An Exploratory Study Using Next-Generation Sequencing to Identify Prothrombotic Variants in Patients with Cerebral Vein Thrombosis.

Prothrombotic hereditary risk factors for cerebral vein thrombosis (CVT) are of clinical interest to better understand the underlying pathophysiology and stratify patients for the risk of recurrence. This study explores prothrombotic risk factors in CVT patients. An initial screening in patients of the outpatient clinic of the Department of Transfusion Medicine and Hemostaseology of the University Hospital Erlangen, Germany, revealed 183 patients with a history of CVT. An initial screening identified a number of common prothrombic risk factors, including Factor V Leiden (rs6025) and Prothrombin G20210A (rs1799963). All patients without relevant findings (58 individuals) were invited to participate in a subsequent genetic analysis of 55 relevant genes using next-generation sequencing (NGS). Three intron variants (ADAMTS13: rs28446901, FN1: rs56380797, rs35343655) were identified to occur with a significantly higher frequency in the CVT patient cohort compared to the general European population. Furthermore, the combined prevalence of at least two of four potentially prothrombic variants (FGA (rs6050), F13A1 (rs5985), ITGB3 (rs5918), and PROCR (rs867186)) was significantly higher in the CVT subjects. The possible impact of the identified variants on CVT is discussed.

Systemic calcitonin gene-related peptide receptor antagonism decreases survival in a porcine model of polymicrobial sepsis: blinded randomised controlled trial.

BACKGROUND: Calcitonin gene-related peptide (CGRP) and procalcitonin, which are overexpressed in sepsis, exert distinct immunomodulatory effects mediated through the CGRP receptor. The CGRP receptor antagonist olcegepant improves survival in murine sepsis. This study evaluated whether CGRP receptor antagonism is similarly beneficial in a porcine model of polymicrobial sepsis. METHODS: We conducted a prospective randomised, controlled, investigator-blinded trial in adult pigs of either sex, that were anaesthetised and ventilated before sepsis was induced by polymicrobial (autologous) faecal peritonitis. After the onset of early septic shock (systolic blood pressure <90 mm Hg or >10% decline from baseline MAP), pigs were resuscitated (i.v. fluid/antibiotics/vasopressors) and randomised to receive either i.v. olcegepant (n=8) or vehicle control (n=8). The primary outcome was time to death, euthanasia required up to 72 h after surgery (according to predefined severe cardiorespiratory failure), or both. Secondary outcomes included haemodynamic changes, and systemic as well as organ inflammation (mRNA expression). RESULTS: Septic shock developed 8.7 h (inter-quartile range, 5.8-11.1 h) after the onset of faecal peritonitis. Olcegepant worsened survival, with 6/8 pigs randomised to the control group surviving 72.0 h (50.9-72.0 h), compared with 3/8 pigs receiving olcegepant surviving 51.3 h (12.5-72.0 h; P=0.01). At 48 h, lower MAP and higher cardiac output occurred in pigs receiving olcegepant. Cardiac, hepatic, and renal injury was not different between pigs randomised to receive olcegepant or vehicle. Olcegepant reduced mRNA expression of several inflammation-related cytokines and CD68+ macrophages in liver but not in lung tissue. CONCLUSIONS: CGRP receptor antagonism with olcegepant was not beneficial in this porcine model of polymicrobial sepsis, which closely mimics human sepsis.

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.

Ion and Water Transport in Neutrophil Granulocytes and Its Impairment during Sepsis.

Neutrophil granulocytes are the vanguard of innate immunity in response to numerous pathogens. Their activity drives the clearance of microbe- and damage-associated molecular patterns, thereby contributing substantially to the resolution of inflammation. However, excessive stimulation during sepsis leads to cellular unresponsiveness, immunological dysfunction, bacterial expansion, and subsequent multiple organ dysfunction. During the short lifespan of neutrophils, they can become significantly activated by complement factors, cytokines, and other inflammatory mediators. Following stimulation, the cells respond with a defined (electro-)physiological pattern, including depolarization, calcium influx, and alkalization as well as with increased metabolic activity and polarization of the actin cytoskeleton. Activity of ion transport proteins and aquaporins is critical for multiple cellular functions of innate immune cells, including chemotaxis, generation of reactive oxygen species, and phagocytosis of both pathogens and tissue debris. In this review, we first describe the ion transport proteins and aquaporins involved in the neutrophil ion-water fluxes in response to chemoattractants. We then relate ion and water flux to cellular functions with a focus on danger sensing, chemotaxis, phagocytosis, and oxidative burst and approach the role of altered ion transport protein expression and activity in impaired cellular functions and cell death during systemic inflammation as in sepsis.

Complement C5a Functions as a Master Switch for the pH Balance in Neutrophils Exerting Fundamental Immunometabolic Effects.

During sepsis, excessive activation of the complement system with generation of the anaphylatoxin C5a results in profound disturbances in crucial neutrophil functions. Moreover, because neutrophil activity is highly dependent on intracellular pH (pHi), we propose a direct mechanistic link between complement activation and neutrophil pHi In this article, we demonstrate that in vitro exposure of human neutrophils to C5a significantly increased pHi by selective activation of the sodium/hydrogen exchanger. Upstream signaling of C5a-mediated intracellular alkalinization was dependent on C5aR1, intracellular calcium, protein kinase C, and calmodulin, and downstream signaling regulated the release of antibacterial myeloperoxidase and lactoferrin. Notably, the pH shift caused by C5a increased the glucose uptake and activated glycolytic flux in neutrophils, resulting in a significant release of lactate. Furthermore, C5a induced acidification of the extracellular micromilieu. In experimental murine sepsis, pHi of blood neutrophils was analogously alkalinized, which could be normalized by C5aR1 inhibition. In the clinical setting of sepsis, neutrophils from patients with septic shock likewise exhibited a significantly increased pHi These data suggest a novel role for the anaphylatoxin C5a as a master switch of the delicate pHi balance in neutrophils resulting in profound inflammatory and metabolic changes that contribute to hyperlactatemia during sepsis.

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.

Allergies - A T cells perspective in the era beyond the TH1/TH2 paradigm.

Allergic diseases have emerged as a major health care burden, especially in the western hemisphere. They are defined by overshooting reactions of an aberrant immune system to harmless exogenous stimuli. The TH1/TH2 paradigm assumes that a dominance of TH2 cell activation and an inadequate TH1 cell response are responsible for the development of allergies. However, the characterization of additional T helper cell subpopulations such as TH9, TH17, TH22, THGM-CSF and their interplay with regulatory T cells suggest further layers of complexity. This review summarizes state-of-the-art knowledge on T cell diversity and their induction, while revisiting the TH1/TH2 paradigm. With respect to these numerous contributors, it offers a new perspective on the pathogenesis of asthma, allergic rhinitis (AR) and atopic dermatitis (AD) incorporating recent discoveries in the field of T cell plasticity.

Early Detection of Junctional Adhesion Molecule-1 (JAM-1) in the Circulation after Experimental and Clinical Polytrauma.

Severe tissue trauma-induced systemic inflammation is often accompanied by evident or occult blood-organ barrier dysfunctions, frequently leading to multiple organ dysfunction. However, it is unknown whether specific barrier molecules are shed into the circulation early after trauma as potential indicators of an initial barrier dysfunction. The release of the barrier molecule junctional adhesion molecule-1 (JAM-1) was investigated in plasma of C57BL/6 mice 2 h after experimental mono- and polytrauma as well as in polytrauma patients (ISS ≥ 18) during a 10-day period. Correlation analyses were performed to indicate a linkage between JAM-1 plasma concentrations and organ failure. JAM-1 was systemically detected after experimental trauma in mice with blunt chest trauma as a driving force. Accordingly, JAM-1 was reduced in lung tissue after pulmonary contusion and JAM-1 plasma levels significantly correlated with increased protein levels in the bronchoalveolar lavage as a sign for alveolocapillary barrier dysfunction. Furthermore, JAM-1 was markedly released into the plasma of polytrauma patients as early as 4 h after the trauma insult and significantly correlated with severity of disease and organ dysfunction (APACHE II and SOFA score). The data support an early injury- and time-dependent appearance of the barrier molecule JAM-1 in the circulation indicative of a commencing trauma-induced barrier dysfunction.

