Membran-Sphingomyelin zu Ceramid abbaut, im Zusammenhang mit einer Meningokokken-Infektion untersucht. Eine Voraussetzung für die Oberflächenaktivität der ASM ist ihre Translokation vom lysosomalen Kompartiment auf die Zelloberfläche, ein Prozess, der derzeit noch wenig verstanden wird. In dieser Studie wurden verschiedene Ansätze verwendet, darunter klassische Invasions- und Adhärenztests, Durchflusszytometrie sowie klassische und superauflösende Immunfluoreszenzmikroskopie (dSTORM). Die Ergebnisse zeigten, dass der lebende, hochpiliatisierte N. meningitidis Stamm 8013/12 eine kalziumabhängige ASM-Translokation in mikrovaskulären Endothelzellen des menschlichen Gehirns (HBMEC) induzierte. Des Weiteren förderte er die Bildung Ceramid-reicher Plattformen (CRPs). Zusätzlich wurden ASM-Translokation und CRP-Bildung beobachtet, nachdem die Zellen mit pili-angereicherten Fraktionen desselben Stammes behandelt worden waren. Die Bedeutung für N. meningitidis in der Pathogenese zeigte sich durch die Hemmung der Calcium-abhängigen ASM-Translokation, wodurch die Zahl der invasiven Bakterien stark reduziert wurde. Ich untersuchte auch die Bedeutung der Glykosphingolipide GM1 und Gb3. Die Ergebnisse zeigten, dass GM1, aber nicht Gb3, eine wichtige Rolle bei der Fähigkeit von N. meningitidis spielt, in Gehirnendothelzellen einzudringen. Durch die Kombination von dSTORM-Bildgebung und mikrobiologischen Ansätzen konnten wir zeigen, dass sich GM1 während der Infektion vermehrt um die Bakterien herum anreicherte und dass diese Interaktion für die Invasion von Meningokokken essenziell ist. Sphingolipide sind nicht nur für ihre positive Wirkung auf Krankheitserreger bekannt. Sphingoidbasen, einschließlich Sphingosin, sind zusätzlich für ihre antimikrobielle Aktivität bekannt. Im letzten Teil dieser Studie wurde ein neuartiger korrelativer licht- und elektronenmikroskopischer Ansatz in der Kombination mit Click-Chemie etabliert, um azidofunktionalisierte Sphingolipide in N. meningitidis genau zu lokalisieren. Das Ergebnis zeigte eine deutliche konzentrationsabhängige Lokalisation entweder in der äußeren Membran (niedrige Konzentration) oder akkumuliert im Zytosol (hohe Konzentration). Dieses Muster konnte durch einen Massenspektrometrischen Ansatz bestätigt werden. Hierfür wurde eine Separation der inneren und äußeren Membran, nach Behandlung mit der niedrigen Konzentration, etabliert. Die verschiedenen Membranfraktionen wurden anschließend auf ihren Gehalt an funktionalisierten Sphingolipiden hin untersucht und bestätigten die lokalisierung in der äußeren Membran. Unsere Daten geben einen ersten Einblick in den zugrundeliegenden Mechanismus der antimikrobiellen Sphingolipide. KW - Neisseria meningitidis KW - Sphingolipide KW - Infektion KW - Pathogenität KW - host-pathogen interaction KW - antimicrobial Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226233 ER - TY - THES A1 - Bauriedl, Saskia Corinna T1 - The influence of riboregulation on fitness and virulence in Neisseria meningitidis T1 - Der Einfluss der Riboregulation auf Fitness und Virulenz von Neisseria meningitidis N2 - Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies. A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx. Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis. Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches. N2 - Neisseria meningitidis (N. meningitidis) ist ein kommensal lebendes Bakterium, welches unter nicht vollständig geklärten Bedingungen auch lebensbedrohliche Infektionen im Menschen wie bakterielle Meningitis und Sepsis verursachen kann. Obwohl experimentell nachgewiesen wurde, dass die Expression kleiner, nicht kodierender RNAs (sRNAs) so-wie des RNA-Chaperons Hfq in Meningokokken physiologisch relevant ist, blieb der Ein-fluss der RNA-basierten Genregulation (Riboregulation) auf die Fitness und Virulenz von N. meningitidis bisher unvollständig verstanden. Daher befasste sich diese Studie durch Kombination verschiedener Hochdurchsatz-Technologien mit dieser Fragestellung. Es wurde differentielle RNA-Sequenzierung (dRNA-seq) angewendet, um das primäre Transkriptom des N. meningitidis Stamms 8013 möglichst genau zu kartieren. Die durch-geführte dRNA-seq-Analyse detektierte 1.625 Transkriptionsstartstellen (TSS) einschließ-lich 65 potentieller sRNAs. Durch Anwendung von Northern-Blot-Analysen konnten an-schließend 20 sRNAs experimentell validiert werden. Darüber hinaus wurde durch Ko-Immunopräzipitation mit Hfq (RIP-seq) ein großes, Hfq-zentriertes, post- transkripti-onelles regulatorisches Netzwerk identifiziert, welches 23 sRNAs und 401 mRNAs um-fasst. Rifampicin-Stabilitätsversuche zeigten, dass durch Hfq-Bindung die Stabilität die-ser sRNAs erhöht wird. Basierend auf diesen Daten konnte die Interaktion zwischen zweier Hfq-gebundener paraloger sRNAs und der prpB mRNA sowohl in vivo als auch in vitro bestätigt werden. Beide sRNAs reprimieren die Translation des PrpB-Genes, wel-ches für eine Methylisocitratlyase kodiert und wahrscheinlich die Kolonisation des menschlichen Nasopharynxs durch Meningokokken begünstigt. Neben dem ausführlich charakterisierten RNA-Chaperon Hfq wurden Proteine mit FinO-Domäne kürzlich als eine neue Familie von RNA-bindenden Proteinen (RBPs) mit regula-torischen Funktionen in verschiedenen Bakterien identifiziert. Sie weisen eine große Bandbreite regulierter Gene auf: Während das Plasmid-kodierte FinO-Protein nur ein ein-zelnes RNA-Paar bindet, stellt das enterobakterielle ProQ-Protein ein globales RBP dar. Um die Wirkungsweise dieser RBP-Familie besser zu verstehen, wurde in vivo untersucht, wie viele RNAs mit dem minimalen ProQ-Protein in N. meningitidis assoziiert sind. Durch Kombination von UV-Crosslinken mit RNA-Sequenzierung (UV-CLIP) konnte die Bin-dung von 16 sRNAs und 166 biologisch diverser mRNAs mit ProQ identifiziert werden, welches daher ebenfalls ein globales RBP in Meningokokken darstellt. Es konnte gezeigt werden, dass ProQ vorwiegend RNA-Regionen mit ausgeprägter Sekundärstruktur bin-det, darunter DNA-Aufnahmesequenzen (DUS) und Rho-unabhängige Transkriptions-terminatoren. Die ProQ-Bindung führt dabei häufig zur Stabilisation der RNAs, was durch Rifampicin-Stabilitätsexperimente nachgewiesen wurde. Wie aufgrund der großen Zahl ProQ-gebundener RNAs zu erwarten, beeinflusste die Deletion des ProQ Proteins die zelluläre Expression von mindestens 244 mRNAs und 80 Proteinen. Phänotypische Analysen deuten darauf hin, dass ProQ sowohl die Toleranz gegenüber oxidativem