MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration.

Early initiated adequate antibiotic treatment is essential in intensive care. Shortening the length of antibiotic susceptibility testing (AST) can accelerate clinical decision-making. Our objective was to develop a simple flow cytometry (FC)-based AST that produces reliable results within a few hours.

Mac-1 Receptor Clustering Initiates Production of Pro-Inflammatory, Antibacterial Extracellular Vesicles From Neutrophils.

Depending on the prevailing environmental conditions, neutrophilic granulocytes release extracellular vesicles (EV) which have either anti-inflammatory effects on other neutrophils or pro-inflammatory and antibacterial effects. In the present study we investigated the molecular mechanisms underlying the biogenesis of functionally heterogenic EVs. We show that selective stimulation of Mac-1 integrin (complement receptor 3) by specific ligands initiates the generation of EVs which are able to impair bacterial growth and to induce the secretion of the pro-inflammatory cytokine IL-8 (aEV).

The Functional Heterogeneity of Neutrophil-Derived Extracellular Vesicles Reflects the Status of the Parent Cell.

Similar to other cell types, neutrophilic granulocytes also release extracellular vesicles (EVs), mainly medium-sized microvesicles/microparticles. According to published data, authors have reached a consensus on the physical parameters (size, density) and chemical composition (surface proteins, proteomics) of neutrophil-derived EVs. In contrast, there is large diversity and even controversy in the reported functional properties.

Neutrophils produce proinflammatory or anti-inflammatory extracellular vesicles depending on the environmental conditions.

Extracellular vesicles (EVs) are important elements of intercellular communication. A plethora of different, occasionally even opposite, physiologic and pathologic effects have been attributed to these vesicles in the last decade. A direct comparison of individual observations is however hampered by the significant differences in the way of elicitation, collection, handling, and storage of the investigated vesicles.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis.

Role of Mac-1 integrin in generation of extracellular vesicles with antibacterial capacity from neutrophilic granulocytes.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils.

New flow cytometry-based method for the assessment of the antibacterial effect of immune cells and subcellular particles.

Techniques currently used for assessment of bacterial count or growth are time-consuming, offer low throughput, or they are complicated or expensive. The aim of the present work was to elaborate a new method that is able to detect the antibacterial effect of cells, subcellular particles, and soluble compounds in a fast, cost, and labor effective way. Our proposed technique is based on flow cytometry (FC) optimized for detection of small particles and on fluorescently labeled bacteria.

Functionally and morphologically distinct populations of extracellular vesicles produced by human neutrophilic granulocytes.

EVs in the microvesicle size range released during spontaneous death of human neutrophils were characterized and their properties compared with previously described EVs with antibacterial effect (aEVs, generated on specific activation) or produced spontaneously (sEVs). The 3 vesicle populations overlapped in size and in part of the constituent proteins were stained with annexin V and were impermeable to PI. However, none of them produced superoxide.

Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.

Aim: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes.

Methods: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration.

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils.

Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes.

Changing world of neutrophils.

Neutrophilic granulocytes are no longer regarded as cells involved only in the last phase of the immune response with one single-although vitally important-task: engulfing and killing of microorganisms marked by immunoglobulin or complement fragments. In recent years, it was shown that neutrophils are actively involved in initiation and organization of the adaptive immune response by releasing various cytokines, interacting with all major types of immune cells, regulating their own lifespan, and participating in the anaphylactic reaction and in several classically nonimmune functions such as hemostasis, atherogenesis, and even insulin resistance. The antibacterial effect is no longer restricted to killing and destruction of microorganisms sequestered in the phagosomal space.

Antibacterial effect of microvesicles released from human neutrophilic granulocytes.

Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties.

p63RhoGEF--a key mediator of angiotensin II-dependent signaling and processes in vascular smooth muscle cells.

The purpose of our study was to investigate the role of endogenous p63RhoGEF in G(q/11)-dependent RhoA activation and signaling in rat aortic smooth muscle cells (RASMCs). Therefore, we studied the expression and subcellular localization in freshly isolated RASMCs and performed loss of function experiments to analyze its contribution to RhoGTPase activation and functional responses such as proliferation and contraction. By this, we could show that p63RhoGEF is endogenously expressed in RASMCs and acts there as the dominant mediator of the fast angiotensin II (ANG II)-dependent but not of the sphingosine-1-phosphate (S(1)P)-dependent RhoA activation.

The oxidation state of phospholipids controls the oxidative burst in neutrophil granulocytes.

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes.