Refine
Year of publication
Document Type
- Doctoral Thesis (42)
- Article (33)
Is part of the Bibliography
- no (75)
Keywords
- - (26)
- Staphylococcus aureus (15)
- Sepsis (9)
- <i>Staphylococcus aureus</i> (4)
- Antikörper (4)
- IgE (4)
- Immunreaktion (4)
- Immunsuppression (4)
- Immunsystem (4)
- allergy (4)
- vaccine (4)
- BALB/c Maus (3)
- Impfstoff (3)
- JSNZ (3)
- Maus (3)
- Regulatorischer T-Lymphozyt (3)
- T-Lymphozyt (3)
- sepsis (3)
- Allergie (2)
- Antibody response (2)
- Apoptose (2)
- Bakteriämie (2)
- COVID-19 (2)
- DEREG (2)
- Foxp3 (2)
- Genotypisierung (2)
- Granulozyt (2)
- Immunoglobulin G (2)
- Immunoglobulin M (2)
- IsdB (2)
- Methylene blue (2)
- Monozyt (2)
- Pathogen inactivation (2)
- S. aureus (2)
- SplB (2)
- Stress (2)
- Superantigen (2)
- Surgery (2)
- T cell (2)
- T-Zellen (2)
- Th2 (2)
- Tumor (2)
- Zelltod (2)
- colonization (2)
- host adaptation (2)
- infection (2)
- innate immunity (2)
- mouse models (2)
- neutrophils (2)
- platelets (2)
- 3-dioxygenase (1)
- <i>S. aureus</i> (1)
- <i>Streptococcus mutans</i> (1)
- <i>Streptococcus oralis</i> (1)
- A20 (1)
- ATA (1)
- Airway inflammation (1)
- Allergen (1)
- Allergic diseases (1)
- Allergy (1)
- Antigenpräsentation (1)
- Antikörperantworten (1)
- B cell response (1)
- B-Zelle (1)
- BALB/c mouse (1)
- Bacteria (1)
- Bacterial allergens (1)
- Bakteriologie (1)
- Bauchfellentzündung (1)
- Bauchspeicheldrüsenkrebs (1)
- Blood compatibility test (1)
- Blood supply (1)
- C57 BL/6 mice (1)
- C57BL/6 Maus (1)
- CASP (1)
- CD38 antibody (1)
- CD4 (1)
- CTLA-4 (1)
- Card-ii-Omics (1)
- Carrier (1)
- Chlamydia (1)
- ClfA (1)
- Coombs test (1)
- Corticosteron (1)
- Cystic Fibrosis (1)
- Cystic fibrosis (1)
- Cytokine (1)
- Cytokines (1)
- Daratumumab (1)
- Demographic change (1)
- Dendritische Zelle (1)
- Diabetes mellitus (1)
- Donor research (1)
- Effects of blood donation (1)
- Enterotoxin Gene Cluster (1)
- Entzündung (1)
- Erkundungsverhalten (1)
- FcγRIIa (1)
- FnBPA (1)
- Fragebogen (1)
- Furunkulose (1)
- GATA1 (1)
- GWA (1)
- Gel card (1)
- GlpQ (1)
- Glucocorticosteroide (1)
- HDI (1)
- HEK cells (1)
- HLA (1)
- HLA-DR (1)
- Histondeacetylase-Inhibitor (1)
- Host Pathogen Interactions (1)
- Human (1)
- Humanbiologie (1)
- IDO1 (1)
- IL-10 (1)
- IL-33 (1)
- IgG (1)
- IgG4 (1)
- IgM (1)
- Immobilisation (1)
- Immunantwort (1)
- Immune modulation (1)
- Immune response (1)
- Immunglobulin G (1)
- Immunglobuline (1)
- Immunisierung (1)
- Immunmodulation (1)
- Immunologie (1)
- Immunregulation (1)
- Immunstimulation (1)
- Immuntherapie (1)
- Indirect antiglobulin test (1)
- Indolamin-2 (1)
- Infektionen (1)
- Infektionsmodelle (1)
- Inflammation (1)
- Inselzelltransplantation (1)
- Kaltes Plasma (1)
- Kolonisierung (1)
- Kynurenin (1)
- Körperbild (1)
- LC-MS/MS (1)
- LPS (1)
- Leukozyten (1)
- Lipoprotein (1)