Stress als auch die Reparatur von DNA-Schäden reguliert, die beide für die vollständige Viru-lenz von N. meningitidis von Bedeutung sind. Zusammenfassend beschreibt diese Arbeit die Koexistenz von zwei großen posttranskrip-tionellen Regulons in N. meningitidis, von denen eines von ProQ und das andere von Hfq kontrolliert wird. Im Rahmen dieser Arbeit wurde die Rolle beider RBPs und ihrer assozi-ierten sRNAs für die bakterielle Virulenz verdeutlicht und hervorgehoben, dass Riboregu-lation sehr wahrscheinlich dazu beiträgt, wie sich Meningokokken an verschiedene Wirts-nischen anpassen. KW - Neisseria meningitidis KW - Small non-messenger RNS KW - Hfq KW - ProQ KW - Non-coding RNA KW - High throughput screening Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192978 ER - TY - JOUR A1 - Bauriedl, Saskia A1 - Gerovac, Milan A1 - Heidrich, Nadja A1 - Bischler, Thorsten A1 - Barquist, Lars A1 - Vogel, Jörg A1 - Schoen, Christoph T1 - The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition JF - Nature Communications N2 - FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence. KW - Neisseria meningitidis KW - natural transformation KW - dual function KW - FinO family KW - HFQ KW - chaperone KW - transcriptome KW - regulator KW - sequence KW - in vivo Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230040 VL - 11 ER - TY - JOUR A1 - Heidrich, Nadja A1 - Bauriedl, Saskia A1 - Barquist, Lars A1 - Li, Lei A1 - Schoen, Christoph A1 - Vogel, Jörg T1 - The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq JF - Nucleic Acids Research N2 - Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of −35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx. KW - RNA KW - Neisseria meningitidis KW - dRNA-seq KW - transcriptome KW - RNA chaperone Hfq Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170828 VL - 45 IS - 10 ER - TY - THES A1 - Ye, Fang T1 - The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori N2 - Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA (ó28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second ó28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori. N2 - Zusammenfassung Die Mechanismen der globalen Kontrolle der Genregulation bei Bakterien sind bisher noch wenig charakterisiert. Unterschiede in der globalen oder lokalen Topologie der chromosomalen DNA spielen wahrscheinlich eine Rolle bei der globalen Kontrolle der Genexpression. Bei Helicobacter pylori, einem Bakterium mit wenigen funktionell definierten Transkriptionsregulatoren, spricht die Struktur einiger Promotoren dafür, daß sie durch die DNA-Topologie kontrolliert werden. Der Promotor des Hauptflagellingens flaA, ein Sigma-28 abhängiger Promotor, hat ein gegenüber dem Konsensuspromotor (15 Nukleotide) verkürztes Spacing von 13 Nukleotiden. Veränderungen der DNA-Superhelizität könnten ein Mechanismus der genspezifischen und globalen transkriptionellen Kontrolle in diesem Bakterium sein. Ziel dieser Untersuchungen war es zu zeigen, ob Veränderungen des globalen Supercoiling-Niveaus einen Einfluß auf die globale Genregulation von H. pylori haben und ob sie sich auf die zeitlich gesteuerte Regulation der Geißelbiosynthese (Beweglichkeitsorganell; essenzieller Virulenz- und Persistenzfaktor von H. pylori) in diesem Organismus auswirkt. In der vorliegenden Arbeit wurde das Niveau der DNA-Superspiralisierung bei H. pylori erstmals durch Visualisierung des Supercoilingzustands von Plasmiden unter unterschiedlichen Wachstumsbedingungen dargestellt. Wir konnten mit dieser Methode zeigen, daß das zelluläre Supercoiling-Niveau bei H. pylori in Abhängigkeit von der Wachstumsphase stark variiert. In Wachstumsphasen mit erhöhtem Supercoiling war auch die Transkription des flaA-Gens erhöht, während die Transkription eines zweiten Sigma-28-abhängigen Gens mit normalem Promotorabstand (HP0472) reduziert war. Dieser Befund stützte die Hypothese, daß die wachstumsphasenabhängige Aktivität dieser Promotoren durch Veränderungen der DNA-Topologie bewirkt wird. Das Supercoiling-Niveau konnte reproduzierbar durch Hemmung der Gyrase mit Novobiocin beeinflusst werden. Die Gegenwart von Novobiocin führte zur DNA-Relaxation und zu einem gleichzeitigen Absinken der Transkription von flaA. Es wurde eine gerichtete Mutagenese der Promotor-Spacer-Region des flaA-Promotors durchgeführt. Die Verlängerung oder Verkürzung des H.pylori flaA-Promotors führte zu einer verminderten Transkription von flaA, sowie zu reduzierter Empfindlichkeit der Promotoraktivität gegenüber Veränderungen des Supercoiling-Niveaus. Unter spezifischen Bedingungen war die Supercoiling-Abhängigkeit umgekehrt im Vergleich zum Wildtyppromotor. Es konnte weiterhin eine inverse transkriptionelle Abhängigkeit zwischen dem gekoppelten Genpaar topA-flaB und flaA durch Analyse der flaA-Promotormutanten nachgewiesen werden. Auch die chromosomal gekoppelten Gene gyrA und flgR sowie topA und flaB waren abhängig vom Supercoiling-Zustand und miteinander koreguliert. Die Analyse des Transkriptoms von H.pylori-Wildtypbakterien mit DNA-Microarrays mit und ohne Novobiocinbehandlung führte zur Identifizierung von zahlreichen Genen (etwa 10% des Gesamttranskriptoms), deren Expression Supercoiling-abhängig war und durch Veränderungen des Supercoilings synchronisiert verändert werden konnte. Unter diesen waren Flagellin-, andere Virulenz-, sowie Grundstoffwechsel-Gene. Diese Befunde weisen auf eine enge Verbindung zwischen der chronologischen Kontrolle der Flagellen-Biogenese und des Metabolismus bei H. pylori, die gemeinsam durch das Supercoiling-Niveau gesteuert werden. Eine definierte Gruppe von Genen konnte bei H.pylori durch Überexpression von Topoisomerase-1 reguliert werden. Das Protein HU beeinflusst ebenfalls das Supercoiling-Niveau von DNA durch seine Fähigkeit, DNA zu biegen. HU wird bei H. pylori durch das Gen hup kodiert und ist während sämtlicher Wachstumsphasen konstitutiv exprimiert. Eine HU-defiziente Mutante wurde konstruiert. Zellen, die kein HU-Protein exprimierten, waren lebensfähig, zeigten aber einen deutlichen Wachstumsdefekt. Unsere Daten weisen daraufhin, daß der Mangel von HU sich dramatisch auf das globale DNA-Supercoiling-Niveau auswirkt, und sprechen für eine wichtige Funktion von HU bei der Kontrolle der DNA-Struktur von H. pylori. Mittels DNA-Microarray-Hybridisierung wurden die Transkriptome von H. pylori-Wildtyp und HU-Mutante miteinander verglichen. Die Ergebnisse zeigen, daß insgesamt 66 Gene in der HU-Mutante differentiell transkribiert