The gross anatomy course: SARS-CoV-2 pandemic-related effects on students' learning, interest in peer-teaching, and students' perception of its importance.

The COVID-19 pandemic required adjustments and limitations in university teaching, thereby challenging teaching concepts in anatomy requiring in-person contact, including the gross anatomy course. Therefore, the present study investigates the impact of COVID-19-associated adjustments on students' perception of the gross anatomy course's importance and quality, students' preferred learning setting and outcome, and their motivation to involve themselves in academic activities, including becoming a future peer-teacher of the course. Using paper-based questionnaires in Ulm, Germany, 397 (response rate: 82.3%) students of the winter term of 2020/2021 were surveyed using quantitative and qualitative items, which were compared with cohorts prior to the pandemic. Students reported a higher global rating on course quality during COVID-19 (pre-COVID-19: 5.3 ± 0.9, during-COVID-19: 5.6 ± 0.7, p < 0.001; 1 = very bad, 6 = very good). Students' perceived importance of the gross anatomy course showed a small but significant increase (pre-COVID-19: 4.2 ± 0.6, during-COVID-19: 4.3 ± 0.6, p < 0.001; 1 = strongly disagree, 6 = strongly agree). Students' motivation to apply as a peer-teacher remained stable, nevertheless, they reported less interest in transferring their knowledge to junior students. Finally, students reported that they spent significantly more learning time alone and their examination grades remained unchanged during the pandemic. Astonishingly, despite radical changes of the teaching environment due to COVID-19, students appreciate the offered teaching and highly valued the gross anatomy course.

Impact of surface coating and systemic anticoagulants on hemostasis and inflammation in a human whole blood model.

BACKGROUND: Surface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties. METHODS: We investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood. Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37°C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays. RESULTS: Using the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness. CONCLUSIONS: The MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification.

An exploratory study investigating the effect of targeted hyperoxemia in a randomized controlled trial in a long-term resuscitated model of combined acute subdural hematoma and hemorrhagic shock in cardiovascular healthy pigs.

Severe physical injuries and associated traumatic brain injury and/or hemorrhagic shock (HS) remain leading causes of death worldwide, aggravated by accompanying extensive inflammation. Retrospective clinical data indicated an association between mild hyperoxemia and improved survival and outcome. However, corresponding prospective clinical data, including long-term resuscutation, are scarce. Therefore, the present study explored the effect of mild hyperoxemia for 24 hours in a prospective randomized controlled trial in a long-term resuscitated model of combined acute subdural hematoma (ASDH) and HS. ASDH was induced by injecting 0.1 ml × kg-1 autologous blood into the subdural space and HS was triggered by passive removal of blood. After 2 hours, the animals received full resuscitation, including retransfusion of the shed blood and vasopressor support. During the first 24 hours, the animals underwent targeted hyperoxemia (PaO2 = 200 - 250 mmHg) or normoxemia (PaO2 = 80 - 120 mmHg) with a total observation period of 55 hours after the initiation of ASDH and HS. Survival, cardiocirculatory stability, and demand for vasopressor support were comparable between both groups. Likewise, humoral markers of brain injury and systemic inflammation were similar. Multimodal brain monitoring, including microdialysis and partial pressure of O2 in brain tissue, did not show significant differences either, despite a significantly better outcome regarding the modified Glasgow Coma Scale 24 hours after shock that favors hyperoxemia. In summary, the present study reports no deleterious and few beneficial effects of mild targeted hyperoxemia in a clinically relevant model of ASDH and HS with long-term resuscitation in otherwise healthy pigs. Further beneficial effects on neurological function were probably missed due to the high mortality in both experimental groups. The present study remains exploratory due to the unavailability of an a priori power calculation resulting from the lack of necessary data.

Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Complement and platelets: prothrombotic cell activation requires membrane attack complex-induced release of danger signals.

Complement activation in the diseases paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) results in cytolysis and fatal thrombotic events, which are largely refractory to anticoagulation and/or antiplatelet therapy. Anticomplement therapy, however, efficiently prevents thrombotic events in PNH and aHUS, but the underlying mechanisms remain unresolved. We show that complement-mediated hemolysis in whole blood induces platelet activation similarly to activation by adenosine 5'-diphosphate (ADP). Blockage of C3 or C5 abolished platelet activation. We found that human platelets failed to respond functionally to the anaphylatoxins C3a and C5a. Instead, complement activation did lead to prothrombotic cell activation in the whole blood when membrane attack complex (MAC)-mediated cytolysis occurred. Consequently, we demonstrate that ADP receptor antagonists efficiently inhibited platelet activation, although full complement activation, which causes hemolysis, occurred. By using an established model of mismatched erythrocyte transfusions in rats, we crossvalidated these findings in vivo using the complement inhibitor OmCI and cobra venom factor. Consumptive complement activation in this animal model only led to a thrombotic phenotype when MAC-mediated cytolysis occurred. In conclusion, complement activation only induces substantial prothrombotic cell activation if terminal pathway activation culminates in MAC-mediated release of intracellular ADP. These results explain why anticomplement therapy efficiently prevents thromboembolisms without interfering negatively with hemostasis.

Multimodal analysis of granulocytes, monocytes, and platelets in patients with cystic fibrosis before and after Elexacaftor-Tezacaftor-Ivacaftor treatment.

Cystic fibrosis (CF) is a monogenetic disease caused by an impairment of the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organs and is associated with acute and chronic inflammation. In 2020, Elexacaftor-Tezacaftor-Ivacaftor (ETI) was approved to enhance and restore the remaining CFTR functionality. This study investigates cellular innate immunity, with a focus on neutrophil activation and phenotype, comparing healthy volunteers with patients with CF before (T1, n = 13) and after six months (T2, n = 11) of ETI treatment. ETI treatment reduced sweat chloride (T1: 95 mmol/l (83|108) vs. T2: 32 mmol/l (25|62), p < 0.01, median, first|third quartile) and significantly improved pulmonal function (FEV1 T1: 2.66 l (1.92|3.04) vs. T2: 3.69 l (3.00|4.03), p < 0.01). Moreover, there was a significant decrease in the biomarker human epididymis protein 4 (T1: 6.2 ng/ml (4.6|6.3) vs. T2: 3.0 ng/ml (2.2|3.7), p < 0.01) and a small but significant decrease in matrix metallopeptidase 9 (T1: 45.5 ng/ml (32.5|140.1) vs. T2: 28.2 ng/ml (18.2|33.6), p < 0.05). Neutrophil phenotype (CD10, CD11b, CD62L, and CD66b) and function (radical oxygen species generation, chemotactic and phagocytic activity) remained largely unaffected by ETI treatment. Likewise, monocyte phenotype and markers of platelet activation were similar at T1 and T2. In summary, the present study confirmed a positive impact on patients with CF after ETI treatment. However, neither beneficial nor harmful effects of ETI treatment on cellular innate immunity could be detected, possibly due to the study population consisting of patients with well-controlled CF.

The effect of sodium thiosulfate on immune cell metabolism during porcine hemorrhage and resuscitation.