- Lymphozyt (1)
- MHC (1)
- MRI (1)
- MS-275 (1)
- MSCRAMM (1)
- Macrophage (1)
- Magen (1)
- Makrophage (1)
- Massenspektrometrie (1)
- Mausmodell (1)
- Mausstammvergleich (1)
- Mood regulation (1)
- Mouse model (1)
- Mukoviszidose (1)
- NETose (1)
- NETosis (1)
- NETs (1)
- NF-kappaB Signalweg (1)
- NF-kappaB pathway (1)
- NK-Cells (1)
- NK-Zellen (1)
- NLRP3 inflammasome (1)
- NOD Maus (1)
- NOD mouse (1)
- NaB (1)
- Natürliche Killerzelle (1)
- Neuroendokrines System (1)
- Neutrophile (1)
- Newcastle disease Virus (1)
- Niereninsuffizienz (1)
- Noradrenalin (1)
- Onkolyse (1)
- Operation (1)
- PBMC (1)
- PF4 (1)
- PTMs (1)
- Panton-Valentin-Leukozidin (1)
- Panton-Valentine Leukocidin (1)
- Peritonitis (1)
- Plasmamedizin (1)
- Polymerase-Kettenreaktion (1)
- Polytrauma (1)
- Postoperative immune suppression (1)
- Psychischer Stress (1)
- RBC antibody screening (1)
- Red blood cell concentrates (1)
- Reserpin (1)
- SAHA (1)
- Schlaganfall (1)
- Serine protease-like proteins (1)
- Solid phase (1)
- Sozialverhalten (1)
- SplD (1)
- Staphyloccus aureus (1)
- Staphylococci (1)
- Surgically induced immune dysfunction (1)
- Sympathikus (1)
- Systemic infection (1)
- T cell superallergen (1)
- T cells (1)
- T-cells (1)
- TLR4 (1)
- TNFAIP3 (1)
- TSST-1 (1)
- Th2 cells (1)
- Therapeutic antibody interference (1)
- Tierversuch (1)
- Toll-like-Rezeptor (1)
- Transkriptomanalyse (1)
- Translokation (1)
- Transportstress (1)
- Trauma (1)
- Tryptophan (1)
- Tryptophanstoffwechsel (1)
- Virotherapie (1)
- Western Blot Verfahren (1)
- Wirtsadaptation (1)
- Wundheilung (1)
- Zystische Fibrose (1)
- aPF4/H antibodies (1)
- activation status (1)
- acute stress (1)
- adaptive Immunantwort (1)
- adaptive Immunität (1)
- adaptive immunity (1)
- adaptives Immunsystem (1)
- adhesion inhibitor (1)
- adjuvants (1)
- akuter Stress (1)
- alpha-toxin (1)
- anti- (1)
- antibody (1)
- antibody repertoire (1)
- antibody response (1)
- antibody-secreting cells (1)
- anti‐PF4/heparin antibodies (1)
- apoptosis (1)
- asthma (1)
- atomic force microscopy (1)
- aurintricarboxylic acid (1)
- autoimmune (1)
- bakterielle Infektion (1)
- bakterielle Translokation (1)
- bleeding (1)
- bleeding tendency (1)
- blood platelets (1)
- blood smear (1)
- body image (1)
- bone marrow transplantation (1)
- case report (1)
- cell elasticity (1)
- cerebral thrombosis (1)
- chemotherapy (1)
- chirurgisch (1)
- clonal complex (1)
- coagulation (1)
- cold physical plasma (1)
- complication (1)
- cystic fibrosis (1)
- cytokines (1)
- dendritic cells (1)
- depressionsähnliches Verhalten (1)
- dirty mouse (1)
- egc superantigens (1)
- egc-Superantigene (1)
- enterotoxin gene cluster (1)
- eosinophils (1)
- epidemiology (1)
- expression (1)
- factor analysis (1)
- foxp3 (1)
- furunculosis (1)
- genotyping (1)
- gut epithelial (1)