werden, darunter Virulenzgene und Gene für viele andere Zellfunktionen. Diese Daten deuten darauf hin, daß auch HU eine wichtige Rolle in der Kontrolle der globalen Genexpression bei H. pylori spielt. Die erhöhte Expression von Hitzestress-Proteinen, verbunden mit einer verminderten Transkription des Ureasegenclusters, könnte auf eine koordinierte Antwort der Bakterien auf Veränderungen der Umweltbedingungen in ihrer spezifischen ökologischen Nische hinweisen. Nach der Publikation der Gesamtgenomsequenzen von H.pylori 26695 und J99 wurden 2 ORFs (HP 0116 und HP 0440) als Topoisomerase-1-Orthologe annotiert. HP 0116 ist das funktionelle H.pylori Topoisomerase-1-Gen. HP 0442 (topA2) wurde nur in wenigen (5 aus 43) Stämmen nachgewiesen. topA2 ist trotz seines seltenen Vorkommens kein Pseudogen und wird in H.pylori transkribiert. Westernblot-Analysen sprechen dafür, daß TopA2 sich antigenetisch von TopA unterscheidet. Das TopA2-Protein unterscheidet sich ebenfalls funktionell von TopA, da ihm ein funktionell essentielles Zinkfingermotiv fehlt. TopA2 konnte außerdem eine TopA-defiziente E.coli-Mutante nicht funktionell komplementieren. Wie bei topA war auch die Transkription von topA2 von der Wachstumsphase abhängig. Eine Funktion von TopA2 bei der Kontrolle der DNA-Topologie konnte bisher nicht nachgewiesen werden, Transkriptomanalysen zeigten aber, dass TopA2 eine klare Regulationsfunktion hat, da die topA2-Mutante gravierende Veränderungen des Transkriptoms gegenüber dem Wildtyp aufwies. Diese Untersuchungen zeigten, daß 46 Gene in der TopA2-Mutante differentiell reguliert wurden, darunter Flagellengene und Ureasegene. Die Ergebnisse sprechen dafür, daß TopA2 ein weiterer wichtiger Regulator von sowohl Flagellenbiosynthese als auch Ureasebildung bei H.pylori sein könnte. KW - Helicobacter pylori KW - Genexpression KW - Flagelline KW - Supercoiling KW - regulation KW - flagella Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-9878 ER - TY - THES A1 - von Saint André - von Arnim, Amélie T1 - The Role of Endosymbiotic Wolbachia Bacteria in the Pathogenesis of River Blindness T1 - Die Rolle des endosymbiontischen Wolbachia Bakteriums in der Pathogenese der Flußblindheit N2 - Introduction: This study investigates the role of Wolbachia bacteria in the pathogenesis of O. volvulus keratitis in a mouse model. Wolbachia bacteria are essential symbionts of most filarial nematodes of importance for mankind. Methods: Using a mouse model for river blindness in which soluble extracts of filarial nematodes are injected in the corneal stroma, changes in stromal thickness and haze of the cornea are observed by in vivo confocal microscopy, followed by immunohistochemical staining for neutrophils and PECAM-1, as well as ELISA of corneal chemokines. Reactions to filarial extracts containing Wolbachia are compared to those without the endosymbiont. Results: The approach of characterizing Wolbachia’s role in river blindness in this study is threefold. Firstly, Wolbachia-depleted extracts from doxycycline treated onchocerciasis patients led to a diminished inflammatory response in corneas of C57BL/6 mice compared to untreated, i.e. Wolbachia containing antigen. The decreased cell recruitment observed with doxycycline treated extracts involved neutrophils, but not eosinophils. This finding demonstrated that the presence of Wolbachia increases neutrophil recruitment. Secondly, extracts from Wolbachia-containing B. malayi revealed markedly more pathology than endosymbiont-free A. viteae antigen. This again pointed at the role of Wolbachia in development of disease. Thirdly, Toll-like Receptor 4 (TLR4) dependence was shown to exist for the inflammatory response to Wolbachia harboring O. volvulus antigen by looking at the corneal pathology in TLR4-mutant C3H/HeJ mice, compared to the wild-type C3H/HeN strain. Investigating further Wolbachia mediated mechanisms of neutrophil recruitment to the cornea, this study also showed that expression of the adhesion molecule PECAM-1 in limbal vessels, as well as upregulation of the CXC chemokines KC and MIP-2 were dependent on the presence of functional TLR4 and Wolbachia respectively. Conclusions: This study indicates that the innate immune system and Wolbachia endobacteria play an important role in the inflammatory response associated with the pathogenesis of onchocerca keratitis, suggesting a complete alteration in our understanding of the immunopathology of filariasis. N2 - Einleitung: Diese Arbeit untersucht die Rolle des Bakteriums Wolbachia in der Pathogenese der Onchozerka volvulus Keratitis anhand eines Mausmodels. Wolbachia sind essentielle endosymbiontische Bakterien, die in den meisten Filariosen, die für die Menschheit von Bedeutung sind, existieren. Methoden: Mit Hilfe eines Mausmodels für die Flußblindheit, in dem lösliche Filarienextrakte in das korneale Stroma von Mäusen injiziert werden, lassen sich Veränderungen in der Stromadicke und –durchsichtigkeit mit in vivo konfokaler Mikroskopie beobachten, gefolgt von immunhistochemischer Färbung von Neutrophilen und PECAM-1, wie auch ELISA von kornealen Chemokinen. Dabei werden Entzündungsreaktionen nach Injektion von Filarienmaterial mit oder ohne Wolbachia verglichen. Resultate: Die Untersuchung von Wolbachia's Rolle in der Flußblindheit erfolgte in drei Schritten. Zunächst führte Antigenmaterial von Wolbachia-freien, mit Doxyzyklin behandelten Onchozerkosepatienten zu geringerer Entzündungsreaktion in der Kornea von C57BL/6 Mäusen verglichen mit Wolbachia-enthaltendem Material. Die verminderte Enzündungszellzahl bei Doxyzyklin-behandelten Extrakten umfasste Neutrophile, aber nicht Eosinophile Granulozyten. Die Anwesenheit von Wolbachia führt daher zu verstärkter Neutrophileneinwanderung. Zweitens erwiesen Wolbachia-enthaltende B. malayi Extrakte eine signifikant verstärkte korneale Pathologie verglichen mit Endosymbiont-freiem A. viteae Antigen. Dieses Ergebnis deutete erneut auf die Rolle von Wolbachia in der Krankheitsentstehung. Drittens wurde anhand von Toll-like Rezeptor 4 (TLR4) mutanten C3H/HeJ Mäusen gezeigt, dass die Entzündungsreaktion, die von Wolbachia-enthaltenden O. volvulus Extrakten hervorgerufen wird, von TLR4 abhängig ist. Weitere Untersuchungen Wolbachia-abhängiger Mechanismen der Neutrophileneinwanderung in die Kornea erwiesen, dass die Expression des Adhäsionsmoleküls PECAM-1 in limbischen Gefäßen, wie auch die Hochregulation der CXC Chemokine KC und MIP-2 von TLR4 und der Anwesenheit von