Über Kalziumsignale und Zytotoxizität menschlicher natürlicher Killerzellen : Calcium signaling and cytotoxicity of human natural killer cells

Calcium signaling is an essential component of immune cell function. Immunocompetent cells employ calcium ions as a second messenger to regulate proliferation, migration and maturation. In addition, intracellular calcium signals are necessary for cytotoxic lymphocytes (CLs) such as CD8+ T-cells and natural killer cells (NK cells) to eliminate target cells. For several decades, the doctrine used to be that this elimination is mainly achieved by forcing a target cell into apoptosis. In recent years, several groups reported that there is at least one further way of target cell killing. Given the right circumstances, CLs are also able to kill by a direct lysis of the target cell’s membrane, a process referred to as ’necrosis’. Our research group, the Department of Biophysics at Saarland University, directed by Prof. Dr. Markus Hoth, investigates the Ca2+ dependence of signaling processes in immune cells. We found that calcium ion influx into killer cells is not only necessary for a successful attack on pathogens and tumor cells, but that extracellular calcium supply can influence a killer cell’s global killing efficiency. The aim of this doctoral thesis was to study influx kinetics of Ca2+ ions in active NK cells at a single-cell level using fluorescence microscopy. The conducted experiments led to two main conclusions: I) Not only do NK cells employ both necrosis and apoptosis when killing certain target cells, but the frequency distributions of both killing types depend highly on the extracellular Ca2+ concentration ([Ca2+]ext). Low concentrations favor the occurrence of apoptosis, while an increase in [Ca2+]ext shifts the balance towards necrotic killing. This shift occurs even after raising Ca2+ ion levels beyond the amount necessary for necrosis induction. II) The shape of Ca2+ influx differs, regarding necrotic and apoptotic killing. Necrosis induction depends on high sustained rises in [Ca2+]int, while NK cells causing apoptosis tend to show low and oscillatory Ca2+ signals. Further experiments were conducted in attempt to reveal the responsible molecular mechanisms. Artificially releasing high amounts of Ca2+ ions into the cytosol of killing NK cells, using a photolabile Ca2+ chelator, suggests that high cytosolic Ca2+ concentrations are necessary but not sufficient for necrosis induction by NK cells. The presented results have contributed to shedding light on the Ca2+ dependence of NK cell function. In his doctoral thesis published in 2016, Christian Backes from our research group demonstrated that the balance of necrosis to apoptosis induced by NK cells greatly affects their total killing competence. The observation that global killing efficiency of CD8+ T-lymphocytes and NK cells depends on [Ca2+]ext can now in parts be explained by the likelihood of necrotic and apoptotic killing processes shifting. Although different Ca2+ influx patterns in active NK cells were previously described by other groups, no exact quantification of these signals has yet been provided. In addition to giving such a quantification, this thesis can provide evidence that these different signal types are clearly linked to distinct outcomes regarding NK cell cytotoxicity in vitro. NK cells play a pivotal role in tumor surveillance and - in case of already developed cancer - keeping malignant cell clones in check. Apoptosis and lysis of target cells may both help achieve a shared aim but will differently affect the anti-tumor immune response. Hence, different Ca2+ signals in tumor-killing NK cells as well as their likelihood of inducing apoptosis or necrosis may also affect tumor development, progression as well as the efficacy of immune therapy and adoptive cell transfer. Many recent studies suggest that tumor cells benefit from necrosis by taking advantage of the resulting microenvironment. If we can deepen our understanding of how different calcium signal types in CLs come about, we could try to modulate killing behaviour to shape a more desirable cellular immune response to malignancies.Kalziumsignale spielen eine Schlüsselrolle in der Funktion unserer Immunabwehr. Immunzellen verwenden diese Signale, um komplexe Prozesse wie Proliferation, Migration und Reifung zu regulieren. Darüber hinaus benötigen Killerzellen wie CD8+-T-Zellen und natürliche Killerzellen (NK-Zellen) hohe zytosolische Kalziumspiegel, um Zielzellen abzutöten. Die Doktrin der letzten Jahrzehnte war, dass dieses Abtöten im