miR-Blood - a small RNA atlas of human blood components.

miR-Blood is a high-quality, small RNA expression atlas for the major components of human peripheral blood (plasma, erythrocytes, thrombocytes, monocytes, neutrophils, eosinophils, basophils, natural killer cells, CD4+ T cells, CD8+ T cells, and B cells). Based on the purified blood components from 52 individuals, the dataset provides a comprehensive repository for the expression of 4971 small RNAs from eight non-coding RNA classes.

Evaluation of the Novel Sepsis Biomarker Host-Derived Delta-like Canonical Notch Ligand 1-A Secondary Analysis of 405 Patients Suffering from Inflammatory or Infectious Diseases.

Sepsis is defined as organ failure caused by dysregulated host response to infection. While early antibiotic treatment in patients with acute infection is essential, treating non-infectious patients must be avoided. Current guidelines recommend procalcitonin (PCT) to guide discontinuation of antibiotic treatment. For initiation of therapy, there is currently no recommended biomarker. In this study, we evaluated Host-Derived Delta-like Canonical Notch Ligand 1 (DLL1), a monocyte membrane ligand that has shown promising results in differentiating infectious from non-infectious critically ill patients. Soluble DLL1 levels were measured in plasma samples of six different cohorts. The six cohorts comprise two cohorts with non-infectious inflammatory auto-immune diseases (Hidradenitis Suppurativa, Inflammatory Bowel Disease), one cohort of bacterial skin infection, and three cohorts of suspected systemic infection or sepsis. In total, soluble DLL1 plasma levels of 405 patients were analyzed. Patients were divided into three groups: inflammatory disease, infection, and sepsis (defined according to the Sepsis-3 definition), followed by the evaluation of its diagnostic performance via Area Under the Receiver Operating Characteristics (AUROC) analyses. Patients of the sepsis group showed significantly elevated plasma DLL1 levels compared to patients with uncomplicated infections and sterile inflammation. However, patients with infections had significantly higher DLL1 levels than patients with inflammatory diseases. Diagnostic performance was evaluated and showed better performance for DLL1 for the recognition of sepsis (AUC: 0.823; CI 0.731-0.914) than C-reactive protein (AUC 0.758; CI 0.658-0.857), PCT (AUC 0.593; CI 0.474-0.711) and White Blood Cell count (AUC 0.577; CI 0.46-0.694). DLL1 demonstrated promising results for diagnosing sepsis and was able to differentiate sepsis from other infectious and inflammatory diseases.

suPAR links a dysregulated immune response to tissue inflammation and sepsis-induced acute kidney injury.

Acute kidney injury (AKI) secondary to sepsis results in poor outcomes and conventional kidney function indicators lack diagnostic value. Soluble urokinase plasminogen activator receptor (suPAR) is an innate immune-derived molecule implicated in inflammatory organ damage. We characterized the diagnostic ability of longitudinal serum suPAR levels to discriminate severity and course of sepsis-induced AKI (SI-AKI) in 200 critically ill patients meeting Sepsis-3 criteria. The pathophysiologic relevance of varying suPAR levels in SI-AKI was explored in a polymicrobial sepsis model in WT, (s)uPAR-knockout, and transgenic suPAR-overexpressing mice. At all time points studied, suPAR provided a robust classification of SI-AKI disease severity, with improved prediction of renal replacement therapy (RRT) and mortality compared with established kidney biomarkers. Patients with suPAR levels of greater than 12.7 ng/mL were at highest risk for RRT or death, with an adjusted odds ratio of 7.48 (95% CI, 3.00-18.63). suPAR deficiency protected mice against SI-AKI. suPAR-overexpressing mice exhibited greater kidney damage and poorer survival through inflamed kidneys, accompanied by local upregulation of potent chemoattractants and pronounced kidney T cell infiltration. Hence, suPAR allows for an innate immune-derived and kidney function-independent staging of SI-AKI and offers improved longitudinal risk stratification. suPAR promotes T cell-based kidney inflammation, while suPAR deficiency improves SI-AKI.

Interleukin-3 protects against viral pneumonia in sepsis by enhancing plasmacytoid dendritic cell recruitment into the lungs and T cell priming.