Wolbachia abhängig sind. Konklusion: Diese Arbeit zeigt, dass das angeborene Immunsystem und Wolbachia eine wichtige Rolle in der Pathogenese der O. volvulus Keratitis spielen, was auf eine neue Verstehensweise der Filariosenimmunpathologie hinweist. KW - Onchozerkose KW - Wolbachia KW - Endosymbiont KW - Filariose KW - Konfokale Mikroskopie KW - Doxyzyklin KW - Toll like Rezeptor 4 KW - Doxycycline KW - Toll like receptor 4 Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-31560 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - The role of patients and healthcare workers Staphylococcus aureus nasal colonization in occurrence of surgical site infection among patients admitted in two centers in Tanzania JF - Antimicrobial Resistance & Infection Control N2 - Background Colonization with Staphylococcus aureus has been identified as a risk for subsequent occurrence of infection. This study investigated the relationship between S. aureus colonization of patients and healthcare workers (HCWs), and subsequent surgical site infections (SSI). Methods Between December 2014 and September 2015, a total of 930 patients and 143 HCWs were enrolled from the Bugando Medical Centre and Sekou Toure hospital in Mwanza, Tanzania. On admission and discharge nasal swabs, with an additional of wound swab for those who developed SSI were collected from patients whereas HCWs were swabbed once. Identification and antimicrobial susceptibility testing were done by VITEK-MS and VITEK-2, respectively. Detection of Panton Valentine leukocidin (PVL) and mecA genes was done by PCR. S. aureus isolates were further characterized by spa typing and Multi-Locus Sequence Typing (MLST). Results Among 930 patients screened for S. aureus on admission, 129 (13.9%) were positive of which 5.4% (7/129) were methicillin-resistant S. aureus (MRSA). Amongst 363 patients rescreened on discharge, 301 patients had been tested negative on admission of whom 29 (9.6%) turned positive after their hospital stay. Three (10.3%) of the 29 acquired S. aureus were MRSA. Inducible Clindamycin resistance occurred more often among acquired S. aureus isolates than among isolates from admission [34.5% (10/29) vs. 17.1% (22/129), P = 0.018]. S. aureus contributed to 21.1% (n = 12) of the 57 cases of investigated SSIs among 536 patients followed. Seven out of eight S. aureus carriage/infection pairs had the same spa and sequence types. The previously reported dominant PVL-positive ST88 MRSA strain with spa type t690 was detected in patients and HCW. Conclusion A significant proportion of patients acquired S. aureus during hospitalization. The finding of more than 90% of S. aureus SSI to be of endogenous source underscores the need of improving infection prevention and control measures including screening and decolonization of high risk patients. KW - S. aureus KW - colonization KW - surgical site infection KW - Tanzania Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224185 VL - 8 ER - TY - THES A1 - Fohmann, Ingo T1 - The Role of Sphingosine 1-phosphate and S1PR1-3 in the Pathophysiology of Meningococcal Meningitis T1 - Die Rolle von Sphingosin 1-Phosphat und S1PR1-3 in der Pathophysiologie der durch Meningokokken ausgelösten Meningitis N2 - Neisseria meningitidis (N. meningitidis) is an obligate human pathogen which causes live-threatening sepsis and meningitis. The fatality rate after meningococcal infection is high and surviving patients often suffer from severe sequelae. To cause meningitis, N. meningitidis must overcome the endothelium of the blood-brain barrier. The bacterium achieves this through the interaction with endothelial surface receptors leading to alternations of the cellular metabolism and signaling, which lastly results in cellular uptake and barrier traversal of N. meningitidis. Sphingosine 1-phosphate (S1P) is a lipid mediator that belongs to the class of sphingolipids and regulates the integrity of the blood-brain barrier through the interaction with its cognate receptors S1P receptors 1-3 (S1PR1-3). In this study, high performance liquid chromatography coupled with mass spectrometry (LC-MS/MS) was used to generate a time-resolved picture of the sphingolipid metabolism in a brain endothelial cell line (hCMEC/D3) upon meningococcal infection. Among various changes, S1P was elevated in the cellular compartment as well as in the supernatant of infected hCMEC/D3s. Analysis of mRNA expression in infected hCMEC/D3s with quantitative real-time polymerase chain reaction (RT-qPCR) revealed that the increase in S1P could be attributed to the enhanced expression of the S1P-generating enzyme sphingosine kinase 1 (SphK1). Antibody-based detection of SphK1 protein or phosphorylation at SphK1 residue Serine 225 in hCMEC/D3 plasma membrane fractions via Western Blot revealed that N. meningitidis also induced SphK1 phospho-activation and recruitment to the plasma membrane. Importantly, recruitment of SphK1 to the plasma membrane increases the probability of substrate encounter, thus elevating SphK activity. Enhanced SphK activity was also reflected on a functional level, as detected by a commercially available ATP depletion assay used for measuring the enzymatic activity of SphK. Infection of hCMEC/D3 cells with pilus-deficient mutants resulted in a lower SphK activation compared to the N. meningitidis wild type strain. hCMEC/D3 treatment with pilus-enriched protein fractions showed SphK activation similar to the infection with living bacteria and could be ascribed to pilus interaction with the membrane-proximal domain of cellular surface receptor CD147. Inhibition of SphK1 or SphK2 through pre-treatment with specific inhibitors or RNA interference reduced uptake of N. meningitidis into hCMEC/D3 cells, as measured with Gentamicin protection assays. Released S1P induced the phospho-activation of epidermal growth factor receptor (EGFR) via S1PR2 activation, whose expression was also increasing during infection. Furthermore, S1PR2 blockage had a preventive effect on bacterial invasion into hCMEC/D3 cells. On the contrary, activation of S1PR1+3 also reduced bacterial uptake, indicating an opposing regulatory role of S1PR1+3 and S1PR2 during N. meningitidis uptake. Moreover, SphK2 inhibition prevented inflammatory cytokine expression as well as release of interleukin-8 after N. meningitidis infection. Taken together, this study demonstrates the central role of S1P and its cognate receptors S1PR1-3 in the pathophysiology of meningococcal meningitis. N2 - Neisseria meningitidis (N. meningitidis) ist ein obligat humanpathogenes Bakterium, welches lebensbedrohliche Sepsis und Meningitis auslöst. Die Todesrate nach einer Meningokokkeninfektion ist hoch und überlebende Patienten leiden oft unter gravierenden Folgeschäden. N. meningitidis muss zuerst das Endothel der Blut-Hirn-Schranke überwinden, um Meningitis auslösen zu können. Das Bakterium erzielt dies durch die Interaktion mit endothelialen Rezeptoren, welche den zellulären Metabolismus und die zellulären Signalwege beeinflusst und letztlich zur zellulären Aufnahme von N. meningitidis und zur Überwindung der Barriere führt. Sphingosine 1-phosphat (S1P) ist ein Lipidmediator, der zur Klasse der Sphingolipide gehört und die Integrität der Blut-Hirn-Schranke durch die Interaktion mit den zugehörigen S1P Rezeptoren 1-3 (S1PR1-3s) beeinflusst. In dieser Arbeit wurde Hochleistungsflüssigkeitschromatographie-gekoppelte Massenspektrometrie (LC-MS/MS) genutzt, um ein zeitlich aufgelöstes Bild des Sphingolipidmetabolismus in einer Hinendothelzelllinie (hCMEC/D3) nach Meningokokkeninfektion zu generieren. Neben zahlreichen Veränderungen zeigte sich ein Anstieg von S1P im zellulären Kompartiment und im Überstand von infizierten hCMEC/D3 Zellen. Die Analyse der mRNA Expression in infizierten hCMEC/D3 Zellen mittels quantitativer Echtzeit-Polymerase-Kettenreaktion (RT-qPCR) offenbarte, dass der Anstieg von S1P auf eine erhöhte Expression der S1P-bildenden Sphingosinkinase 1 (SphK1) zurückzuführen war. Die ntikörperbasierte Detektion des Proteins SphK1 oder dessen Phosphorylierung an Serin 225 in den Membranfraktionen von hCMEC/D3 Zellen mittels Western Blot zeigte, dass N. meningitidis außerdem die Phospho-Aktivierung und Membrantranslokation von SphK1 induzierte. Die Plasmamembrantranslokation von SphK1 erhöht die Wahrscheinlichkeit auf das Substrat Sphingosine zu treffen und verstärkt somit die SphK-Aktivität. Die erhöhte SphK-Aktivität zeigte sich auch auf funktioneller Ebene, wie mittels eines ATP-Verbrauchs-Assays zur Messung der SphK-Aktivität nachgewiesen werden konnte. Die Infektion von hCMEC/D3 Zellen mit Pilus-defizienten Mutanten resultierte in einer geringeren SphK-Aktivierung im Vergleich zum Wildtypstamm. Die Behandlung von hCMEC/D3 Zellen mit Pilus-aufgereinigten Fraktionen zeigte eine SphK-Aktivierung, die mit der Aktivierung durch lebende Bakterien vergleichbar war und der Interaktion des Pilus mit der membranproximalen Domäne des zellulären Oberflächenrezeptors CD147 zugeordnet werden konnte. Die Inhibition von SphK1 und SphK2 durch die Vorbehandlung mit spezifischen Inhibitoren oder RNA-Interferenz reduzierte die Aufnahme von N. meningitidis in hCMEC/D3 Zellen, wie mittels Gentamicin Protection Assay nachgewiesen wurde. Das freigesetzte S1P induzierte die Phospho-Aktivierung des epidermalen Wachstumsfaktorrezeptors (EGFR) durch die Aktivierung von S1PR2, welcher während der Infektion vermehrt exprimiert wurde. Die Blockierung von S1PR2 hatte einen präventiven Effekt auf die bakterielle Invasion in hCMEC/D3 Zellen. Im Gegenzug reduzierte die Aktivierung von S1PR1+3 ebenfalls die bakterielle Aufnahme, was auf eine gegensätzliche regulatorische Rolle von S1PR1+3 und S1PR2 während der Aufnahme von N. meningitidis in hCMEC/D3 Zellen hindeutet. Darüber hinaus verhinderte die Inhibition von SphK2 die Expression von inflammatorischen Cytokinen sowie die Freisetzung von Interleukin-8 nach Infektion mit N. meningitidis. Zusammenfassend zeigt diese Arbeit die zentrale Rolle von S1P und den zugehörigen Rezeptoren S1PR1-3 in der Pathophysiologie der durch Meningokokken ausgelösten Meningitis. KW - Blut-Hirn-Schranke KW - Sphingosinkinase KW - Sphingolipide KW - Bakterielle Infektion KW - Sphingosine 1-phosphate KW - Sphingosine 1-phosphate receptor KW - Epidermal growth factor receptor KW - CD147 KW - S1P KW - S1PR KW - Meningococci KW - Basigin KW - EGFR KW - Meningitis cerebrospinalis epidemica KW - Meningitis, Meningococcal Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369764 ER - TY - JOUR A1 - Streck, Laura Elisa A1 - Forster, Johannes A1 - von Hertzberg-Boelch, Sebastian Philipp A1 - Reichel, Thomas A1 - Rudert, Maximilian A1 - Rueckl, Kilian T1 - The role of synovial fluid aspiration in shoulder joint infections JF - BMC Musculoskeletal Disorders N2 - Background Joint aspiration with analysis of synovial fluid white blood cell count (WBC) and microbiological culture is a widely established aspect in the diagnosis of shoulder joint infections (SJI). In case of a two stage revision for SJI, joint aspiration before re−/implantation of a total shoulder arthroplasty (TSA) was used to rule out persistent infection for years but its value is under debate. Shoulder specific data on all aspects is rare. The current study aims to answer the following research questions: Does joint aspiration have an insufficient predictive value in the diagnosis of SJI in (1) initial workup and (2) before definite arthroplasty with polymethylmethacrylate (PMMA)-Spacer in place? Methods This retrospective evaluation investigates 35 patients that were treated for SJI with a two staged implantation of a TSA after debridement and implantation of an PMMA-Spacer. Joint aspirations were performed preoperatively (PA) and before re−/implantation of the prosthesis while spacer was in place (interstage aspiration, IA). Samples were taken for microbiological culture and analysis of WBC. Sensitivity and specificity were calculated with reference to intraoperative microbiological samples. Receiver Operating Characteristic (ROC), Area-Under-Curve analysis (AUC) and calculation of the Youden index were performed to find optimum cut-off for WBC. Results The sensitivity of microbiological cultures from PA was 58.3% and the specificity was 88.9%. The mean WBC was 27,800 leucocytes/mm3 (range 400-96,300). The maximum Youden index (0.857) was a cut-off of 2600 leucocytes/mm3 with a sensitivity of 85.7% and a specificity of 100.0%. The sensitivity and specificity of IA were 0.0% and 88.5%, respectively. Conclusions Preoperative aspiration is likely to miss Cutibacteria spp. and CoNS and cannot rule out infection for sure. However, we recommend it for its advantages of targeted antibiotic therapy in case of germ identification. Empiric antibiotic therapy should cover Cutibacteria and CoNS even if aspiration showed negative microbiological cultures. In contrast, the diagnostic value of interstage aspiration does not qualify for its