Wesentlichen auf einem einzigen Mechanismus basiert: der Aktivierung des programmierten Zelltodes, der Apoptose der Zielzelle. Seit einiger Zeit mehren sich jedoch die Hinweise, dass es noch mindestens einen weiteren Mechanismus gibt. Unter gewissen Voraussetzungen können Killerzellen ihre Opfer auch durch eine direkte Lyse der Zellmembran töten. Dieser Prozess wird gemeinhin als "Nekrose" bezeichnet. Unter der Leitung von Prof. Dr. Markus Hoth erforscht unsere biophysikalische Arbeitsgruppe seit Jahren die Bedeutung von Kalziumsignalen für eine Vielzahl von Immunzellen. Wissenschaftler unserer Abteilung fanden heraus, dass ein Kalziumeinstrom in Killerzellen nicht nur notwendig für das Abtöten von Bakterien und Tumorzellen ist, sondern dass das extrazelluläre Kalziumionenangebot auch einen Einfluss auf die absolute Killingeffizienz ausübt. Das Ziel der vorliegenden Doktorarbeit war, die Kinetik dieser Kalziumströme in aktiven NK-Zellen mittels Fluoreszenzmikroskopie auf Einzelzellebene zu erforschen. Die in diesem Rahmen durchgeführten Experimente lieferten zwei wichtige Erkenntnisse: I) Zusätzlich zur Bestätigung, dass NK-Zellen sowohl Apoptose als auch Nekrose in geeigneten Zielzellen induzieren, stellte sich heraus, dass die statistische Verteilung beider Killingtypen stark vom extrazellulären Kalziumangebot abhängt. Niedrige Konzentrationen an Kalziumionen begünstigen das Auftreten von Apoptosen, während steigende Kalziumspiegel zu mehr und mehr Nekrosen führen. Dieser Einfluss zeigt sich auch noch bei extrazellulären Kalziumkonzentrationen, die weit über dem für Nekrose notwendigen Maß liegen. II) NK-Zellen zeigen verschiedene Arten von Kalziumsignalen, je nachdem, welchen Typ Zytotoxizität (i.e. Nekrose oder Apoptose) sie in ihrer Zielzelle induzieren. Die Lyse der Zelle bedurfte dabei stets eines hohen und anhaltenden Einstroms von Kalziumionen, während Killerzellen, die Apoptose hervorriefen, niedrigere und oszillierende Kalziumströme zeigten. Weitere Experimente zielten darauf ab, die molekularen Mechanismen für diese Unterschiede in den Signalen aufzudecken. Indem große Mengen an Kalziumionen mittels eines photolabilen Chelators künstlich in Killerzellen freigesetzt wurden, konnte gezeigt werden, dass hohe Kalziumeinströme zwar notwendig, aber sehr wahrscheinlich nicht hinreichend für das nekrosebasierte Abtöten sind. Die vorliegenden Ergebnisse haben dazu beigetragen, die Kalziumabhängigkeit der Funktion von NK-Zellen besser zu verstehen. In seiner 2016 publizierten Doktorarbeit konnte Christian Backes aus unserer Arbeitsgruppe zeigen, dass die Balance von Nekrose- zu Apoptoseinduktion einen starken Einfluss auf das globale Killingpotential von NK-Zellen ausübt. Mit Hilfe der hier gewonnenen Erkenntnis, dass diese Balance vom extrazellulären Kalziumangebot abhängt, kann die Beeinflussung der Effizienz natürlicher Killerzellen durch die Verfügbarkeit von Kalziumionen nun in Teilen erklärt werden. Verschiedenartige Muster von Kalziumströmen in aktiven NK-Zellen wurden in der Vergangenheit bereits von anderen Arbeitsgruppen postuliert, jedoch nicht exakt quantifiziert. Neben einer solchen Quantifizierung kann die vorliegende Arbeit erstmals beweisen, dass diese Signalmuster deutlich mit den verschiedenen Killingarten assoziiert sind, zu denen NK-Zellen fähig sind. Natürliche Killerzellen zählen zu den wichtigsten Effektoren der Tumorüberwachung. Auch im Falle von bereits entstandenen Tumoren tragen sie essenziell dazu bei, den Tumor in Schach zu halten. Unmittelbar führen Apoptoseinduktion und Lyse zwar zum gleichen Ergebnis - dem Tod der Tumorzelle -, beide Prozesse beinflussen jedoch die gegen den Tumor gerichtete folgende Immunantwort auf stark unterschiedliche Weise. Es ist anzunehmen, dass die Frage, wie NK-Zellen Tumorzellen eliminieren und welche Kalziumsignale sie dabei präsentieren, auch einen Einfluss auf die Wirksamkeit von Chemotherapien, immunmodulierenden Therapien und adoptivem Zelltransfer haben könnte. Viele aktuelle Studien legen nahe, dass Tumorzellen von einem bestimmten immunologischen Mikromilieu profitieren. Ein solches Milieu scheint insbesondere durch nekrotische Zellen begünstigt zu werden. Ein tiefer gehendes Verständnis davon, wie verschiedene Kalziumsignale in NK-Zellen zustande kommen, könnte es erlauben, sie pharmakologisch zu beeinflussen, um das Tötungsverhalten von NK-Zellen zu modifizieren. Auf diese Weise könnte ein immunologisches Mikromilieu im Tumorgewebe erreicht werden, das bösartige Zellen schädigt, ohne gleichzeitig die Vermehrung benachbarter Tumorzellen zu stimuliere

