Butyrylcholinesterase activity in patients with postoperative delirium after cardiothoracic surgery or percutaneous valve replacement- an observational interdisciplinary cohort study.

BACKGROUND AND OBJECTIVES: Postoperative delirium is a frequent and severe complication after cardiac surgery. Activity of butyrylcholinesterase (BChE) has been discussed controversially regarding a possible role in its development. This study aimed to investigate the relevance of BChE activity as a biomarker for postoperative delirium after cardiac surgery or percutaneous valve replacement. METHODS: A total of 237 patients who received elective cardiothoracic surgery or percutaneous valve replacement at a tertiary care centre were admitted preoperatively. These patients were tested with the Montreal Cognitive Assessment investigating cognitive deficits, and assessed for postoperative delirium twice daily for three days via the 3D-CAM or the CAM-ICU, depending on their level of consciousness. BChE activity was measured at three defined time points before and after surgery. RESULTS: Postoperative delirium occurred in 39.7% of patients (n = 94). Univariate analysis showed an association of pre- and postoperative BChE activity with its occurrence (p = 0.037, p = 0.001). There was no association of postoperative delirium and the decline in BChE activity (pre- to postoperative, p = 0.327). Multivariable analysis including either preoperative or postoperative BChE activity as well as age, MoCA, type 2 diabetes mellitus, coronary heart disease, type of surgery and intraoperative administration of red-cell concentrates was performed. Neither preoperative nor postoperative BChE activity was independently associated with the occurrence of postoperative delirium (p = 0.086, p = 0.484). Preoperative BChE activity was lower in older patients (B = -12.38 (95% CI: -21.94 to -2.83), p = 0.011), and in those with a history of stroke (B = -516.173 (95% CI: -893.927 to -138.420), p = 0.008) or alcohol abuse (B = -451.47 (95% CI: -868.38 to -34.55), p = 0.034). Lower postoperative BChE activity was independently associated with longer procedures (B = -461.90 (95% CI: -166.34 to -757.46), p = 0.002), use of cardiopulmonary bypass (B = -262.04 (95% CI: -485.68 to -38.39), p = 0.022), the number of administered red cell-concentrates (B = -40.99 (95% CI: -67.86 to -14.12), p = 0.003) and older age (B = -9.35 (95% CI: -16.04 to -2.66), p = 0.006). CONCLUSION: BChE activity is not independently associated with the occurrence of postoperative delirium. Preoperative BChE values are related to patients' morbidity and vulnerability, while postoperative activities reflect the severity, length and complications of surgery.

Circulating biomarkers of the CS4P and CLIP scores are not altered in a pig model of acute cardiogenic shock and additional short-term circulatory support.

BACKGROUND: Cardiogenic shock (CS) is the leading cause of death in patients with myocardial infarction with a mortality rate greater than 50%. Recently, the CS 4 Proteins (CS4P) and CLIP scores have been developed to predict survival in CS patients. However, their impact in acute CS and additional short-term left ventricular (LV) circulatory support as prognostic markers is currently not known. METHODS AND RESULTS: CS was induced in a porcine model by injecting microsphere particles into the left main coronary artery. Mechanical circulatory support was performed by additional percutaneous LV unloading using an Impella microaxial flow-pump for 30 minutes. Serum samples were collected at baseline, following the onset of CS, and additional LV unloading. Serum levels of biomarkers of the CS4P (beta-2-microglobulin, ALDOB, L-FABP, SerpinG1) and the CLIP scores (Cystatin C, Lactate, Interleukin-6, NT-proBNP) were neither different at any time point investigated nor did they correlate with cardiac output. CONCLUSION: The CS4P and CLIP scores do not reflect immediate whole-body dysregulation in acute CS and have not been able to predict the potential reversal following additional short-term mechanical support by LV unloading in our experimental model. The impact of both scores as prognostic markers after the immediate onset of CS and following additional short-term LV unloading to identify patients at greatest risk remains to be determined.

GSK3 as a Master Regulator of Cellular Processes.

Since its initial purification and characterization as an enzyme negatively regulating glycogen synthase activity [...].

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

Omicron infection-associated T- and B-cell immunity in antigen-naive and triple-COVID-19-vaccinated individuals.