routine use. KW - revision arthroplasty KW - periprosthetic joint infection KW - white blood cell count KW - septic KW - microbiological culture KW - interstage aspiration Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300795 VL - 23 ER - TY - THES A1 - Münstermann, Marcel T1 - The roles of the anaphylatoxin receptors during invasive disease as well as mucosal colonization caused by \(Neisseria\) \(meningitidis\) T1 - Die Rolle der Anaphylatoxinrezeptoren während invasiver Infektion sowie mukosaler Kolonisation verursacht durch \(Neisseria\) \(meningitidis\) N2 - The human specific gram-negative bacterium Neisseria meningitidis (Nme, meningococci) is a common colonizer of the upper respiratory tract. Upon becoming invasive, Nme can cause meningitis and life-threatening sepsis. The most important immune defense mechanism in invasive meningococcal disease (IMD) is the complement mediated killing of bacteria. The complement cascade is activated through different pathogen associated patterns and finally leads to the lysis of the bacteria by the membrane attack complex. In addition to the direct bacterial killing, the complement system is also an important player in different inflammatory processes. A hallmark of IMD is an overreaction of the immune system and the release of the potent anaphylatoxins C3a and C5a by the complement system is an important factor hereby. There are three anaphylatoxin receptors (ATRs), the C3aR, the C5aR1 and the C5aR2, capable of detecting these anaphylatoxins. It has already been shown that blocking the ATR C5aR1 strongly benefitted the outcome of IMD in a murine sepsis model. However, the roles of ATRs C3aR and C5aR2 in IMD are still unclear. This work aims to analyze the role of these ATRs in meningococcal sepsis and to identify possible underlying mechanisms. Furthermore, a possible involvement of the complement system, the ATRs and the type II CRISPR/Cas system on nasopharyngeal colonization is analyzed. In vivo depletion experiments showed that without neutrophils or monocytes/macrophages the complement system alone was not able to clear a low dose Nme infection, which highlights the importance of cellular components in IMD. Analyzing the role of the ATRs in knock-out mice with high dose Nme infections, revealed that the lack of C5aR2, like the lack of C5aR1, was beneficial for the outcome of meningococcal induced sepsis. In contrast, the lack of C3aR in knock-out mice was detrimental. The positive outcome associated with the C5aRs could be reproduced by using an antagonist against both C5aRs or an antagonist specifically against C5aR1 in WT mice. These findings are giving hope to future therapeutic applications. Next, a possible contribution of neutrophils to this positive outcome was analyzed. Absence of C5aR1 led to a decrease of degranulation by neutrophils in a murine whole blood model, while the other ATRs showed no effect. Neutrophil analysis in human whole blood, on the other hand, revealed a reduced oxidative burst and IL-8 secretion upon inhibition of all three ATRs. A functional difference between the C5aRs and the C3aR in neutrophils was observed in phagocytosis, which was reduced upon C3aR inhibition, but was unaltered with C5aR1 or C5aR2 inhibition. Possible underlying mechanisms in the phosphorylation of ERK1/2 were analyzed in bone marrow derived macrophages isolated from ATR knock-out mice. The later phosphorylation of ERK1/2 in macrophages without C5aR1 or C5aR2 expression might explain, why blocking the C5aRs is beneficial for the outcome of IMD in mice. In contrast to these findings, the colonization of the nasopharynx in huCEACAM 1 expressing mice by Nme did not seem to depend on the Complement system factors C3 and C5 nor the ATRs. Additionally, no difference in the colonization could be observed in this model using Nme mutants lacking different parts of the type 2 CRISPR/Cas system. Conclusively, this work highlights the importance of the complement system, the ATRs and the cellular components in IMD. Contrariwise, these factors did not play a role in the analyzed nasopharyngeal infection model. The beneficial effects of C5aR1 and C5aR2 lack/inhibition in IMD might have medicinal applications, which could support the standard therapies of IMD in the future. N2 - Das human spezifische pathogene Gram-negative Bakterium Neisseria meningitidis (Nme, Meningokokken) ist ein Kommensale angesiedelt im Nasopharynx. Bei invasiver Erkrankung können Nme Meningitis oder eine lebensbedrohliche Sepsis verursachen. Die wichtigste Verteidigung des Immunsystems in invasiver Meningokokken-Erkrankung (IMD) ist die Abtötung von Bakterien durch das Komplementsystem. Die Komplementkaskade wird durch verschiedene pathogenassoziierte Muster in Gang gesetzt und resultiert in dem Aufbau des Membranangriffskomplex, welcher die Bakterien schließlich lysiert. Darüber hinaus spielt das Komplementsystem auch eine wichtige Rolle in verschiedenen inflammatorischen Prozessen im Körper. Ein charakteristisches Merkmal von IMD ist eine übermäßige Reaktion des Immunsystems und dabei ist die Freisetzung der Anaphylatoxine C3a und C5a, durch das Komplementsystem, ein wichtiger Faktor. Es gibt drei Anaphylatoxin Rezeptoren (ATR), den C3aR, den C5aR1 und den C5aR2, welche die jeweiligen Anaphylatoxine erkennen. In murinen Modellen wurde bereits gezeigt, dass die Inhibition des C5aR1 einen positiven Einfluss auf den Verlauf von IMD hat. Im Kontrast dazu sind die Rollen der ATRs C3aR und C5aR2 in IMD weiter unklar. Diese Arbeit hat als Ziel, die Rolle der ATRs in Meningokokken induzierter Sepsis zu untersuchen und mögliche zugrundeliegende Mechanismen zu finden. Des Weiteren soll ein möglicher Einfluss des Komplementsystems, der ATRs und des Typ II CRISPR/Cas Systems auf die Kolonisation durch Nme im Nasopharynx untersuchen werden. In vivo Depletions-Versuche zeigten, dass ohne Neutrophile oder Monozyten/Makrophagen das Komplementsystem allein nicht in der Lage war eine Nme-Infektion mit einer niedrigen Infektionsdosis zu beseitigen. Dies zeigt die Wichtigkeit von Immunzellen neben dem Komplementsystem in IMD. Experimente mit hohen Nme-Dosen in ATR knock-out Mäusen zeigten, dass die fehlende Expression von C5aR2, wie die von C5aR1, sich positiv auf den Ausgang von IMD auswirkte. Im Gegensatz dazu, verschlimmerte das Fehlen des C3aR Rezeptors den Ausgang der IMD. Die positive Wirkung in den C5aR knock-out Mäusen, konnte auch mit der Gabe von einem gegen beide C5aRs oder einem