- habitat (1)
- haemorrhage (1)
- hereditary thrombocytopenias (1)
- host-pathogen-interaction (1)
- immune evasion (1)
- immune evasion cluster (1)
- immune polarization (1)
- immunoediting (1)
- immunofluorescence (1)
- immunoglobulin g (1)
- immunoproteomics (1)
- immunosuppression (1)
- immunosupression (1)
- indoleamine 2,3-dioxygenase (1)
- infective endocarditis (1)
- infective endocarditis, antibody response, monoclonal antibodies, S. aureus, SplB, ClfA, FnBPA, Card-ii-Omics (1)
- inherited platelet defects (1)
- inherited platelet disorders (1)
- innate lymphoid cells (1)
- interaction (1)
- islet transplantation (1)
- jep Gene (1)
- kynurenine pathway (1)
- laboratory (1)
- length (1)
- leucocytes (1)
- livestock (1)
- major surgery (1)
- mass spectrometry (1)
- methyltryptophan (1)
- mixed chimerism (1)
- monoclonal antibodies (1)
- monocyte (1)
- mouse (1)
- multi-species biofilm (1)
- multiple myeloma (1)
- mupirocin (1)
- nanoindentation (1)
- nasale Besiedlung (1)
- neutralisierende Antikörper (1)
- neutralizing antibodies (1)
- nose (1)
- oxidation-specific epitopes (1)
- pathogen-specific IgG (1)
- peritonitis (1)
- phagocyte oxidase (1)
- phagocytes (1)
- phosphoproteomics (1)
- pig (1)
- plasma medicine (1)
- platelet factor 4 (1)
- platelet pathophysiology (1)
- platelet transfusion proteomics (1)
- pneumonia (1)
- polymicrobial sepsis (1)
- polyreactive antibodies (1)
- precision medicine (1)
- proteomic analysis (1)
- questionnaire (1)
- rat (1)
- re-establishment of macrophages (1)
- reactive oxygen species (1)
- regulatory T cells (1)
- regulatory t cells (1)
- reserpine (1)
- restraint stress (1)
- sensitization (1)
- serology (1)
- shame boundary (1)
- signaling (1)
- sodium (1)
- solid tumor models (1)
- splenectomy (1)
- stiffness (1)
- superantigen (1)
- superantigens (1)
- sympathetic nervous system (1)
- sympathische Denervierung (1)
- synthetic heparins (1)
- targeted proteomics (1)
- targeted therapy (1)
- therapeutics (1)
- thrombocytopenia (1)
- toxic shock syndrome (1)
- transcriptome (1)
- transportation stress (1)
- tryptophan metabolites (1)
- tumor surveillance (1)
- type 2 immune response (1)
- type 2 immunity (1)
- virulence (1)
- weight loss (1)
- wildling (1)
- wound healing (1)
- zweidimensionale Westernblots (1)
- γδ T cells (1)
Institute
- Institut für Immunologie u. Transfusionsmedizin - Abteilung Immunologie (75) (remove)
Publisher
- MDPI (12)
- Frontiers Media S.A. (10)
- S. Karger AG (5)
- Wiley (3)
- AO Research Institute Davos (1)
- American Society for Microbiology (ASM) (1)
- BMJ Publishing Group (1)
Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus, SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus-naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins’ polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response.
