TY - THES A1 - Krampert, Laura T1 - Dynamics of cardiac neutrophil diversity in murine myocardial infarction T1 - Dynamik der Diversität kardialer neutrophiler Granulozyten im Mausmodell Herzinfarkt N2 - After myocardial infarction, an inflammatory response is induced characterized by a sterile inflammation, followed by a reparative phase in order to induce cardiac healing. Neutrophils are the first immune cells that enter the ischemic tissue. Neutrophils have various functions in the ischemic heart, such as phagocytosis, production of reactive oxygen species or release of granule components. These functions can not only directly damage cardiac tissue, but are also necessary for initiating reparative effects in post-ischemic healing, indicating a dual role of neutrophils in cardiac healing after infarction. In recent years, evidence has been growing that neutrophils show phenotypic and functional differences in distinct homeostatic and pathogenic settings. Preliminary data of my working group using single-cell RNA-sequencing revealed the time- dependent heterogeneity of neutrophils, with different populations showing distinct gene expression profiles in ischemic hearts of mice, including the time-dependent appearance of a SiglecFhigh neutrophil population. To better understand the dynamics of neutrophil heterogeneity in the ischemic heart, my work aimed to validate previous findings at the protein level, as well as to investigate whether the distinct neutrophil populations show functional differences. Furthermore, in vivo depletion experiments were performed in order to modulate circulating neutrophil levels. Hearts, blood, bone marrow and spleens were processed and analyzed from mice after 1 day and 3 days after the onset of cardiac ischemia and analyzed using flow cytometry. Results showed that the majority of cardiac neutrophils isolated at day 3 after myocardial infarction were SiglecFhigh, whereas nearly no SiglecFhigh neutrophils could be isolated from ischemic hearts at day 1 after myocardial infarction. No SiglecFhigh neutrophils could be found in the blood, spleen and bone marrow either after 1 day or 3 days after myocardial infarction, indicating that the SiglecFhigh state of neutrophils is unique to the ischemic cardiac tissue. When I compared SiglecFhigh and SiglecFlow neutrophils regarding their phagocytosis activity and ROS production, SiglecFhigh neutrophils showed a higher phagocytosis ability than their SiglecFlow counterparts, as well as higher ROS production capacity. In vivo depletion experiments could not achieve successful and efficient depletion of cardiac neutrophils either 1 day or 3 days after myocardial infarction, but led to a shift of a higher percentage of SiglecFhigh expressing neutrophils in the depletion group. Bone marrow neutrophil levels only showed partial depletion at day 3 after MI. Regarding blood neutrophils, depletion efficiently reduced circulating neutrophils at both time points, 1 and 3 days after MI. To summarize, this work showed the time-dependent presence of different neutrophil states in the ischemic heart. The main population of neutrophils isolated 3 days after MI showed a high expression of SiglecF, a unique state that could not be detected at different time points or other organs. These SiglecFhigh neutrophils showed functional differences regarding their phagocytosis ability and ROS production. Further investigation is needed to reveal what role these SiglecFhigh neutrophils could play within the ischemic heart. To better target neutrophil depletion in vivo, more efficient or different anti-neutrophil strategies are needed. N2 - Nach Myokardinfarkt kommt es zu einer Entzündungsantwort, die durch eine sterile Entzündung und nachfolgende reparative Phase gekennzeichnet ist, um eine kardiale Heilung zu initiieren. Neutrophile