spezifisch gegen C5aR1 gerichteten Antagonisten in WT Mäusen beobachtet werden. Diese Ergebnisse geben Hoffnung auf eine mögliche zukünftige therapeutische Applikation. Als nächstes wurde eine mögliche Beteiligung von Neutrophilen an dem positiven Ausgang von IMD in Abhängigkeit von den ATRs untersucht. Eine fehlende C5aR1 Expression führte zu einer verminderten Degranulation durch Neutrophile in dem verwendeten murinen Vollblutmodel, wohingegen die fehlende Expression der anderen ATRs keinen Effekt zeigte. Im Gegensatz dazu, zeigten Versuche mit humanem Vollblut einen verminderten Oxidativen Burst sowie eine verminderte Ausschüttung von IL-8 bei der Blockade von allen drei ATRs. Ein Unterschied zwischen den C5aRs und dem C3aR zeigte sich hingegen in der Phagozytose, welche mit C3aR Inhibierung reduziert war, aber unverändert nach der Inhibierung von C5aR1 oder C5aR2 blieb. Mögliche zugrundeliegende Mechanismen in der Phosphorylation von ERK1/2 wurden anschließend in Knochenmark-gereiften Makrophagen von ATR knock-out Mäusen untersucht. Ohne C5aR1 oder C5aR2 Expression wurde eine verzögerte Phosphorylierung von ERK1/2 in den Makrophagen beobachtet, was erklären könnte warum die Blockade von C5aRs den Ausgang von Meningokokken induzierter Sepsis in Mäusen positiv beeinflusst. Im Gegensatz zu diesen Ergebnissen wurde die Kolonisation des Nasopharynx durch Nme in huCEACAM-1 exprimierenden Mäusen, weder durch die Komplementfaktoren C3 und C5 noch durch die ATRs beeinflusst. Zusätzlich konnte auch kein Unterschied in der Besiedelung des Nasopharynx durch Nme-Mutanten, die verschiedene Mutationen des Typ 2 CRISPR/Cas Systems besaßen, beobachtet werden. Diese Arbeit zeigt die Wichtigkeit des Komplementsystems, der ATRs und der Immunzellen in IMD. Zusätzlich zeigt diese Arbeit, dass das Komplementsystem und die ATRs jedoch keine Auswirkungen auf die Kolonisation des Nasopharynx in Mäusen haben. Die äußerst positive Auswirkung auf IMD, wenn C5aR1 und C5aR2 nicht gebildet oder blockiert werden, könnte medizinisch von Bedeutung sein und eventuell in der Zukunft die Standarttherapie bei IMD unterstützen. KW - Anaphylatoxine KW - Komplement C3a KW - Komplement C5a KW - Neisseria meningitidis KW - Komplement KW - anaphylatoxin receptors KW - invasive meningococcal diseases KW - Anaphylatoxinrezeptoren Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-269759 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Rauschendorfer, Theresa A1 - Rodríguez, Luis Zanon A1 - Krohne, Georg T1 - The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis N2 - Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. KW - Cestoda KW - Echinococcus KW - Neoblast KW - Germinative cell KW - Stem cell KW - Nanos KW - Argonaute KW - Mucin KW - Alkaline phosphatase Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110315 ER - TY - JOUR A1 - Mottola, Austin A1 - Ramírez-Zavala, Bernardo A1 - Hünninger, Kerstin A1 - Kurzai, Oliver A1 - Morschhäuser, Joachim T1 - The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans JF - Molecular Microbiology N2 - The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall. KW - cell wall KW - zinc cluster transcription factor KW - Candida albicans KW - protein kinases Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259583 VL - 116 IS - 2 ER - TY - JOUR A1 - Ampattu, Biju Joseph A1 - Hagmann, Laura A1 - Liang, Chunguang A1 - Dittrich, Marcus A1 - Schlüter, Andreas A1 - Blom, Jochen A1 - Krol, Elizaveta A1 - Goesmann, Alexander A1 - Becker, Anke A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Schoen, Christoph T1 - Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence JF - BMC Genomics N2 - Background: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. Results: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. Conclusions: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis. KW - neisseria meningitidis KW - MITE KW - virulenceregulatory evolution KW - systems biology KW - metabolism KW - cryptic KW - genetic variation KW - stringent response KW - relA Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157534 VL - 18 IS - 282 ER - TY - JOUR A1 - Tappe, Dennis A1 - Meyer, Michael A1 - Oesterlein, Anett A1 - Jaye, Assan A1 - Frosch, Matthias A1 - Schoen, Christoph A1 - Pantchev, Nikola T1 - Transmission of Armillifer armillatus Ova at Snake Farm, The Gambia, West Africa JF - Emerging Infectious Diseases N2 - Visceral pentastomiasis caused by Armillifer armillatus larvae was diagnosed in 2 dogs in The Gambia. Parasites were subjected to PCR; phylogenetic analysis confirmed relatedness with branchiurans/crustaceans. Our investigation highlights transmission of infective A. armillatus ova to dogs and, by serologic evidence, also to 1 human, demonstrating a public health concern. KW - Pentastomiasis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142804 N1 - All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. VL - 17 IS - 2 ER - TY - THES A1 - Swiderek, Halina T1 - Typing and genome comparison of Neisseria meningitidis by DNA-microarrays T1 - Typisierung und Genom-Vergleich of Neisseria meningitidis mit DNA-microarrays N2 - In the present thesis, two projects on the use of microarray technology for molecular epidemiology of Neisseria meningitidis have been followed. The first one evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of N. meningitidis adopting the multilocus sequence typing (MLST) concept. The number of oligonucleotides needed to cover all known polymorphisms was much lower compared to the number needed if a tiling strategy would have been chosen. Initial experiments using oligonucleotides 28-32 nucleotides in length, revealed that the applied hybridisation protocols were highly specific. However, despite of several optimisation steps, the rate of misidentification of oligonucleotides remained >1.8% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. This finding led to the assumption that the high density of polymorphic sites and extensive GC-content variations at N. meningitidis MLST loci hindered the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design. In the 1980s, the ET-15 clone emerged within the ST-11 complex of N. meningitidis. This new clone was associated with severe meningococcal disease and outbreaks world-wide. Therefore, the goal of the second project was to identify genetic differences between ET-15 strains and other ST-11 strains using whole genome microarray technology. Three genes encoding hypothetical proteins were