Does experience matter? Assessing user motivations to accept a vehicle-to-grid charging tariff

Vehicle-to-grid (V2G) could be a cornerstone to ensure the efficient integration of a large number of electric vehicles (EVs) and the resulting electricity demand into the energy system. However, successful V2G adoption requires direct interaction with the EV user. To explore user preferences and requirements in the context of a V2G charging tariff, we conducted a survey (N = 1196). We assess users’ minimum range requirements and willingness to pay for a V2G charging tariff and relate them to users’ experience with EVs. By building a mediation model, we evaluate the importance of three charging strategies to guide users’ minimum range requirements and expected monetary savings. The results reveal EV owners’ preference for a climate-neutral charging strategy, leading to a higher readiness to accept lower minimum ranges and lower monetary savings. These results are especially important to aggregators, aiming to design profitable business models, while accounting for user requirements and preferences

Acute Respiratory Distress Syndrome due to Mycoplasma pneumoniae Misinterpreted as SARS-CoV-2 Infection

Background. In 2020, a novel coronavirus caused a global pandemic with a clinical picture termed COVID-19, accounting for numerous cases of ARDS. However, there are still other infectious causes of ARDS that should be considered, especially as the majority of these pathogens are specifically treatable. Case Presentation. We present the case of a 36-year-old gentleman who was admitted to the hospital with flu-like symptoms, after completing a half-marathon one week before admission. As infection with SARS-CoV-2 was suspected based on radiologic imaging, the hypoxemic patient was immediately transferred to the ICU, where he developed ARDS. Empiric antimicrobial chemotherapy was initiated, the patient deteriorated further, therapy was changed, and the patient was transferred to a tertiary care ARDS center. As cold agglutinins were present, the hypothesis of an infection with SARS-CoV-2 was then questioned. Bronchoscopic sampling revealed Mycoplasma (M.) pneumoniae. When antimicrobial chemotherapy was adjusted, the patient recovered quickly. Conclusion. Usually, M. pneumoniae causes mild disease. When antimicrobial chemotherapy was adjusted, the patient recovered quickly. The case underlines the importance to adhere to established treatment guidelines, scrutinize treatment modalities, and not to forget other potential causes of severe pneumonia or ARDS

Comparison of Serial and Parallel Connections of Membrane Lungs against Refractory Hypoxemia in a Mock Circuit

Extracorporeal membrane oxygenation (ECMO) is an important rescue therapy method for the treatment of severe hypoxic lung injury. In some cases, oxygen saturation and oxygen partial pressure in the arterial blood are low despite ECMO therapy. There are case reports in which patients with such instances of refractory hypoxemia received a second membrane lung, either in series or in parallel, to overcome the hypoxemia. It remains unclear whether the parallel or serial connection is more effective. Therefore, we used an improved version of our full-flow ECMO mock circuit to test this. The measurements were performed under conditions in which the membrane lungs were unable to completely oxygenate the blood. As a result, only the photometric pre- and post-oxygenator saturations, blood flow and hemoglobin concentration were required for the calculation of oxygen transfer rates. The results showed that for a pre-oxygenator saturation of 45% and a total blood flow of 10 L/min, the serial connection of two identical 5 L rated oxygenators is 17% more effective in terms of oxygen transfer than the parallel connection. Although the idea of using a second membrane lung if refractory hypoxia occurs is intriguing from a physiological point of view, due to the invasiveness of the solution, further investigations are needed before this should be used in a wider clinical setting