In cystic fibrosis (CF) infectious and allergic airway inflammation cause pulmonary exacerbations that destroy the lungs. Staphylococcus aureus is a common long-term colonizer and cause of recurrent airway infections in CF. The pathogen is also associated with respiratory allergy; especially the staphylococcal serine protease-like proteins (Spls) can induce type 2 immune responses in humans and mice. We measured the serum IgE levels specific to 7 proteases of S. aureus by ELISA, targeting 5 Spls (76 CF patients and 46 controls) and the staphopains A and B (16 CF patients and 46 controls). Then we compared cytokine release and phenotype of T cells that had been stimulated with Spls between 5 CF patients and 5 controls. CF patients had strongly increased serum IgE binding to all Spls but not to the staphopains. Compared to healthy controls, their Spl-stimulated T cells released more type 2 cytokines (IL-4, IL-5, IL-13) and more IL-6 with no difference in the secretion of type 1- or type 3 cytokines (IFNγ, IL-17A, IL-17F). IL-10 production was low in CF T cells. The phenotype of the Spl-exposed T cells shifted towards a Th2 or Th17 profile in CF but to a Th1 profile in controls. Sensitization to S. aureus Spls is common in CF. This discovery could explain episodes of allergic inflammation of hitherto unknown causation in CF and extend the diagnostic and therapeutic portfolio.
Our goal was to provide a comprehensive overview of the antibody response to Staphylococcus aureus antigens in the general population as a basis for defining disease-specific profiles and diagnostic signatures. We tested the specific IgG and IgA responses to 79 staphylococcal antigens in 996 individuals from the population-based Study of Health in Pomerania. Using a dilution-based multiplex suspension array, we extended the dynamic range of specific antibody detection to seven orders of magnitude, allowing the precise quantification of high and low abundant antibody specificities in the same sample. The observed IgG and IgA antibody responses were highly heterogeneous with differences between individuals as well as between bacterial antigens that spanned several orders of magnitude. Some antigens elicited significantly more IgG than IgA and vice versa. We confirmed a strong influence of colonization on the antibody response and quantified the influence of sex, smoking, age, body mass index, and serum glucose on anti-staphylococcal IgG and IgA. However, all host parameters tested explain only a small part of the extensive variability in individual response to the different antigens of S. aureus.
Infections are often caused by pathobionts, endogenous bacteria that belong to the microbiota. Trauma and surgical intervention can allow bacteria to overcome host defences, ultimately leading to sepsis if left untreated. One of the main defence strategies of the immune system is the production of highly specific antibodies. In the present proof-of-concept study, plasma antibodies against 9 major pathogens were measured in sepsis patients, as an example of severe systemic infections. The binding of plasma antibodies to bacterial extracellular proteins was quantified using a semi-automated immunoblot assay. Comparison of the pathogen-specific antibody levels before and after infection showed an increase in plasma IgG in 20 out of 37 tested patients. This host-directed approach extended the results of pathogen-oriented microbiological and PCR diagnostics: a specific antibody response to additional bacteria was frequently observed, indicating unrecognised poly-microbial invasion. This might explain some cases of failed, seemingly targeted antibiotic treatment.
Staphylococcus aureus(S. aureus) is a pathobiont of humans as well as a multitude of animalspecies. The high prevalence of multi-resistant and more virulent strains ofS. aureusnecessitatesthe development of new prevention and treatment strategies forS. aureusinfection. Major advancestowards understanding the pathogenesis ofS. aureusdiseases have been made using conventionalmouse models, i.e., by infecting naïve laboratory mice with human-adaptedS. aureusstrains. However,the failure to transfer certain results obtained in these murine systems to humans highlights thelimitations of such models. Indeed, numerousS. aureusvaccine candidates showed promising resultsin conventional mouse models but failed to offer protection in human clinical trials. These limitationsarise not only from the widely discussed physiological differences between mice and humans, but alsofrom the lack of attention that is paid to the specific interactions ofS. aureuswith its respectivehost. For instance, animal-derivedS. aureuslineages show a high degree of host tropism and carry arepertoire of host-specific virulence and immune evasion factors. Mouse-adaptedS. aureusstrains,humanized mice, and microbiome-optimized mice are promising approaches to overcome theselimitations and could improve transferability of animal experiments to human trials in the future.