Introduction: Sodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers. Methods: After 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA). Results: PBMCs showed significantly higher mitochondrial O2 uptake and lower O 2 • - production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis. Conclusion: In a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. O 2 • - concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods.

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.

Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). Results: 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.

Randomised controlled monocentric trial to compare the impact of using professional actors or peers for communication training in a competency-based inverted biochemistry classroom in preclinical medical education.

OBJECTIVE: In medical education, biochemistry topics are usually knowledge based, and students often are unaware of their clinical relevance. To improve students' awareness of the relevance, we integrated communication skills training into biochemistry education. No studies before have examined the difference between peer and standardised patient (SP) role plays where students explain the biochemical background of a disease in patient-centred language. Therefore, we evaluated whether students' self-perceived competency in Canadian Medical Education Directives for Specialists (CanMEDS) roles and their opinion of the quality of role play differ if the layperson is played by peers or SPs. METHODS: We randomly assigned medical students in a preclinical semester to one of the two groups. The groups used predefined scripts to role play a physician-parent consultation with either a peer (peer group) or an SP (SP group) in the parent role. Students then assessed the activity's effects on their competency in CanMEDS roles and motivation and the relevance of the role play. To determine whether students achieved biochemistry learning goals, we evaluated results of a biochemistry exam. RESULTS: Students' self-perceived competency improved in both groups. The SP group rated their competency in the roles 'Scholar' and 'Professional' significantly higher than the peer group did. The peer group rated their competency in the role of 'Medical Expert' significantly higher if they played the role of the parent rather than physician or observer. The SP group agreed more that they were motivated by the role play and wanted to receive more role play-based teaching. The SP group perceived the role play as being realistic and rated the feedback discussion as more beneficial. The examination results were the same in both groups. CONCLUSION: We showed that role plays in a biochemistry seminar improve students' self-perceived competency. The use of SPs has some advantages, such as being more realistic.

The H2S Donor Sodium Thiosulfate (Na2S2O3) Does Not Improve Inflammation and Organ Damage After Hemorrhagic Shock in Cardiovascular Healthy Swine.

We previously demonstrated marked lung-protective properties of the H2S donor sodium thiosulfate (Na2S2O3, STS) in a blinded, randomized, controlled, long-term, resuscitated porcine model of swine with coronary artery disease, i.e., with decreased expression of the H2S-producing enzyme cystathionine-γ-lyase (CSE). We confirmed these beneficial effects of STS by attenuation of lung, liver and kidney injury in mice with genetic CSE deletion (CSE-ko) undergoing trauma-and-hemorrhage and subsequent intensive care-based resuscitation. However, we had previously also shown that any possible efficacy of a therapeutic intervention in shock states depends both on the severity of shock as well as on the presence or absence of chronic underlying co-morbidity. Therefore, this prospective, randomized, controlled, blinded experimental study investigated the effects of the STS in cardiovascular healthy swine. After anesthesia and surgical instrumentation, 17 adult Bretoncelles-Meishan-Willebrand pigs were subjected to 3 hours of hemorrhage by removal of 30% of the blood volume and titration of the mean arterial pressure (MAP) ≈ 40 ± 5 mmHg. Afterwards, the animals received standardized resuscitation including re-transfusion of shed blood, fluids, and, if needed, continuous i.v. noradrenaline to maintain MAP at pre-shock values. Animals were randomly allocated to either receive Na2S2O3 or vehicle control starting 2 hours after initiation of shock until 24 hours of resuscitation. The administration of Na2S2O3 did not alter survival during the observation period of 68 hours after the initiation of shock. No differences in cardio-circulatory functions were noted despite a significantly higher cardiac output, which coincided with significantly more pronounced lactic acidosis at 24 hours of resuscitation in the Na2S2O3 group. Parameters of liver, lung, and kidney function and injury were similar in both groups. However, urine output was significantly higher in the Na2S2O3 group at 24 hours of treatment. Taken together, this study reports no beneficial effect of Na2S2O3 in a clinically relevant model of hemorrhagic shock-and-resuscitation in animals without underlying chronic cardiovascular co-morbidity.