Since early 2022, various Omicron variants have dominated the SARS-CoV-2 pandemic in most countries. All Omicron variants are B-cell immune escape variants, and antibodies induced by first-generation COVID-19 vaccines or by infection with earlier SARS-CoV-2 variants largely fail to protect individuals from Omicron infection. In the present study, we investigated the effect of Omicron infections in triple-vaccinated and in antigen-naive individuals. We show that Omicron breakthrough infections occurring 2-3.5 months after the third vaccination restore B-cell and T-cell immune responses to levels similar to or higher than those measured 14 days after the third vaccination, including the induction of Omicron-neutralizing antibodies. Antibody responses in breakthrough infection derived mostly from cross-reacting B cells, initially induced by vaccination, whereas Omicron infections in antigen-naive individuals primarily generated B cells binding to the Omicron but not the Wuhan spike protein. Although antigen-naive individuals mounted considerable T-cell responses after infection, B-cell responses were low, and neutralizing antibodies were frequently below the limit of detection. In summary, the detection of Omicron-associated B-cell responses in primed and in antigen-naive individuals supports the application of Omicron-adapted COVID-19 vaccines, but calls into question their suitability if they also contain/encode antigens of the original Wuhan virus.

Simple and safe digitoxin dosing in heart failure based on data from the DIGIT-HF trial.

BACKGROUND: The present study aimed to develop a simple dosing score when starting the cardiac glycoside digitoxin in heart failure with reduced ejection fraction (HFrEF) employing first data from the randomized, double-blinded DIGIT-HF trial. METHODS AND RESULTS: In DIGIT-HF, digitoxin was started with a dose of 0.07 mg once daily (o.d.) in all patients. For score derivation, 317 patients were analyzed who had been randomized to digitoxin. In these patients, after scheduled determination of serum levels at study week 6, the digitoxin dose had remained unchanged or had been reduced to 0.05 mg o.d. (97% of patients) to achieve serum concentrations within a predefined range (10.5-23.6 nmol/l). In logistic regression analyses, sex, age, body mass index (BMI), and estimated glomerular filtration rate (eGFR) were associated with need for dose reduction and, therefore, selected for further developing the dosing score. Optimal cut-points were derived from ROC curve analyses. Finally, female sex, age ≥ 75 years, eGFR < 50 ml/min/1.73 m2, and BMI < 27 kg/m2 each were assigned one point for the digitoxin dosing score. A score of ≥ 1 indicated the need for dose reduction with sensitivity/specificity of 81.6%/49.7%, respectively. Accuracy was confirmed in a validation data set including 64 patients randomized to digitoxin yielding sensitivity/specificity of 87.5%/37.5%, respectively. CONCLUSION: In patients with HFrEF, treatment with digitoxin should be started at 0.05 mg o.d. in subjects with either female sex, eGFR < 50 ml/min/1.73m2, BMI < 27 kg/m2, or age ≥ 75 years. In any other patient, digitoxin may be safely started at 0.07 mg o.d.

Cardiovascular risk in bipolar disorder - A case for the hypothalamus-pituitary-adrenal axis?

BACKGROUND: Unipolar major depressive disorder (MDD) and bipolar disorder (BD) are associated with elevated mortality risk secondary to natural causes. Cardiovascular disease (CVD) constitutes the most prevalent underlying condition. Patients with BD display higher CVD-associated excess mortality than MDD patients. Epicardial adipose tissue (EAT) volume, a known predictor of premature CV morbidity and adrenal gland (AG) volume, an indicator for chronic hypothalamus-pituitary-adrenal (HPA) axis activation, were compared in BD and MDD patients. METHODS: Magnetic resonance imaging was performed to assess EAT and AG volume in age-, gender-, and body mass index (BMI)-matched MDD (N = 27) and BD (N = 27) patients. Ten-year CV mortality risk and diabetes risk were assessed by PROCAM, ESC-SCORE, and FINDRISK, respectively; metabolic syndrome (MetS) was determined following NCEP/ATP III criteria. RESULTS: Cardiometabolic risk scores and frequency of MetS were comparable, and scores of cardiometabolic risk indices did not significantly differ in both groups. After adjustment for age, BMI, and physical activity, EAT and AG volumes were significantly higher in BD compared to MDD. Partial correlation analyses showed a significant positive association of EAT and AG volumes in BD but not in the MDD. LIMITATIONS: The modest sample size warrants confirmation in a larger cohort and the cross-sectional design does not allow for temporal or causal inferences. CONCLUSION: Our study indicates increased EAT accumulation in BD patients. This was associated with HPA axis dysregulation. Therapeutic lifestyle interventions that reduce EAT volume should be considered in clinical BD management.