Granulozyten sind die ersten Immunzellen, die das ischämische Gewebe infiltrieren. Neutrophile Granulozyten haben verschiedene Funktionen im ischämischen Herz, wie beispielsweise Phagozytose, Produktion reaktiver Oxigenspezies oder Entleerung von Granulae. Diese Funktionen können nicht nur das kardiale Gewebe direkt schädigen, sondern sind auch notwendig, um die reparative Phase zu initiieren, was auf eine duale Rolle der neutrophilen Granulozyten hinsichtlich kardialer Heilung hinweist. Evidenz der letzten Jahre zeigt, dass neutrophile Granulzyten phänotypische und funktionelle Unterschiede zeigen, sowohl in Homöostase als auch in verschiedenen pathologischen Umgebungen. Vorangehende Ergebnisse meiner Arbeitsgruppe von Einzelzellsequenzierungen zeigten die zeitabhängige Heterogenität neutrophiler Granulozyten, die in verschiedenen Populationen und mit unterschiedlichen Expressions-Mustern in ischämischen Mäuseherzen auftauchten. Unter anderem zeigte sich das zeitabhängige Auftauchen einer SiglecFhigh NeutrophilenPopulation. Um die Dynamik der Neutrophilen-Heterogenität im ischämischen Herz besser zu verstehen, war es das Ziel meiner Arbeit, vorangehende Ergebnisse auf Proteinbasis zu bestätigen, und die verschiedenen Neutrophilen-Populationen hinsichtlich möglicher funktioneller Unterschiede zu untersuchen. Zusätzlich wurden in-vivo Depletionsversuche durchgeführt, um das Vorkommen zirkulierender neutrophiler Granulozyten zu modulieren und mögliche Änderungen hinsichtlich des Vorkommens neutrophiler Granulozyten im Herzen zu untersuchen. Herz, Blut, Knochenmark und Milz von Mäusen, die einen 1 oder 3 Tage alten Myokardinfarkt hatten, wurden prozessiert und mittels Durchflusszytometrie analysiert. Es zeigte sich, dass der überwiegende Anteil kardialer neutrophiler Granulozyten die an Tag 3 nach Myokardinfarkt isoliert wurden SiglecFhigh waren, wohingegen quasi keine SiglecFhigh neutrophile Granulozyten an Tag 1 nach Myokardinfarkt gefunden werden konnten. Es konnten keine SiglecFhigh neutrophile Granulozyten in Blut, Knochenmark oder Milz gefunden werden, weder 1 noch 3 Tage nach Myokardinfarkt, was darauf hinweist, dass der SiglecFhigh Status neutrophiler Granulozyten spezifisch für ischämisches Herzgewebe ist. Im Vergleich von SiglecFhigh und SiglecFlow neutrophilen Granulozyten hinsichtlich der Phagozytose und Produktion reaktiver Oxigenspezies, zeigten SiglecFhigh neutrophile Granulozyten sowohl eine höhere Phagozytose als auch eine höhere Produktion reaktiver Oxigenspezies. In vivo Depletionsversuche konnten keine komplette und effiziente Depletion kardialer neutrophiler Granulozyten sowohl an Tag 1 als auch an Tag 3 nach Myokardinfarkt erzielen, führten jedoch zu einem höheren Prozentsatz an SiglecFhigh neutrophilen Granulozyten in der Depletionsgruppe. Neutrophile Granulozyten im Knochenmark konnten nur an Tag 3 teilweise depletiert werden. Mit Blick auf neutrophile Granulozyten im Blut führte die Depletion zu einer effizienten Reduktion zirkulierender neutrophiler Granulozyten sowohl an Tag 1 als auch an Tag 3 nach Myokardinfarkt. Zusammenfassend zeigt diese Arbeit die zeitabhängige Präsenz verschiedener neutrophiler Granulozyten im ischämischen Herzen. Der größte Anteil neutrophiler Granulozyten, die an Tag 3 isoliert wurden, zeigten eine hohe Expression von SiglecF, einem spezifischen und einzigartigem Phänotypus, der weder zu anderen Zeitpunkten noch in anderen Organen gefunden wurde. Diese SiglecFhigh neutrophilen Granulozyten zeigten funktionelle Unterschiede hinsichtlich ihrer Phagozytose und Produktion reaktiver Oxigenspezies. Weitere Untersuchungen sind notwendig um aufzuzeigen, welche Rolle diese SiglecFhigh neutrophilen Granulozyten im ischämischen Herzen spielen könnten.Um eine effizientere Depletion neutrophiler Granulozyten in vivo zu erzielen, sind andere oder