Sepsis ist die dritthäufigste Todesursache in Deutschland und verursacht jährliche Krankenhauskosten von mehr als 8 Mrd. €. Über die Pathophysiologie ist noch immer vieles unbekannt. Bei der Bekämpfung von extrazellulären Infektionserregern spielt vor allem das humorale Immunsystem eine wichtige Rolle, da die von B-Zellen/ Plasmazellen gebildeten Antikörper wichtige antiinfektive Agenzien darstellen. Dennoch ist die Rolle der B-Zellen bei einer Sepsis nicht gut verstanden. Ergebnisse aus Mausmodellen, aber auch aus klinischen Studien mit Sepsispatienten zeigen einerseits die vermehrte Apoptose von B-Zellen, anderseits wurde auch eine polyklonale B-Zellaktivierung beschrieben, die mit einem unspezifischen Anstieg der Antikörperkonzentrationen im Blut einhergeht.
In dieser Arbeit sollte untersucht werden, ob während einer systemischen bakteriellen Infektion, wie der Sepsis, auch eine Erreger-spezifische Antikörperantwort ausgebildet wird. Mit Hilfe von zwei serologischen Assays wurde die Antikörperantwort von Sepsispatienten gegen extrazelluläre Proteine von 16 typischen Sepsiserregern bestimmt. Anhand von Plasmaproben aus zwei prospektiven Studien konnte die Antikörperkinetik von einem Zeitpunkt vor der klinischen Diagnose bis maximal 16 Tage nach Diagnose ermittelt werden.
Mittels eines Simple Western Assays - einem semi-quantitativen Immunoblot-Assay - wurde zunächst die Erreger-spezifische Antikörperantwort von Patienten mit einer vorliegenden mikrobiologischen Erregerdiagnose untersucht. 54 % der Patienten zeigten eine spezifische humorale Immunantwort gegen den mikrobiologisch diagnostizierten Erreger, wohingegen die Antikörperspiegel für das Kontrollantigen TT unverändert blieben.
Zur Untersuchung der zweiten Patientenkohorte wurde ein Bead-basierter Suspensions-Array auf Grundlage der xMAP-Technologie (Luminex®) entwickelt. Der Infection Array ermöglichte die gleichzeitige Quantifizierung der spezifischen Antikörperantwort gegen 16 verschiedene Erreger. Bei 64 der 76 untersuchten Patienten wurden Anstiege der IgG-Antikörper gegen einen oder mehrere dieser Erreger beobachtet. In 62,5 % der Fälle stimmten diese Anstiege mit der mikrobiologischen Diagnose überein. Bei 20/64 Patienten wurden signifikante Anstiege der IgG-Spiegel spezifisch für einen oder zwei Erreger nachgewiesen, in 44/64 Fällen wurden Anstiege gegen mehr als zwei Erreger beobachtet. Bei Letzteren richtete sich die Antikörperantwort hauptsächlich gegen Enterokokken und Enterobacteriaceae, was primär auf zwei Ursachen zurückgeführt werden kann: (i) Ein Großteil dieser Patienten hatte einen intraabdominellen Infektionsfokus. Polymikrobielle Infektionen durch endogene Darmbakterien, typischerweise verursacht durch eine Darmruptur oder die Insuffizienz einer chirurgischen Darmnaht, sind hierbei ein plausibler Befund, der der mikrobiologischen Diagnostik offenbar häufig entgeht. (ii) Außerdem können Sepsis-bedingte Organstörungen zu einer gesteigerten Darmpermeabilität führen, die wiederum die Translokation intestinaler Bakterien erleichtert.
Die Ergebnisse dieser Arbeit lassen den Schluss zu, dass die beobachteten Antikörperreaktionen auf eine Antigen-spezifische Memoryantwort zurückzuführen sind. In etwa 2/3 der Fälle wird eine Sepsis endogen durch Bakterien des eigenen Mikrobioms verursacht. Entsprechend war es nicht überraschend, dass gegen alle untersuchten Erreger bereits vor der Infektion und auch bei gesunden Kontrollpersonen basale antibakterielle IgG-Spiegel gemessen wurden. Zudem waren die IgG-Anstiege oft bereits zwischen Tag 0 und Tag 8 zu beobachten. Bei einer Primärantwort mit dem Erreger würde die Aktivierung der Zellen und der Klassenwechsel der Antikörper deutlich mehr Zeit erfordern.
Die Untersuchung der Erreger-spezifischen Antikörperantwort hat gezeigt, dass ein serologischer Assay Rückschlüsse auf den Infektionserreger zulässt. Außerdem zeigen die Daten, dass auch Kommensale wie Darmbakterien das Immunsystem prägen, was wiederum Einfluss auf die humorale Immunantwort während einer Infektion haben kann. Dieser Aspekt wird bei Mausmodellen oft vernachlässigt, kann aber entscheidend für die Translation der Ergebnisse aus Tierversuchen auf den Menschen sein. Aber auch diagnostisch bietet der Infection Array Einsatzmöglichkeiten. Im Gegensatz zur konventionellen Erregerdiagnostik ist die Serologie robust gegenüber einer bereits begonnenen Antibiotikagabe, und sie könnte dabei helfen, zwischen einer Kontamination und dem Infektionserreger zu unterscheiden, z. B. im Fall von KNS wie S. epidermidis. Ebenso wäre der Einsatz bei Biofilm-assoziierten Infektionen wie z. B. Protheseninfektionen oder Endokarditis denkbar. Hier besteht die Infektion oft bereits lange asymptomatisch, bevor sie klinisch diagnostiziert wird. Bei Diagnose bestehen meist bereits erhöhte Antikörperspiegel, die sich von denen gesunder Individuen unterscheiden. Ein serologischer Test könnte hier invasive Eingriffe, um an Material für die mikrobiologische Diagnose heranzukommen, reduzieren und die Sensitivität der Erregerdiagnostik erhöhen. Durch den Einsatz rekombinanter Proteine kann die Spezifität des Assays in der Zukunft erhöht werden. Zu diesem Zweck wurden in dieser Arbeit bereits erste immunogene Proteine identifiziert. Durch die Verwendung rekombinanter Proteine wäre zudem zukünftig die Erweiterung des Erregerpanels um typische, aber womöglich schwerkultivierbare Erreger möglich. Damit könnte die Sepsisforschung Neuland betreten.
Exploring Virulence Factors and Alternative Therapies against Staphylococcus aureus Pneumonia
(2020)
Direct monitoring of drug‐induced mechanical response of individual cells by atomic force microscopy
(2020)
Abstract
Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cells’ states. Disturbances including methyl‐β‐cyclodextrin (MβCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the real‐time response of single cells to these drugs in their physiological condition. Here, nanoindentation‐based atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MβCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for real‐time measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cell´s elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.
Abstract
Background
Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli.
Objectives
To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets.
Methods
Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed.
Results
Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS.
Conclusions
Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.
Abstract
Background
Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin‐induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6‐, 8‐, 10‐, and 12‐mer size and a hypersulfated 12‐mer (S12‐mer).
Methods
We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti‐PF4/heparin antibodies.
Results
The synthetic heparin‐like compounds display stronger binding characteristics to PF4 than animal‐derived heparins of corresponding lengths. Upon complexation with PF4, 6‐mer and S12‐mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8‐, 10‐, and 12‐mer heparins. Anti‐PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ≤ 8‐mer than with complexes formed between PF4 and heparins ≥ 10‐mer. Addition of one sulfate group to the 12‐mer resulted in a S12‐mer, which showed substantial changes in its binding characteristics to PF4.
Conclusions
We provide a template for characterizing interactions of newly developed heparin‐based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.
Neue Antibiotika und Präventionsmaßnahmen gegen S. aureus sind aufgrund der starken Ausbreitung multiresistenter S. aureus-Stämme dringend erforderlich. Zur Entwicklung von Therapie- und Präventionsmaßnahmen werden geeignete Infektionsmodellen benötigt, die die klinische Situation möglichst exakt widerspiegeln. Da die Spezies S. aureus stark wirtsspezifisch ist, könnten wirtsadaptierte S. aureus-Stämme hierbei äußerst hilfreich sein. In der Infektionsforschung werden vor allem Mausmodelle verwendet. Da bisher jedoch angenommen wurde, dass Mäuse keine natürlichen Wirte von S. aureus sind, sind S. aureus-Forscher davon ausgegangen, dass Mäuse kein geeignetes Modell darstellen. Das wurde durch unsere und andere Arbeitsgruppen allerdings in den letzten Jahren widerlegt. Wir konnten zeigen, dass Labor- und Wildmäuse mit S. aureus besiedelt sind.
Im Rahmen dieser Arbeit sollte geklärt werden, ob murine Infektionsmodelle durch die Verwendung von mausadaptierten S. aureus-Stämmen optimiert werden können. Aus über 250 S. aureus-Stämmen, die aus Labor und Wildmäusen isoliert wurden, wurden vier mausadaptierte S. aureus-Isolate ausgewählt und mit dem humanen S. aureus-Isolat Newman in einem Pneumonie- und Bakteriämiemodell vergleichen. Diese Stämme wiesen einen repräsentativen spa-Typ sowie typischen Phagenmuster und Virulenzgene auf. Zudem waren sie in der Lage, murines Plasma zu koagulieren und in murinem Vollblut zu replizieren.
Es zeigte sich, dass das murine Isolat S. aureus DIP sowohl im Pneumonie- als auch im Bakteriämiemodell deutlich virulenter war als das humane Isolat Newman und die anderen getesteten mausadaptierten Stämme. Nach kürzester Zeit starben alle Tiere, die mit S. aureus DIP infiziert wurden. Wurde die Infektionsdosis im Vergleich zu Newman um 90 % reduziert, waren die bakterielle Last, der Belastungsscore, sowie die Zytokin- und Chemokinkonzentrationen nach Infektion mit S. aureus DIP bzw. S. aureus Newman vergleichbar. Im Besiedlungsmodell konnte gezeigt werden, dass die mausadaptierten Stämme S. aureus JSNZ sowie S. aureus DIP in der Lage sind, Mäuse über einen Zeitraum von 7 Tagen stabil zu besiedeln. Mäuse, die mit S. aureus Newman besiedelt waren, konnten den Stamm innerhalb dieses Zeitraums eliminieren. Die Genomsequenzierung der in vivo verwendeten S. aureus Stämme zeigte, dass lediglich S. aureus DIP für das Leukozidin LukMF‘ kodiert. Das lässt vermuten, dass die Präsenz des Virulenzfaktors für die gesteigerte Virulenz von S. aureus DIP verantwortlich sein könnte.
Des Weiteren sollten in dieser Arbeit ein Besiedlungsmodell mit murinen S. aureus-Isolaten etabliert und die beteiligten Immunzellen quantifiziert werden. Es zeigte sich, dass Mäuse mit murinen S. aureus-Isolaten bis zu 7 Tage besiedelt werden können wohingegen S. aureus Newman zu diesem Zeitpunkt nur noch in 20 % der Tiere nachweisbar war. Zudem konnte bei der intranasalen Besiedlung mit einer hohen Dosis S. aureus DIP [1 × 10^8 CFU] gezeigt werden, dass sowohl Th17-Zellen als auch γδ-T-Zellen nach 7 Tagen IL-17A, IL-17F und IL-22 produzieren. Jedoch konnte die Zytokinproduktion nur in Tieren nachgewiesen werden, die einen hohen Belastungsscore aufwiesen. Da nach 24 Stunden bei Tieren mit hohem Belastungsscore auch Bakterien in der Lunge detektiert wurde, ist anzunehmen, dass S. aureus diese Tiere nicht nur besiedelt, sondern bei ihnen auch eine Atemwegsinfektion verursacht hatte. Durch den geringen prozentualen Anteil an ILCs in den zervikalen Lymphknoten war es nicht möglich Rückschlüsse auf deren Zytokinproduktion zu ziehen. Somit gelang es zwar ein murines S. aureus-Besiedlungsmodell zu etablieren, jedoch kann keine Aussage zu den beteiligten Zellen des Immunsystems getroffen werden.