identified to be present in all ET-15 strains but absent in other ST-11 strains. This finding together with unpublished observation from our group suggested that several genome alterations occurred before the clonal expansion of the ET-15 clone started. The role that these three genes play in the pathogenicity of the ET-15 clone is unclear. The genome comparisons revealed furthermore that studies of the ET-15 clone displayed approximately two-fold less gene content variation than ST-11 strains not belonging to the ET-15 clone. This finding is in accordance with the recent emergence and clonal expansion of the ET-15 variant. N2 - In der vorliegenden Doktorarbeit wurden zwei Projekte zum Einsatz der microarray-Technologie in der molekularen Epidemiologie von Neisseria meningitidis bearbeitet. Im Rahmen des ersten Projektes wurde die Einsatzmöglichkeit der microarray-Technologie unter Verwendung von Oligonukleotiden, die von bekannten Polymorphismen abgeleitet waren, für die Typisierung von N. meningitidis nach dem Prinzip der Multilokus-Sequenztypisierung (MLST) untersucht. Um alle bekannten Polymorphismen abzudecken, wurde im Vergleich zu der so genannten tiling-Methode eine deutlich geringere Anzahl an Oligonukleotiden benötigt. Vorversuche mit Oligonukleotiden einer Länge von 28-32 Nukleotiden zeigten, dass unter den angewandten Hybridisierungsbedingungen eine hohe Spezifität erzielt werden konnte. Dennoch lag die durchschnittliche Fehlidentifikationsrate der Oligonukleotide von microarrays, die die genetische Diversität von drei MLST-Loci repräsentierten, trotz mehrerer Optimierungsschritte der Oligonukleotide über 1,8%. Es ist anzunehmen, dass die hohe Dichte an Polymorphismen und die große Variation des GC-Gehaltes der MLST-Loci von N. meningitidis den erfolgreichen Einsatz eines MLST-microarrays mit Oligonukleotiden, die von bekannten Polymorphismen abgeleitet waren, verhinderten. In den achtziger Jahren des letzten Jahrhunderts tauchte der ET-15-Klon als Abkömmling des Sequenztyp (ST-) 11-Komplexes von N. meningitidis auf. Dieser neue Klon ist assoziiert mit schweren Meningokokkenerkrankungen und weltweiten Erkrankungsausbrüchen. Deshalb war es das Ziel des zweiten Projektes dieser Arbeit, genetische Unterschiede zwischen ET-15-Stämmen und anderen ST-11-Stämmen mit Hilfe von Gesamtgenom-microarrays zu identifizieren. Drei Gene, die hypothetische Proteine kodieren, waren in allen ET-15-Stämmen vorhanden, während sie in den anderen ST-11-Stämmen fehlten. Dieses Ergebnis, zusammen mit weiteren bisher nicht publizierten Beobachtungen aus unserer Arbeitsgruppe, deutet daraufhin, dass vor der klonalen Ausbreitung des ET-15-Klons mehrere Veränderungen im Genom auftraten. Die Bedeutung dieser drei Gene für die Pathogenität des ET-15-Klons ist unklar. Die Genomvergleiche zeigten weiterhin, dass Stämme des ET-15-Klons eine nur halb so große genetische Variabilität aufwiesen wie die ST-11-Stämme, die nicht zum ET-15-Klon gehörten. Dieses Ergebnis steht in Einklang mit der erst kürzlich erfolgten klonalen Expansion der ET-15-Meningokokken. KW - Neisseria meningitis KW - Typisierung KW - Genanalyse KW - Neisseria meningitidis KW - MLST KW - Typisierung KW - Genom-Vergleich KW - ET-15 clone KW - Neisseria meningitidis KW - MLST KW - typing KW - genome comparison KW - ET-15 clone Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-13374 ER - TY - JOUR A1 - Schreiber, Laura M. A1 - Lohr, David A1 - Baltes, Steffen A1 - Vogel, Ulrich A1 - Elabyad, Ibrahim A. A1 - Bille, Maya A1 - Reiter, Theresa A1 - Kosmala, Aleksander A1 - Gassenmaier, Tobias A1 - Stefanescu, Maria R. A1 - Kollmann, Alena A1 - Aures, Julia A1 - Schnitter, Florian A1 - Pali, Mihaela A1 - Ueda, Yuichiro A1 - Williams, Tatiana A1 - Christa, Martin A1 - Hofmann, Ulrich A1 - Bauer, Wolfgang A1 - Gerull, Brenda A1 - Zernecke, Alma A1 - Ergün, Süleyman A1 - Terekhov, Maxim T1 - Ultra-high field cardiac MRI in large animals and humans for translational cardiovascular research JF - Frontiers in Cardiovascular Medicine N2 - A key step in translational cardiovascular research is the use of large animal models to better understand normal and abnormal physiology, to test drugs or interventions, or to perform studies which would be considered unethical in human subjects. Ultrahigh field magnetic resonance imaging (UHF-MRI) at 7 T field strength is becoming increasingly available for imaging of the heart and, when compared to clinically established field strengths, promises better image quality and image information content, more precise functional analysis, potentially new image contrasts, and as all in-vivo imaging techniques, a reduction of the number of animals per study because of the possibility to scan every animal repeatedly. We present here a solution to the dual use problem of whole-body UHF-MRI systems, which are typically installed in clinical environments, to both UHF-MRI in large animals and humans. Moreover, we provide evidence that in such a research infrastructure UHF-MRI, and ideally combined with a standard small-bore UHF-MRI system, can contribute to a variety of spatial scales in translational cardiovascular research: from cardiac organoids, Zebra fish and rodent hearts to large animal models such as pigs and humans. We present pilot data from serial CINE, late gadolinium enhancement, and susceptibility weighted UHF-MRI in a myocardial infarction model over eight weeks. In 14 pigs which were delivered from a breeding facility in a national SARS-CoV-2 hotspot, we found no infection in the incoming pigs. Human scanning using CINE and phase contrast flow measurements provided good image quality of the left and right ventricle. Agreement of functional analysis between CINE and phase contrast MRI was excellent. MRI in arrested hearts or excised vascular tissue for MRI-based histologic imaging, structural imaging of myofiber and vascular smooth muscle cell architecture using high-resolution diffusion tensor imaging, and UHF-MRI for monitoring free radicals as a surrogate for MRI of reactive oxygen species in studies of oxidative stress are demonstrated. We conclude that UHF-MRI has the potential to become an important precision imaging modality in translational cardiovascular research. KW - ultrahigh-field MRI KW - large animal models KW - translational research KW - research infrastructure KW - heart KW - organoid KW - pig KW - cardiovascular MRI Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317398 SN - 2297-055X VL - 10 ER -