effizientere Anti-Neutrophilen Strategien notwendig. KW - Neutrophiler Granulozyt KW - neutrophils KW - Entzündung KW - Herzinfarkt KW - inflammation KW - myocardial infarction Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349576 ER - TY - JOUR A1 - Boeckel, Hannah A1 - Karsten, Christian M. A1 - Göpel, Wolfgang A1 - Herting, Egbert A1 - Rupp, Jan A1 - Härtel, Christoph A1 - Hartz, Annika T1 - Increased expression of anaphylatoxin C5a-receptor-1 in neutrophils and natural killer cells of preterm infants JF - International Journal of Molecular Sciences N2 - Preterm infants are susceptible to infection and their defense against pathogens relies largely on innate immunity. The role of the complement system for the immunological vulnerability of preterm infants is less understood. Anaphylatoxin C5a and its receptors C5aR1 and -2 are known to be involved in sepsis pathogenesis, with C5aR1 mainly exerting pro-inflammatory effects. Our explorative study aimed to determine age-dependent changes in the expression of C5aR1 and C5aR2 in neonatal immune cell subsets. Via flow cytometry, we analyzed the expression pattern of C5a receptors on immune cells isolated from peripheral blood of preterm infants (n = 32) compared to those of their mothers (n = 25). Term infants and healthy adults served as controls. Preterm infants had a higher intracellular expression of C5aR1 on neutrophils than control individuals. We also found a higher expression of C5aR1 on NK cells, particularly on the cytotoxic CD56\(^{dim}\) subset and the CD56\(^-\) subset. Immune phenotyping of other leukocyte subpopulations revealed no gestational-age-related differences for the expression of and C5aR2. Elevated expression of C5aR1 on neutrophils and NK cells in preterm infants may contribute to the phenomenon of “immunoparalysis” caused by complement activation or to sustained hyper-inflammatory states. Further functional analyses are needed to elucidate the underlying mechanisms. KW - preterm infants KW - C5a KW - C5aR1 KW - neutrophils KW - NK cells KW - innate immunity KW - sepsis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-321196 SN - 1422-0067 VL - 24 IS - 12 ER - TY - JOUR A1 - Strinitz, Marc A1 - Pham, Mirko A1 - März, Alexander G. A1 - Feick, Jörn A1 - Weidner, Franziska A1 - Vogt, Marius L. A1 - Essig, Fabian A1 - Neugebauer, Hermann A1 - Stoll, Guido A1 - Schuhmann, Michael K. A1 - Kollikowski, Alexander M. T1 - Immune cells invade the collateral circulation during human stroke: prospective replication and extension JF - International Journal of Molecular Sciences N2 - It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R\(^2\) = 0.09696, p = 0.0373) and (4) greater infarct extent (R\(^2\) = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment. KW - ischemic stroke KW - cerebral ischemia KW - mechanical thrombectomy KW - large vessel occlusion KW - leukocytes KW - neutrophils KW - collateral circulation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284281 SN - 1422-0067 VL - 22 IS - 17 ER - TY - JOUR A1 - Essig, Fabian A1 - Babilon, Lilith A1 - Vollmuth, Christoph A1 - Kollikowski, Alexander M. A1 - Pham, Mirko A1 - Solymosi, László A1 - Haeusler, Karl Georg A1 - Kraft, Peter A1 - Stoll, Guido A1 - Schuhmann, Michael K. T1 - High mobility group box 1 protein in cerebral thromboemboli JF - International Journal of Molecular Sciences N2 - High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6–6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke. KW - acute ischemic stroke KW - thromboemboli KW - HMGB1 KW - neutrophils KW - platelets KW - immunohistochemistry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265568 SN - 1422-0067 VL - 22 IS - 20 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 - Essig, Fabian A1 - Kollikowski, Alexander M. A1 - Pham, Mirko A1 - Solymosi, László