Microembolic signal monitoring in patients with HeartMate 3 and HeartWare left ventricular assist devices: Association with antithrombotic treatment and cerebrovascular events.

BACKGROUND: In patients with left ventricular assist devices (LVADs), ischemic and hemorrhagic stroke are dreaded complications. Predictive markers for these events are lacking. This study aimed to investigate the prevalence and predictive value of microembolic signals (MES) for stroke, detected by Transcranial Doppler sonography (TCD) in patients with HeartMate 3 (HM 3) or HeartWare (HW). METHODS: A thirty-minute bilateral TCD monitoring of the middle cerebral artery (MCA) was performed in 62 outpatients with LVAD (HM 3 N = 31, HW N = 31) and 31 healthy controls. Prevalence and quantity of MES were investigated regarding clinical and laboratory parameters. Cerebrovascular events (CVE) were recorded on follow-up at 90 and 180 days. RESULTS: MES were detected in six patients with HM 3, three patients with HW, and three controls. Within the LVAD groups, patients on monotherapy with vitamin-K-antagonist (VKA) without antiplatelet therapy were at risk for a higher count of MES (negative binomial regression: VKA: 1; VKA + ASA: Exp(B) = 0.005, 95%CI 0.001-0.044; VKA + clopidogrel: Exp(B) = 0.012, 95%CI 0.002-0.056). There was no association between the presence of MES and CVE or death on follow-up (p > 0.05). CONCLUSION: For the first time, the prevalence of MES was prospectively investigated in a notable outpatient cohort of patients with HM 3 and HW. Despite optimized properties of the latest LVAD, MES remain detectable depending on antithrombotic therapy. No association between MES and CVE could be detected.

Stroke, Seizures, Hallucinations and Postoperative Delirium as Neurological Complications after Cardiac Surgery and Percutaneous Valve Replacement.

(1) Background: Neurological complications such as acute ischemic stroke or postoperative delirium are frequent after cardiac surgery or percutaneous valve replacement. This study aimed to identify corresponding risk factors. (2) Methods: 297 patients with percutaneous valve replacement or cardiac surgery were postoperatively screened for neurological complications such as delirium, stroke, seizures and hallucinations twice daily for three days. Pre- and perioperative risk factors were evaluated in a multivariate model. (3) Results: Neurological complications occurred in 43.8% (n = 130) as composed of delirium (43.43%, n = 129), stroke (2.7%, n = 8), seizures (1.35%, n = 4) and real hallucinations (3.36%, n = 10). Multiple logistic regression revealed an association of neurological complications with lower Montreal Cognitive Assessment scores (Exp(B) 2.042; 95% CI, 1.183−3.525, p = 0.010), older age (Exp(B) 1.071; 95% CI, 1.036−1.107, p < 0.001), red blood cell transfusions until postoperative day 3 (Exp(B) 1.157; 95% CI, 1.030−1.300, p = 0.014), history of heart failure (Exp(B) 1.985; 95% CI, 1.130−3.487, p = 0.017) and increased CRP levels (Exp(B) 1.004; 95% CI, 1.000−1.008, p = 0.037). (4) Conclusions: Postoperative delirium remains a frequent complication after cardiac surgery, while stroke and seizures occur rarely. A preoperative risk profile including older age, history of heart failure and cognitive impairment was identified for a complicated postoperative course. However, the impact of an intense inflammatory response must not be neglected.

Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Circulating Inflammatory Biomarkers in Early Prediction of Stroke-Associated Infections.

(1) Background: Patients with acute ischaemic stroke (AIS) are at high risk for stroke-associated infections (SAIs). We hypothesised that increased concentrations of systemic inflammation markers predict SAIs and unfavourable outcomes; (2) Methods: In 223 patients with AIS, blood samples were taken at ≤24 h, 3 d and 7d after a stroke, to determine IL-6, IL-10, CRP and LBP. The outcome was assessed using the modified Rankin Scale at 90 d. Patients were thoroughly examined regarding the development of SAIs; (3) Results: 47 patients developed SAIs, including 15 lower respiratory tract infections (LRTIs). IL-6 and LBP at 24 h differed, between patients with and without SAIs (IL-6: p < 0.001; LBP: p = 0.042). However, these associations could not be confirmed after adjustment for age, white blood cell count, reduced consciousness and NIHSS. When considering the subgroup of LRTIs, in patients who presented early (≤12 h after stroke, n = 139), IL-6 was independently associated with LRTIs (OR: 1.073, 95% CI: 1.002−1.148). The ROC-analysis for prediction of LRTIs showed an AUC of 0.918 for the combination of IL-6 and clinical factors; (4) Conclusions: Blood biomarkers were not predictive for total SAIs. At early stages, IL-6 was independently associated with outcome-relevant LRTIs. Further studies need to clarify the use of biochemical markers to identify patients prone to SAIs.