Advances in single crystal growth and annealing treatment of electron-doped HTSC

High quality electron-doped HTSC single crystals of $\rm Pr_{2-x}Ce_{x}CuO_{4+\delta}$ and $\rm Nd_{2-x}Ce_{x}CuO_{4+\delta}$ have been successfully grown by the container-free traveling solvent floating zone technique. The optimally doped $\rm Pr_{2-x}Ce_{x}CuO_{4+\delta}$ and $\rm Nd_{2-x}Ce_{x}CuO_{4+\delta}$ crystals have transition temperatures $T_{\rm c}$ of $25$\,K and $23.5$\,K, respectively, with a transition width of less than $1$\,K. We found a strong dependence of the optimal growth parameters on the Ce content $x$. We discuss the optimization of the post-growth annealing treatment of the samples, the doping extension of the superconducting dome for both compounds as well as the role of excess oxygen. The absolute oxygen content of the as-grown crystals is determined from thermogravimetric experiments and is found to be $\ge 4.0$. This oxygen surplus is nearly completely removed by a post-growth annealing treatment. The reduction process is reversible as demonstrated by magnetization measurements. In as-grown samples the excess oxygen resides on the apical site O(3). This apical oxygen has nearly no doping effect, but rather influences the evolution of superconductivity by inducing additional disorder in the CuO$_{2}$ layers. The very high crystal quality of $\rm Nd_{2-x}Ce_{x}CuO_{4+\delta}$ is particularly manifest in magnetic quantum oscillations observed on several samples at different doping levels. They provide a unique opportunity of studying the Fermi surface and its dependence on the carrier concentration in the bulk of the crystals.Comment: 19 pages, 7 figures, submitted to Eur. Phys. J.

Respiratory Physiology of COVID-19 and Influenza Associated Acute Respiratory Distress Syndrome

There is ongoing debate whether lung physiology of COVID-19-associated acute respiratory distress syndrome (ARDS) differs from ARDS of other origin. Objective: The aim of this study was to analyze and compare how critically ill patients with COVID-19 and Influenza A or B were ventilated in our tertiary care center with or without extracorporeal membrane oxygenation (ECMO). We ask if acute lung failure due to COVID-19 requires different intensive care management compared to conventional ARDS. Methods: 25 patients with COVID-19-associated ARDS were matched to a cohort of 25 Influenza patients treated in our center from 2011 to 2021. Subgroup analysis addressed whether patients on ECMO received different mechanical ventilation than patients without extracorporeal support. Results: Compared to Influenza-associated ARDS, COVID-19 patients had higher ventilatory system compliance (40.7 mL/mbar [31.8–46.7 mL/mbar] vs. 31.4 mL/mbar [13.7–42.8 mL/mbar], p = 0.198), higher ventilatory ratio (1.57 [1.31–1.84] vs. 0.91 [0.44–1.38], p = 0.006) and higher minute ventilation at the time of intubation (mean minute ventilation 10.7 L/min [7.2–12.2 L/min] for COVID-19 vs. 6.0 L/min [2.5–10.1 L/min] for Influenza, p = 0.013). There were no measurable differences in P/F ratio, positive end-expiratory pressure (PEEP) and driving pressures (∆P). Respiratory system compliance deteriorated considerably in COVID-19 patients on ECMO during 2 weeks of mechanical ventilation (Crs, mean decrease over 2 weeks −23.87 mL/mbar ± 32.94 mL/mbar, p = 0.037) but not in ventilated Influenza patients on ECMO and less so in ventilated COVID-19 patients without ECMO. For COVID-19 patients, low driving pressures on ECMO were strongly correlated to a decline in compliance after 2 weeks (Pearson’s R 0.80, p = 0.058). Overall mortality was insignificantly lower for COVID-19 patients compared to Influenza patients (40% vs. 48%, p = 0.31). Outcome was insignificantly worse for patients requiring veno-venous ECMO in both groups (50% mortality for COVID-19 on ECMO vs. 27% without ECMO, p = 0.30/56% vs. 34% mortality for Influenza A/B with and without ECMO, p = 0.31). Conclusion: The pathophysiology of early COVID-19-associated ARDS differs from Influenza-associated acute lung failure by sustained respiratory mechanics during the early phase of ventilation. We question whether intubated COVID-19 patients on ECMO benefit from extremely low driving pressures, as this appears to accelerate derecruitment and consecutive loss of ventilatory system compliance

Killer immunoglobulin-like receptor 2DS5 is associated with recovery from coronavirus disease 2019