Rationale: Sepsis, a global health burden, is often complicated by viral infections leading to increased long-term morbidity and mortality. Interleukin-3 (IL-3) has been identified as an important mediator amplifying acute inflammation in sepsis; however, its function in the host response to viral infections during sepsis remains elusive. Objectives: To investigate the role of IL-3 during viral pneumonia in sepsis. Methods: We included septic patients from two different cohorts and used in vitro and in vivo assays. The obtained data were substantiated using a second model (SARS-CoV-2 infections). Measurements and main results: Low plasma IL-3 levels were associated with increased herpes simplex virus (HSV) airway infections in septic patients, resulting in reduced overall survival. Likewise, Il-3-deficient septic mice were more susceptible to pulmonary HSV-1 infection and exhibited higher pulmonary inflammation than control mice. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating plasmacytoid dendritic cells (pDCs) into the airways and by enhancing pDC-mediated T cell activation upon viral stimulation. Interestingly, the ability of IL-3 to improve adaptive immunity was confirmed in patients with SARS-CoV-2 infections. Conclusion: Our study identifies IL-3 as a predictive disease marker for viral reactivation in sepsis and reveals that IL-3 improves antiviral immunity by enhancing the recruitment and the function of pDCs.

A 29-MRNA HOST RESPONSE WHOLE-BLOOD SIGNATURE IMPROVES PREDICTION OF 28-DAY MORTALITY AND 7-DAY INTENSIVE CARE UNIT CARE IN ADULTS PRESENTING TO THE EMERGENCY DEPARTMENT WITH SUSPECTED ACUTE INFECTION AND/OR SEPSIS.

ABSTRACT: Background: Risk stratification of emergency department patients with suspected acute infections and/or suspected sepsis remains challenging. We prospectively validated a 29-messenger RNA host response classifier for predicting severity in these patients. Methods: We enrolled adults presenting with suspected acute infections and at least one vital sign abnormality to six emergency departments in Greece. Twenty-nine target host RNAs were quantified on NanoString nCounter and analyzed with the Inflammatix Severity 2 (IMX-SEV-2) classifier to determine risk scores as low, moderate, and high severity. Performance of IMX-SEV-2 for prediction of 28-day mortality was compared with that of lactate, procalcitonin, and quick sequential organ failure assessment (qSOFA). Results: A total of 397 individuals were enrolled; 38 individuals (9.6%) died within 28 days. Inflammatix Severity 2 classifier predicted 28-day mortality with an area under the receiver operator characteristics curve of 0.82 (95% confidence interval [CI], 0.74-0.90) compared with lactate, 0.66 (95% CI, 0.54-0.77); procalcitonin, 0.67 (95% CI, 0.57-0.78); and qSOFA, 0.81 (95% CI, 0.72-0.89). Combining qSOFA with IMX-SEV-2 improved prognostic accuracy from 0.81 to 0.89 (95% CI, 0.82-0.96). The high-severity (rule-in) interpretation band of IMX-SEV-2 demonstrated 96.9% specificity for predicting 28-day mortality, whereas the low-severity (rule-out) band had a sensitivity of 78.9%. Similarly, IMX-SEV-2 alone accurately predicted the need for day-7 intensive care unit care and further boosted overall accuracy when combined with qSOFA. Conclusions: Inflammatix Severity 2 classifier predicted 28-day mortality and 7-day intensive care unit care with high accuracy and boosted the accuracy of clinical scores when used in combination.

Delta-like canonical Notch ligand 1 is predictive for sepsis and acute kidney injury in surgical intensive care patients.