Autoantibodies against NMDAR subunit NR1 disappear from blood upon anesthesia.

Anesthetics penetrate the blood-brain-barrier (BBB) and - as confirmed preclinically - transiently disrupt it. An analogous consequence in humans has remained unproven. In mice, we previously reported that upon BBB dysfunction, the brain acts as 'immunoprecipitator' of autoantibodies against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB). We thus hypothesized that during human anesthesia, pre-existing NMDAR1-AB will specifically bind to brain. Screening of N = 270 subjects undergoing general anesthesia during cardiac surgery for serum NMDAR1-AB revealed N = 25 NMDAR1-AB seropositives. Only N = 14 remained positive post-surgery. No changes in albumin, thyroglobulin or CRP were associated with reduction of serum NMDAR1-AB. Thus, upon anesthesia, BBB opening likely occurs also in humans.

Dysregulated Methylation Patterns in Exon IV of the Brain-Derived Neurotrophic Factor (BDNF) Gene in Nicotine Dependence and Changes in BDNF Plasma Levels During Smoking Cessation.

Introduction: Several studies reported dysregulated protein levels of brain-derived neurotrophic factor (BDNF) in smokers and during cessation. However, the epigenetic regulation of the BDNF gene has not yet been investigated. We measured the plasma levels of BDNF and the epigenetic regulation of exon IV of the BDNF gene in smokers compared to healthy controls over a cessation period of 14 days. Method: We measured BDNF plasma levels and BDNF promoter methylation in 49 smokers and 51 non-smokers at baseline, day 7, and day 14 of smoking cessation. Mean methylation levels of 11 Cytosine Guanosine dinucleotides of exon IV of the BDNF gene were determined via bisulfite sequencing. Results: BDNF plasma and methylation levels were significantly lower in healthy controls when compared with smokers across all time points. BDNF levels for smokers decreased significantly during the cessation period. Comparing the sexes, female smokers showed significantly lower plasma BDNF levels than healthy controls at baseline and over 14 days of cessation. Male and female smokers showed significantly higher mean methylation rates than non-smokers at baseline. In male smokers, mean methylation levels decreased significantly during the cessation period. Conclusion: Our findings replicate the findings of previous studies that BDNF plasma levels are altered in smokers. Furthermore, BDNF expression and gene methylation are altered during the first 14 days of cessation. Our novel findings of dysregulated methylation patterns in exon IV of the BDNF gene further support the thesis that BDNF plays a role in nicotine dependence.

Alteration of serum leptin and LEP/LEPR promoter methylation in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder based on a loss of paternally expressed but maternally imprinted genes in chromosome region 15q11-13. PWS individuals typically show insatiable appetite with subsequent obesity representing the major mortality factor unless food intake is inhibited. The neurobiological basis of PWS-typical hyperphagia has remained poorly understood. Many PWS-typical abnormalities are based on hypothalamic dysregulation, a region in which hunger and satiety are hormonally regulated, with the hormone leptin being a main long-term regulator of satiety. Previous studies in PWS have inconsistently shown leptin alterations solely in early childhood, without investigating the leptin system on an epigenetic level. The present study investigates serum leptin levels (S-leptin) and DNA methylation of the leptin (LEP) and leptin receptor gene (LEPR) promoter in 24 individuals with PWS compared to 13 healthy controls matched for sex, age, and body mass index (BMI) and relates the results to the extent of hyperphagia in PWS. S-Leptin levels were obtained by Enzyme-linked Immunosorbent Assay. LEP/LEPR-promoter DNA methylation was assessed by bisulfite-sequencing, hyperphagia by Hyperphagia Questionnaire for Clinical Trials (HQ-CT). PWS and control groups differed significantly in S-leptin levels with higher S-leptin in PWS. Methylation analysis showed significant differences in mean promoter methylation rate both for LEP and LEPR with a lower methylation rate in PWS. LEPR, but not LEP methylation correlated significantly with S-leptin levels. S-leptin and both LEP and LEPR methylation did not correlate with HQ-CT scores in PWS. The present study is the first to show significantly elevated S-leptin levels in an adult PWS cohort combined with an altered, downregulated LEP and LEPR promoter methylation status compared to sex-, age- and BMI-matched controls. Analogous to previous studies, no link to the behavioral dimension could be drawn. Overall, the results suggest an increased leptin dysregulation in PWS, whereby the findings partly mirror those seen in non-syndromic obesity.