Background Despite numerous advances in the identification of risk factors for the development of severe coronavirus disease 2019 (COVID-19), factors that promote recovery from COVID-19 remain unknown. Natural killer (NK) cells provide innate immune defense against viral infections and are known to be activated during moderate and severe COVID-19. Killer immunoglobulin-like receptors (KIR) mediate NK cell cytotoxicity through recognition of an altered MHC-I expression on infected target cells. However, the influence of KIR genotype on outcome of patients with COVID-19 has not been investigated so far. We retrospectively analyzed the outcome associations of NK cell count and KIR genotype of patients with COVID-19 related severe ARDS treated on our tertiary intensive care unit (ICU) between February and June 2020 and validated our findings in an independent validation cohort of patients with moderate COVID-19 admitted to our tertiary medical center. Results Median age of all patients in the discovery cohort (n = 16) was 61 years (range 50–71 years). All patients received invasive mechanical ventilation; 11 patients (68%) required extracorporeal membrane oxygenation (ECMO). Patients who recovered from COVID-19 had significantly higher median NK cell counts during the whole observational period compared to patients who died (121 cells/µL, range 16–602 cells/µL vs 81 cells/µL, range 6–227 cells/µL, p-value = 0.01). KIR2DS5 positivity was significantly associated with shorter time to recovery (21.6 ± 2.8 days vs. 44.6 ± 2.2 days, p-value = 0.01). KIR2DS5 positivity was significantly associated with freedom from transfer to ICU (0% vs 9%, p-value = 0.04) in the validation cohort which consisted of 65 patients with moderate COVID-19. Conclusion NK cells and KIR genotype might have an impact on recovery from COVID-19

TNF-related apoptosis-inducing ligand, interferon gamma-induced protein 10, and C-reactive protein in predicting the progression of SARS-CoV-2 infection : a prospective cohort study

Background: Early prognostication of COVID-19 severity will potentially improve patient care. Biomarkers, such as TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP), might represent possible tools for point-of-care testing and severity prediction. Methods: In this prospective cohort study, we analyzed serum levels of TRAIL, IP-10, and CRP in patients with COVID-19, compared them with control subjects, and investigated the association with disease sever ity. Results: A total of 899 measurements were performed in 132 patients (mean age 64 years, 40.2% females). Among patients with COVID-19, TRAIL levels were lower (49.5 vs 87 pg/ml, P = 0.0142), whereas IP-10 and CRP showed higher levels (667.5 vs 127 pg/ml, P <0.001; 75.3 vs 1.6 mg/l, P <0.001) than healthy controls. TRAIL yielded an inverse correlation with length of hospital and intensive care unit (ICU) stay, Simplified Acute Physiology Score II, and National Early Warning Score, and IP-10 showed a positive cor relation with disease severity. Multivariable regression revealed that obesity (adjusted odds ratio [aOR] 5.434, 95% confidence interval [CI] 1.005-29.38), CRP (aOR 1.014, 95% CI 1.002-1.027), and peak IP-10 (aOR 1.001, 95% CI 1.00-1.002) were independent predictors of in-ICU mortality

Autoantibodies to muscarinic acetylcholine receptors found in patients with primary biliary cirrhosis

<p>Abstract</p> <p>Background</p> <p>Autoantibodies to the human muscarinic acetylcholine receptor of the M3 type (hmAchR M3) have been suggested to play an etiopathogenic role in Sjögren's syndrome. Primary biliary cirrhosis (PBC) often is associated with this syndrome. Therefore, we studied the co-presence of hmAchR M3 autoantibodies in patients with PBC.</p> <p>Methods</p> <p>Frequency of hmAchR M3 autoantibodies was assessed by Western blotting analysis as well as by an ELISA using a 25-mer peptide of the 2^ndextracellular loop of hmAchR M3. Co-localization of hmAchR M3/PBC-specific autoantibodies was studied by confocal laser scanning microscopy. Finally, sera from patients with PBC as well as from healthy controls were tested.</p> <p>Results</p> <p>Western blotting analysis as well as results from ELISA testing revealed a significantly enhanced IgG reactivity in PBC patients in contrast to healthy controls. Co-localization of autoantibodies with the hmAchR M3 receptor-specific autoantibodies was observed in 10 out of 12 PBC-patients but none of the 5 healthy controls. Antibodies of the IgM type were not found to be affected.</p> <p>Conclusions</p> <p>For the first time, our data demonstrate the presence of autoantibodies to the hmAchR M3 in PBC patients. These findings might contribute to the understanding of the pathogenesis of this disease. Further studies have to focus on the functionality of hmAchR M3 autoantibodies in PBC patients.</p