A1 - Stoll, Guido A1 - Haeusler, Karl Georg A1 - Kraft, Peter A1 - Schuhmann, Michael K. T1 - Immunohistological analysis of neutrophils and neutrophil extracellular traps in human thrombemboli causing acute ischemic stroke JF - International Journal of Molecular Sciences N2 - Ischemic stroke caused by thromboembolic occlusion of large cerebral arteries, such as the internal carotid (ICA) and/or the middle cerebral artery (MCA), is treated by mechanical thrombectomy (MT). MT allows salvage of the vessel-occluding thrombemboli, which most frequently originate from the left atrium or the left ventricle of the heart or from sites of plaque rupture within large arteries above the heart. Clot composition may influence the efficacy of (intravenous) thrombolysis and MT, respectively. We analyzed 37 human thrombemboli obtained from acute ischemic stroke patients during MT with special emphasis on histological staining of neutrophils and neutrophil extracellular traps (NETs). We found neutrophils as the main cellular component of cerebral thrombemboli but encountered considerable morphological heterogeneity. Neutrophils accumulated in the border region of fibrin-rich structures indicating possible interaction of neutrophils with distinct structural thrombembolus components. Web-like NETs were found in 35 of 37 thrombemboli in varying amounts. NETs were almost exclusively found within fibrin-rich areas. Importantly, stroke etiology, age and present oral anticoagulation was associated with morphological patterns and the amount of neutrophils. Correlation of histological data and imaging data revealed that relative Hounsfield units of cerebral thrombemboli positively correlated with the amount of red blood cells. In summary, our results demonstrate that neutrophils and NETs are substantial constituents of cerebral thrombemboli and contribute to their structural complexity. KW - acute ischemic stroke KW - thrombemboli KW - neutrophils KW - NETs KW - immunohistochemistry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236192 SN - 1422-0067 VL - 21 IS - 19 ER - TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - JOUR A1 - Herrmann, Johannes A1 - Muenstermann, Marcel A1 - Strobel, Lea A1 - Schubert-Unkmeir, Alexandra A1 - Woodruff, Trent M. A1 - Gray-Owen, Scott D. A1 - Klos, Andreas A1 - Johswich, Kay O. T1 - Complement C5a receptor 1 exacerbates the pathophysiology of N. meningitidis sepsis and is a potential target for disease treatment JF - mBio N2 - Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1\(^{-/-}\) mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1\(^{-/-}\) mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. Importance: The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease. KW - C5aR1 KW - whole-blood model KW - Neisseria meningitidis KW - anaphylatoxins KW - complement system KW - inflammation KW - invasive disease KW - mouse model KW - neutrophils KW - sepsis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175792 VL - 9 IS - 1 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Pachel, Christina A1 - Mathes, Denise A1 - Bayer, Barbara A1 - Dienesch, Charlotte A1 - Wangorsch, Gaby A1 - Heitzmann, Wolfram A1 - Lang, Isabell A1 - Ardehali, Hossein A1 - Ertl, Georg A1 - Dandekar, Thomas A1 - Wajant, Harald A1 - Frantz, Stefan T1 - Exogenous Administration of a Recombinant Variant of TWEAK Impairs Healing after Myocardial Infarction by Aggravation of Inflammation JF - PLoS ONE N2 - Background: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factorinducible 14 (Fn14) are upregulated after myocardial infarction (MI) in both humans and mice. They modulate inflammation and the extracellular matrix, and could therefore be important for healing and remodeling after MI. However, the function of TWEAK after MI remains poorly defined. Methods and results: Following ligation of the left coronary artery, mice were injected twice per week with a recombinant human serum albumin conjugated variant of TWEAK (HSA-Flag-TWEAK), mimicking the activity of soluble TWEAK. Treatment with HSA-Flag-TWEAK resulted in significantly increased mortality in comparison to the placebo group due to myocardial rupture. Infarct size, extracellular matrix remodeling, and apoptosis rates were not different after MI. However, HSA-Flag-TWEAK treatment increased infiltration of proinflammatory cells into the myocardium. Accordingly, depletion of neutrophils prevented cardiac ruptures without modulating all-cause mortality. Conclusion: Treatment of mice with HSA-Flag-TWEAK induces myocardial healing defects after experimental MI. This is mediated by an exaggerated neutrophil infiltration into the myocardium. KW - apoptosis KW - myocardial infarction KW - neutrophils KW - cytokines KW - inflammation KW - myocardium KW - heart KW - extracellular matrix Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129889 VL - 8 IS - 11 ER - TY - JOUR A1 - Meier, Friedegund A1 - Gross, Eva A1 - Klotz, Karl-Norbert A1 - Ruzicka, Thomas T1 - Leukotriene B4 receptors on neutrophils in patients with psoriasis and atopic exzema N2 - Polymorphonuclear leukocyte (PMNL) infiltration is an important characteristic in psoriatic lesions. Elevated concentrations of the chemoattractant eicosanoid leukotriene B4 (L TB4) are present in psoriatic skin. Its chemotactic activity is mediated via high affinity receptors on PMNL. The goal of our work was to ascertain whether PMNL infiltration in psoriasis can be accounted for by functional abnormalities of the circulating PMNL due to alterations in the LTB4 receptor density or affinity (or both). No significant difference was found between patients with psoriasis, healthy controls and patients with another inflammatory dermatosis (atopic eczema) with regard to the binding parameters of LTB4 receptors on PMNL. Our findings suggest that PMNL accumulation in psoriatic skin may be the result of an excess of cutaneous hemoattractant rather than the increased readiness of psoriatic PMNL to migrate towards L TB4 due to altered LTB4 receptor density or affinity. KW - Dermatologie KW - Venerologie KW - Pharmakologie KW - Pharmazie KW - LTB4 receptor KW - neutrophils KW - psoriasis KW - atopic eczema Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86265 ER - TY - THES A1 - Hornbach, Anke T1 - Aktivierungsmuster humaner neutrophiler Granulozyten nach Kontakt mit den pathogenen Pilzen Candida albicans und Aspergillus fumigatus T1 - Activation patterns of human neutrophils after contact with the pathogenic fungi Aspergillus fumigatus und Candida albicans N2 - Humane neutrophile Granulozyten spielen eine wichtige Rolle in der Immunabwehr invasiver Infektionen durch die humanpathogenen Pilze Candida albicans und Aspergillus fumigatus. Das Ziel der hier vorliegenden Arbeit bestand in einer Charakterisierung der Interaktion beider Pilzspezies mit neutrophilen Granulozyten, mit Fokussierung auf die unterschiedlichen Effektormechanismen dieser Zellen. C. albicans exprimiert eine Reihe von Aspartatproteasen, welche mit der Virulenz des Erregers assoziiert sind und zu Adhäsion, Gewebeinvasion und Immunevasion beitragen können. In dieser Arbeit wurde die Rolle der Aspartatproteasen Sap1-6, Sap9 und Sap10 in der Interaktion mit neutrophilen Granulozyten analysiert. Es konnte gezeigt werden, dass, im Gegensatz zu anderen Aspartatproteasen, das zelloberflächenassoziierte GPI-verankerte Enzym Sap9 einen maßgeblichen Einfluss auf die Erkennung von C. albicans durch neutrophile Granulozyten hat. SAP9-Expression ist erforderlich, um die gerichtete Motilität (Chemotaxis) neutrophiler Granulozyten zu C. albicans-Keimschläuchen hin zu induzieren. Dieser Prozess stellt eine Grundvoraussetzung zur effektiven Aktivierung neutrophiler Granulozyten darstellt. Die Chemotaxis neutrophiler Granulozyten kann durch autologe Sekretion des Zytokins IL-8 verstärkt werden. Es konnte jedoch kein Einfluss von SAP9 auf die IL-8 Sekretion beobachtet werden. Allerdings führte die Deletion von SAP9 zu reduzierter Freisetzung von reaktiven Sauerstoffspezies (engl. reactive oxygen species, ROS) in neutrophilen Granulozyten. Die mit der ROS-Generierung in Verbindung stehende und durch C. albicans induzierte Apoptose neutrophiler Granulozyten war ebenfalls vermindert. In Konfrontationsassays war die Abtötung einer SAP9-Deletionsmutante verglichen mit dem Wildtyp reduziert. Die Degranulation stellt neben der Produktion von ROS einen weiteren wichtigen Effektormechanismus zur Abtötung von Mikroben dar, jedoch verlief die Freisetzung von Elastase ebenso unabhängig von SAP9 wie die durch neutrophile Granulozyten ausgelöste Wachstumsinhibition von Keimschläuchen. Die hier präsentierten Daten verbinden die Aktivität der Protease Sap9, der zuvor bereits eine Rolle in der Immunevasion von C. albicans zugeschrieben wurde, mit der Initiation der protektiven angeborenen Immunität. Wie C. albicans stimuliert auch A. fumigatus die Aktivität der neutrophilen Granulozyten. Microarray- Analysen mit Fokus auf dem Zytokinprofil neutrophiler Granulozyten während der Interaktion mit A. fumigatus-Hyphen offenbarten, dass nur wenige Zytokine im Lauf der Infektion hochreguliert wurden. Zusammenfassend konnte gezeigt werden, dass die Sap-Granulozyten-Interaktion neue molekulare Mechanismen zur Aktivierung dieser Zellen birgt. Zudem brachten die Microarray Analysen die Erkenntnis, dass die de novo-Zytokinsynthese durch A. fumigatus nur geringfügig beeinflusst wird und eine schnelle Abtötung des Pilzes offenbar im Vordergrund steht. N2 - Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defence against invasive infections caused by the pathogenic fungi Candida albicans and Aspergillus fumigatus. The aim of this work was to further characterize the interaction of both fungi with PMNs with a focus on diverse PMN effector functions. C. albicans expresses a set of aspartic proteases which are associated with virulence and can contribute to adhesion, tissue invasion and immune evasion. Here, the role of the aspartic proteases Sap1-6, Sap9 and Sap10 in the interaction with human neutrophils was analysed. It could be demonstrated that, in contrast to other aspartic proteases, the cell surface associated GPI-anchored enzyme Sap9 has a major impact on recognition of C. albicans by PMNs. SAP9 expression is required for the effective induction of PMN chemotaxis towards C. albicans filaments, an essential prerequisite of effective PMN activation. Targeted motility can be enhanced by autologous secretion of IL-8, but no influence of SAP9 on the process of IL-8 secretion could be observed here. However, deletion of SAP9 led to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs. Likewise, C. albicans induced apoptosis triggered by ROI formation was decreased. In confrontation assays, killing of a SAP9 deletion mutant was reduced in comparison to the wild-type. Beside ROI production, degranulation represents an additional important effector mechanism contributing to microbial killing, however, the release of elastase in response to C. albicans was found to be not dependent on SAP9, as well as the PMN-mediated growth inhibition of germ tubes. The data presented here clearly implicate Sap9 protease activity, which was already attributed to immune evasion before, with the initiation of protective innate immunity. Like C. albicans, A. fumigatus evokes PMN antifungal activity. Custom-made microarray analyses focusing on the cytokine profile of PMNs during interaction with A. fumigatus hyphae revealed that only