Endoscopic Bariatric Treatment with Duodenal-Jejunal Bypass Liner Improves Non-invasive Markers of Non-alcoholic Steatohepatitis.

PURPOSE: People with obesity often develop non-alcoholic fatty liver disease (NAFLD) and are at high risk of progression to non-alcoholic steatohepatitis (NASH). Few therapies are effective other than bariatric surgery. We therefore analyzed data from duodenal-jejunal bypass liner (DJBL) patients regarding steatosis, fibrosis, and NASH. METHODS: Consecutive DJBL patients with type 2 diabetes underwent standardized assessments up to device removal at 48 weeks. These included aspartate and alanine transaminase (AST, ALT), controlled attenuation parameter (CAP, for steatosis), and liver stiffness measurement (LSM, for fibrosis). The NAFLD fibrosis score (NFS), fibrosis-4 score (FIB4), and enhanced liver fibrosis (ELF) test were also used to assess fibrosis and the Fibroscan-AST (FAST) score to assess NASH. Mixed models were used and missing data were accounted for with multiple imputation. RESULTS: Thirty-two patients (18 female, mean age 55.1, mean BMI 40.2 kg/m2) were included. After 48 weeks, the change compared to baseline with 95% CI was a factor 0.74 (0.65 to 0.84) for AST, 0.63 (0.53 to 0.75) for ALT, and a difference of - 0.21 (- 0.28 to - 0.13) for FAST, all with p < 0.001. Fibrosis based on LSM, NFS, and ELF did not change whereas FIB4 exhibited slight improvement. Eight DJBL were explanted early due to device-related complications and eight complications led to hospitalization. CONCLUSIONS: One year of DJBL therapy is associated with relevant improvements in non-invasive markers of steatosis and NASH, but not fibrosis, and is accompanied by a substantial number of complications. Given the lack of alternatives, DJBL deserves further attention.

Impact of fasting on stress systems and depressive symptoms in patients with major depressive disorder: a cross-sectional study.

Major depressive disorder (MDD) is frequently associated with poor response to treatment. Common antidepressants target neurotransmission and neuronal plasticity, which require adequate energy supply. As imaging studies indicate disturbances in central energy metabolism, and caloric restriction improves neuroplasticity and impacts mood and cognition, correction of energy status might increase the effectiveness of antidepressant treatments and reduce the psychopathological symptoms of depression. Metabolic parameters, stress hormones, and brain-derived neurotrophic factor (BDNF) levels were assessed in serum of depressed inpatients (MDD, N = 21) and healthy volunteers (Ctrl, N = 28) before and after a 72 h fasting period during which only water was consumed. Depression severity was assessed by Beck's Depression Inventory (BDI)-2 sum-score and cognitive-affective and somatic sub-scores. Fasting similarly impacted metabolic parameters and stress systems in both groups. Fasting elevated BDI-2 sum-scores and somatic sub-scores in Ctrl. In MDD, fasting increased somatic-, but decreased cognitive-affective symptoms. Sub-group analyses based on BDI-2 sum-scores pre-fasting showed that cognitive-affective symptoms decreased in patients with moderate/severe but not in those with mild symptoms. This was associated with differential changes in BDNF levels. In conclusion, fasting improved cognitive-affective sub-scores in MDD patients with moderate/severe symptoms that had not responded to prior therapy. Interventions that modulate energy metabolism might directly improve cognitive-affective symptoms and/or augment therapeutic efficacy in moderate-to-severely depressed patients.

Insights Into Immunothrombotic Mechanisms in Acute Stroke due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

Safety and efficacy of prophylactic and therapeutic vaccine based on live-attenuated Listeria monocytogenes in hepatobiliary cancers.

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies.