Modulation of neutrophil activity by soluble complement cleavage products — an in-depth analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies

Performance of the New FlashCam-based Camera in the 28\,m Telescope of H.E.S.S

In October 2019, the central 28 m telescope of the H.E.S.S. experiment has been upgraded with a new camera. The camera is based on the FlashCam design which has been developed in view of a possible future implementation in the Medium-Sized Telescopes of the Cherenkov Telescope Array (CTA), with emphasis on cost and performance optimization and on reliability. The fully digital design of the trigger and readout system makes it possible to operate the camera at high event rates and to precisely adjust and understand the trigger system. The novel design of the front-end electronics achieves a dynamic range of over 3,000 photoelectrons with only one electronics readout circuit per pixel. Here we report on the performance parameters of the camera obtained during the first year of operation in the field, including operational stability and optimization of calibration algorithms.Comment: Proceedings of the 37th International Cosmic Ray Conference (ICRC 2021

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5–1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties

Inflammatory macrophages in patients with fatigue

Background: Hyperinflammatory, so-called M1 macrophages play a major role in chronic inflammatory diseases often linked to impaired well-being as well as fatigue and sarkopenia. Cytokines such as IL-1, IL-6 play a role in polarizing the differentiation into M1 macrophages and simultanously inhibit the differentiation of anti-inflammatory M2 macrophages. Methods: We enriched blood derived macrophages from peripheral blood and characterized their phenotypes by flow cytometry. The purinergic receptor P2 × 7 was tested by patch clamping and ion flux measurement. Microparticle and exosome release was induced by exogenous ATP-stimulation and qualified by trans-electorn microscopy as well as by nanosizer measurements. Results: M1 macrophages typically lacked surface CD163 and P2 × 7, but M2 macrophages expressed both markers. ATP stimulation induced cell death in M1 but microparticle and exosome release in M2 macrophages. Ion channel measurement confirmed the hypersensitivity of M1 and impaired ion flux by ATP. These result imply that chronic inflammatory diseases linked to highly elevated M1 type macrophages are highly sensitive to exogenous ATP, the most important danger signal in physical and psychiatric trauma. By contrast, anti-inflammatory, M2 macrophages mediate Calcium-signaling and exosome release upon ATP stimulation. MiRNA expression analysis further demonstrated that Let7b-5p discriminates between M1 and M2 macrophage polarization. Conclusion: M1 macrophage and microglia polarization may explain the detrimental response against ATP related trauma in a number of inflammatory conditions which may fuel into fatigue and sarkopenia

Doubling survival and improving clinical outcomes using a left ventricular assist device instead of chest compressions for resuscitation after prolonged cardiac arrest : a large animal study

INTRODUCTION: Despite improvements in pre-hospital and post-arrest critical care, sudden cardiac arrest (CA) remains one of the leading causes of death. Improving circulation during cardiopulmonary resuscitation (CPR) may improve survival rates and long-term clinical outcomes after CA. METHODS: In a porcine model, we compared standard CPR (sCPR; n =10) with CPR using an intravascular cardiac assist device without additional chest compressions (iCPR; n =10) following 10 minutes of electrically induced ventricular fibrillation (VF). In a separate crossover experiment, 10 additional pigs were subjected to 10 minutes of VF and 6 minutes of sCPR; the iCPR device was then implanted if a return of spontaneous circulation (ROSC) was not achieved using sCPR. Animals were evaluated in respect to intra- and post-arrest hemodynamics, survival, functional outcome and cerebral and myocardial lesions following CPR. We hypothesized that iCPR would result in more frequent ROSC and better functional recovery than sCPR. RESULTS: iCPR produced a mean flow of 1.36 ± 0.02 L/min, leading to significantly higher coronary perfusion pressure (CPP) values during the early period of CPR (22 ± 10 mmHg vs. 9 ± 5 mmHg, P ≤0.01, 1 minute after start of CPR; 20 ± 11 mmHg vs. 10 ± 7 mmHg, P =0.03, 2 minutes after start of CPR), resulting in high ROSC rates (100% in iCPR vs. 50% in sCPR animals; P =0.03). iCPR animals showed significantly lower serum S100 levels at 10 and 30 minutes following ROSC (3.5 ± 0.6 ng/ml vs. 7.4 ± 3.0 ng/ml 30 minutes after ROSC; P ≤0.01), as well as superior clinical outcomes based on overall performance categories (2.9 ± 1.0 vs. 4.6 ± 0.8 on day 1; P ≤0.01). In crossover experiments, 80% of animals required treatment with iCPR after failed sCPR. Notably, ROSC was still achieved in six of the remaining eight animals (75%) after a total of 22.8 ± 5.1 minutes of ischemia. CONCLUSIONS: In a model of prolonged cardiac arrest, the use of iCPR instead of sCPR improved CPP and doubled ROSC rates, translating into improved clinical outcomes.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH(i)) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH(i), cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation

Complement C5a Alters the Membrane Potential of Neutrophils during Hemorrhagic Shock

Background. Polymorphonuclear granulocytes (PMN) play a crucial role in host defense. Physiologically, exposure of PMN to the complement activation product C5a results in a protective response against pathogens, whereas in the case of systemic inflammation, excessive C5a substantially impairs neutrophil functions. To further elucidate the inability of PMN to properly respond to C5a, this study investigates the role of the cellular membrane potential of PMN in response to C5a. Methods. Electrophysiological changes in cellular and mitochondrial membrane potential and intracellular pH of PMN from human healthy volunteers were determined by flow cytometry after exposure to C5a. Furthermore, PMN from male Bretoncelles-Meishan-Willebrand cross-bred pigs before and three hours after severe hemorrhagic shock were analyzed for their electrophysiological response. Results. PMN showed a significant dose- and time-dependent depolarization in response to C5a with a strong response after one minute. The chemotactic peptide fMLP also evoked a significant shift in the membrane potential of PMN. Acidification of the cellular microenvironment significantly enhanced depolarization of PMN. In a clinically relevant model of porcine hemorrhagic shock, the C5a-induced changes in membrane potential of PMN were markedly diminished compared to healthy littermates. Overall, these membrane potential changes may contribute to PMN dysfunction in an inflammatory environment