few cytokines are upregulated during the course of infection. In summary it could be demonstrated that the interaction between Saps and PMNs is revealing novel molecular mechanisms which lead to an activation of these cells. Furthermore, microarray analyses lead to the awareness that de novo cytokine synthesis of PMNs is barely influenced by A. fumigatus, pointing out that instant killing of the fungus might be of higher importance. KW - Neutrophile Granulozyten KW - Aspergillus fumigatus KW - Candida albicans KW - Immunreaktion KW - neutrophile Granulozyten KW - Aspergillus fumigatus KW - Candida albicans KW - neutrophils KW - Aspergillus fumigatus KW - Candida albicans Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-53520 ER - TY - THES A1 - Fischer, Anna T1 - Die Regulation aktivierender und hemmender Signalwege in humanen neutrophilen Granulozyten durch cAMP- und cGMP-erhöhende Vasodilatatoren T1 - Regulation of activatory and inhibitory signalling pathways in neutrophils mediated by cAMP- and cGMP-elevating vasolidators N2 - Neutrophile Granulozyten sind wichtige Effektorzellen des menschlichen Immunsystems. Eine Suppression der Neutrophilen kann zur Immundeffizienz mit Gefahr für bakterielle Erkrankungen und maligne Tumoren führen, eine Überstimulation dieser Zellen ist jedoch an der Entstehung der Autoimmunerkrankungen beteiligt. Im Rahmen dieser Arbeit wurden aktivierende und hemmende Wege untersucht, die für zukünftige Strategien in Prävention und Therapie dieser Erkrankungen eine wichtige Rolle spielen. Neutrophile Granulozyten enthalten VASP in hoher Konzentration. VASP ist ein bereits gut charakterisiertes Substrat der cAMP-PK und der cGMP-PK. Phosphorylierungsversuche mit cAMP-erhöhenden Substanzen ergaben eine rasche, reversible Phosphorylierung dieses Proteins an Ser-157 und Ser-239 in intakten humanen Neutrophilen Granulozyten. Versuche mit cGMP-erhöhenden Substanzen zeigten jedenfalls eine Phosphorylierung am Ser-157, jedoch keine Phosphorylierung am Ser-239. Diese Ergebnisse unterstreichen deutlich das Vorhandensein und die physiologische Funktion der cAMP-PK bezüglich der Phosphorylierung von VASP, stellen jedoch die Funktion der cGMP-PK in humanen Neutrophilen Granulozyten in Frage. Basierend auf der Methode der Immunfluoreszenz wurde gezeigt, dass VASP bei der Adhärenz der Neutrophilen eine entscheidende Rolle spielt. So ist mit Hilfe der spezifischen monoklonalen Antikörper eine Phosphorylierung am Ser-157 und am Ser-239 nach der Adhäsion der Neutrophilen an die Objektträger nachgewiesen worden. Nach zusätzlicher Stimulation mit PG-E1 zeigte sich kein wesentlicher Phosphorylierungsanstieg in adhärierten Neutrophilen. Zahlreiche chemotaktische Faktoren wie fMLP führen zur Phosphorylierung der p42/p44-, p38-MAPK sowie auch der PKB. Diese intrazellulären Signalmoleküle spielen eine zentrale Rolle bei der Neutrophilenaktivierung. Da es bereits von hemmenden Einflussen der Vasodilatatoren auf die Aktivierung der Neutrophilen Granulozyten berichtet worden ist, wurde in der vorliegenden Arbeit der Einfluss von cAMP- und cGMP-erhöhenden Substanzen auf die fMLP-induzierte Phosphorylierung der p42/p44-, p38-MAPK sowie der PKB untersucht. Flolan, ein cAMP-erhöhender Vasodilatator führte zur signifikanten Hemmung der fMLP-induzierten p42/p44-, p38- sowie PKB-Phosphorylierung. cGMP-erhöhender Vasodilatator SNP zeigte jedoch keinen Einfluss auf die fMLP-induzierte Aktivierung dieser Signalmoleküle. Physiologisch vorkommende cAMP-erhöhende Substanzen besitzen im menschlichen Körper eine wichtige regulatorische Funktion, die Neutrophile Granulozyten vor der „Überstimulation“ bewahrt. N2 - Neutrophil granulocytes are critical effector cells in human humoral and innate immunity and play a vital role in phagozytosis and bacterial killing. Patients with deficient function