Zusammenfassend konnte gezeigt werden, dass Labormäuse mit mausadaptierten S. aureus-Stämmen länger besiedelt werden können als mit dem humanen Referenzstamm Newman. Zudem konnte mit Hilfe des mausadaptierten Stammes S. aureus DIP die Infektionsdosis im Pneumonie- und Bakteriämiemodell erheblich reduziert werden. Somit gelang es Mausmodelle durch die Verwendung von mausadaptierten S. aureus-Stämmen zu optimieren, auch wenn das nicht auf alle getesteten Isolate zutrifft. Durch die Anpassung an den murinen Wirt stellen mausadaptierte S. aureus-Stämme wie DIP und JSNZ ein physiologischeres Modell der Pathogen-Wirts-Interaktion dar. Die Verwendung eines solchen Stammes ermöglicht es ein besseres Verständnis für Infektionsprozesse und die Pathogen-Wirt-Interaktionen zu erlangen und dadurch eventuell neue Therapiemöglichkeiten zu entwickeln.
Es ist zu berücksichtigen, dass auch die Verwendung mausadaptierter S. aureus-Stämme in murinen Besiedlungs- und Infektionsmodellen lediglich ein Modell darstellt, welches Vor- und Nachteile hat. Daher ist es essenziell, dass Wissenschaftler die Grenzen jedes Modellsystems kennen und das richtige Infektionsmodell (oder eine Kombination davon) auswählen, um ihre Forschungsfragen zu beantworten.
Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.
Oxidation-Specific Epitopes (OSEs) Dominate the B Cell Response in Murine Polymicrobial Sepsis
(2020)
In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis.
Although antigen-specific priming of antibody responses is impaired during sepsis, there is nevertheless a strong increase in IgM and IgG serum concentrations. Using colon ascendens stent peritonitis (CASP), a mouse model of polymicrobial abdominal sepsis, we observed substantial increases in IgM as well as IgG of all subclasses, starting at day 3 and peaking 2 weeks after sepsis induction. The dominant source of antibody-secreting cells was by far the spleen, with a minor contribution of the mesenteric lymph nodes. Remarkably, sepsis induction in splenectomized mice did not change the dynamics of the serum IgM/IgG reaction, indicating that the marginal zone B cells, which almost exclusively reside in the spleen, are dispensable in such a setting. Hence, in systemic bacterial infection, the function of the spleen as dominant niche of antibody-producing cells can be compensated by extra-splenic B cell populations as well as other lymphoid organs. Depletion of CD4+ T cells did not affect the IgM response, while it impaired IgG generation of all subclasses with the exception of IgG3. Taken together, our data demonstrate that the robust class-switched antibody response in sepsis encompasses both T cell-dependent and -independent components.
Staphylococcus aureussuperantigens (SAgs) are among the most potent T cell mitogensknown.They stimulate large fractions of T cells by cross-linking their T cell receptor withmajor histocompatibility complex class-II molecules on antigen presenting cells, resulting in Tcell proliferation and massive cytokine release. To date, 26 different SAgs have been described in thespeciesS. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcalenterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoningand toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. Inaddition, there is growing evidence that SAgs are involved in allergic diseases. This review providesan overview on recent epidemiological data on the involvement ofS. aureusSAgs and anti-SAg-IgEin allergy, demonstrating that being sensitized to SEs—in contrast to inhalant allergens—is associatedwith a severe disease course in patients with chronic airway inflammation. The mechanisms by whichSAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, wediscuss known and hypothetical pathways by which SAgs can drive an atopic disease