What factors motivate male and female Generation Z students to become engaged as peer teachers? A mixed-method study among medical and dental students in the gross anatomy course.

Peer-teaching is widely established in anatomy teaching and offers well-described advantages. Nevertheless, at Ulm University, Germany, a reduction in the number of peer teacher applicants for the dissection course was observed. This study examined factors related to the attractiveness of a position as a peer teacher for Generation Z students. Participants of the gross anatomy course were asked to evaluate factors influencing the attractiveness of a peer teacher position using a six-point Likert scale. Additionally, open-ended questions were analyzed qualitatively. Sex-specific subgroup analysis was performed comparing students of low and high motivation to apply for a tutorship. Of the 374 students who participated in this study (response rate 53%), 38% stated that they were intending to apply as peer teachers. Data indicated that students displayed intrinsic motivation to apply for a tutorship because of the opportunity to improve their anatomy knowledge and/or their pleasure in teaching. In contrast, extrinsic factors like remuneration of the tutorship and its relevance for their curriculum vitae were least important. Anatomy educators underestimated the demotivating factor of the workload associated with the tutorship and encouraged students less frequently to apply than peer teachers. Only minor sex-specific differences could be identified. Nevertheless, female students were encouraged less frequently to apply than their male peers. In summary, Generation Z students apply as peer teachers because they are enthusiastic about the task. To motivate students to commit to extracurricular activities like a tutorship, anatomy educators should actively encourage students-particularly females-more frequently to apply.

The effect of targeted hyperoxemia in a randomized controlled trial employing a long-term resuscitated, model of combined acute subdural hematoma and hemorrhagic shock in swine with coronary artery disease: An exploratory, hypothesis-generating study.

Controversial evidence is available regarding suitable targets for the arterial O2 tension (PaO2) after traumatic brain injury and/or hemorrhagic shock (HS). We previously demonstrated that hyperoxia during resuscitation from hemorrhagic shock attenuated cardiac injury and renal dysfunction in swine with coronary artery disease. Therefore, this study investigated the impact of targeted hyperoxemia in a long-term, resuscitated model of combined acute subdural hematoma (ASDH)-induced brain injury and HS. The prospective randomized, controlled, resuscitated animal investigation consisted of 15 adult pigs. Combined ASDH plus HS was induced by injection of 0.1 ml/kg autologous blood into the subdural space followed by controlled passive removal of blood. Two hours later, resuscitation was initiated comprising re-transfusion of shed blood, fluids, continuous i.v. noradrenaline, and either hyperoxemia (target PaO2 200 - 250 mmHg) or normoxemia (target PaO2 80 - 120 mmHg) during the first 24 h of the total of 54 h of intensive care. Systemic hemodynamics, intracranial and cerebral perfusion pressures, parameters of brain microdialysis and blood biomarkers of brain injury did not significantly differ between the two groups. According to the experimental protocol, PaO2 was significantly higher in the hyperoxemia group at the end of the intervention period, i.e., at 24 h of resuscitation, which coincided with a higher brain tissue PO2. The latter persisted until the end of observation period. While neurological function as assessed using the veterinary Modified Glasgow Coma Score progressively deteriorated in the control group, it remained unaffected in the hyperoxemia animals, however, without significant intergroup difference. Survival times did not significantly differ in the hyperoxemia and control groups either. Despite being associated with higher brain tissue PO2 levels, which were sustained beyond the intervention period, targeted hyperoxemia exerted neither significantly beneficial nor deleterious effects after combined ASDH and HS in swine with pre-existing coronary artery disease. The unavailability of a power calculation and, thus, the limited number of animals included, are the limitations of the study.