of neutrophils commonly suffer from repeated bacterial infections. Moreover, neutrophils are active in immunosurveillance against tumours. However, the capacity for bacterial killing carries with it an implicit capacity for host tissue destruction, as observed in autoimmune disease. This study is focussed on the characterization of activatory and inhibitory signalling pathways in neutrophils which will allow new approaches in the therapy of cancer, inflammatory and autoimmune diseases. Neutrophils contain high concentrations of VASP (vasodilator-stimulated phosphoprotein). VASP is an established substrate of both cAMP- and cGMP- dependent protein kinases. This study demonstrates that cAMP-elevating drugs cause reversible VASP phosphorylation at serine 157 and at serine 239 in intact human neutrophils, whereas cGMP-elevating drugs cause VASP phosphorylation only at serine 157 but not at serine 239. This indicates that cAMP-dependent protein kinases play a functional role in neutrophils, the physiologic role of cGMP-dependent protein kinases in these cells still remains unresolved. Furthermore, immunofluorescence microscopy demonstrated VASP phosphorylation at serine 157 and serine 239 during neutrophil adhesion. After stimulation of adherent neutrophils with PG-E1 no increase in VASP phosphorylation could be observed compared to unstimulated adherent cells. Multiple chemotactic factors including fMLP cause phosphorylation and activation of p42/p44-MAPK, p38-MAPK and protein kinase B (Akt-kinase, PKB). These are intracellular signalling molecules which play an important role in neutrophil activation. This study investigates the effects of cAMP- and cGMP-elevating drugs on the fMLP-induced phosphorylation of p42/p44-MAPK, p38-MAPK and PKB. The cAMP-elevating prostacyclin I2 (Flolan™) shows inhibition of fMLP-induced phosphorylation of p42/p44-MAPK, p38-MAPK and PKB. The cGMP-elevating drug sodium nitroprusside did not influence fMLP-induced phosphorylation of these signalling molecules. Therefore, physiological cAMP-elevating substances seem to play a regulatory role and prevent neutrophil granulocytes from “overstimulation”. KW - Neutrophile KW - VASP KW - MAPK KW - Vasodilatatoren KW - neutrophils KW - VASP KW - MAPK KW - vasodilators Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-13331 ER - TY - THES A1 - Mittermeier, Agnes Barbara T1 - Beeinflussung der Migration humaner neutrophiler Granulozyten durch Ca 2+-empfindliche K +-Kanäle (hIK1) T1 - K-channel dependent migration of human neutrophil granulocytes N2 - Mittels RT-PCR konnte gezeigt werden, dass humane neutrophile Granulozyten auf mRNA-Ebene hIK1-Kanäle exprimieren. Die spezifischen hIK1-Kanalblocker Clotrimazol und Charybdotoxin führten zu einer dosisabhängigen Verringerung der Migrationsgeschwindigkeit Neutrophiler. Eine Hemmung der hIK1-Kanäle mittels Clotrimazol und Charybdotoxin fürte zu einer signifikanten Zellschwellung. Die mittels Hypotonie erzwungene Zellschwellung führte zu einer signifikanten Reduktion der Migrationsgeschwindigkeit Neutrophiler. Diese Ergebnisse zeigen, dass die Migration humaner neutrophiler Granulozyten von der Aktivität der hIK1-Kanäle abhängig ist. N2 - Recently it was found that the activity of a Ca-sensitive K-channel (IK1) is required for migration of epithelial cells since it modulates their actin cytoskeleton and cell volume. Here it was tested whether migration of human neutrophils also depends on IK1-channel activity. For this reason neutrophils were isolated from healthy donors and migration monitored by means of videomicroscopy. Application of specific IK1 blockers slowed down migration dose-dependently. KW - K-Kanäle KW - Neutrophile KW - Migration KW - Potassium channels KW - neutrophils KW - migration Y1 - 2003 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-7172 ER -