The early identification of sepsis in surgical intensive care patients is challenging due to the physiological postoperative alterations of vital signs and inflammatory biomarkers. Soluble Delta-like protein 1 (sDLL1) may be a potential discriminatory biomarker for this purpose. For this reason, this study aimed to evaluate sDLL1 for the identification of sepsis in a cohort of surgical intensive care patients. This study comprises a secondary analysis of a prospective observational study including 80 consecutive patients. The study groups included 20 septic shock patients, 20 patients each undergoing major abdominal surgery (MAS) and cardiac artery bypass surgery (CABG), and 20 matched control subjects (CTRL). The surveillance period was 72 h. The plasma concentration of sDLL1 was measured with ELISA. The plasma levels of sDLL1 were significantly elevated in septic patients compared to both surgical cohorts (septic vs. all postoperative time points, data are shown as median and interquartile range [IQR]; septic shock: 17,363 [12,053-27,299] ng/mL, CABG 10,904 [8692-16,250] ng/mL; MAS 6485 [4615-9068] ng/mL; CTRL 5751 [3743-7109] ng/mL; septic shock vs. CABG: p < 0.001; septic shock vs. MAS: p < 0.001). ROC analysis showed a sufficient prediction of sepsis with limited specificity (AUCROC 0.82 [0.75-0.82], sensitivity 84%, specificity 68%). The plasma levels of sDLL correlated closely with renal parameters (creatinine: correlation coefficient = 0.60, r2 = 0.37, p < 0.0001; urea: correlation coefficient = 0.52, r2 = 0.26, p < 0.0001), resulting in a good predictive performance of sDLL1 for the identification of acute kidney injury (AKI; AUCROC 0.9 [0.82-0.9], sensitivity 83%, specificity 91%). By quantifying the plasma concentration of sDLL1, sepsis can be discriminated from the physiological postsurgical inflammatory response in abdominal and cardiac surgical patients. However, sDLL1 has only limited specificity for the detection of sepsis in cardiac surgical patients, which may be explained by impaired renal function. Based on these findings, this study identifies the predictive value of sDLL1 for the detection of AKI, making it a potential biomarker for surgical intensive care patients.Trial registration DRKS00013584, Internet Portal of the German Clinical Trials Register (DRKS), registration date 11.07.2018, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013584 .

Monocyte Metabolism and Function in Patients Undergoing Cardiac Surgery.

Objective: Cardiopulmonary bypass (CPB) can lead to systemic inflammation, which is associated with higher morbidity. Therefore, we investigated the metabolism of isolated blood monocytes before and after CPB compared to healthy controls. Methods: In this prospective, monocentric, observational study, we included 30 patients undergoing CPB and 20 controls. We isolated monocytes from heparinized blood and investigated their metabolism by using Seahorse technology before (t0), 4 h (t4), and 24 h (t24) after the start of the CPB. We also examined programmed cell death 1 ligand (PD-L1), PD-L2, V-domain Ig suppressor of T cell activation (VISTA), and human leukocyte antigen-DR isotype (HLA-DR) using fluorescence-activated cell sorting analysis. Additionally, we investigated plasma cytokine levels in patients without and after ex vivo stimulation. Results: CPB-induced inflammatory responses are shown by significantly elevated plasma interleukin-6 levels in the CPB group compared to baseline and controls [t0: 0 ng/ml (95%CI 0-0 ng/ml); t4: 0.16 ng/ml (95%CI 0.1-0.197 ng/ml), p < 0.0001; t24: 0.11 ng/ml (95% CI 0.1-0.16 ng/ml), p < 0.0001, and controls: 0 ng/ml (95% CI 0-0 ng/ml)]. The cytokine release in the ex vivo stimulation is reduced for lipopolysaccharide stimulation at t4 [t0: 35.68 ng/ml (95% CI 22.17-46.57 ng/ml) vs. t4: 15.02 (95% CI 10.25-24.78 ng/ml), p < 0.0001]. Intracellular metabolism of monocytes after CPB showed a protracted shift to aerobic glycolysis [t0: 179.2 pmol/min (95% CI 138.0-205.1 pmol/min) vs. t24: 250.1 pmol/min (95% CI 94.8-300.2 pmol/min), p < 0.0001]. Additionally, we observed an altered metabolism in monocytes in patients undergoing cardiac surgery compared to controls even before any surgical procedure [t0: 179.2 pmol/min (95% CI 138.0-205.1) vs. controls 97.4 (95% CI 59.13-144.6 pmol/min), p = 0.0031]. Conclusion: After CPB, patients' monocytes show a shift in metabolism from oxidative phosphorylation to aerobic glycolysis, which is associated with energy-demanding and proinflammatory processes. This is the first study to show changes in monocyte immunometabolism in cardiac surgery. Monocytes of patients undergoing cardiac surgery were leaning toward aerobic glycolysis even before any surgical procedure was conducted. Leaving the question of the pathophysiological mechanisms for future studies to be investigated and paving the way for potential therapy approaches preventing inflammatory effects of CPB.

Concurrent Change in Serum Cholinesterase Activity and Midregional-Proadrennomedullin Level Could Predict Patient Outcome following Liver Transplantation.

BACKGROUND: After liver transplantation (LTX), patients are susceptible to opportunistic infections resulting in reduced outcomes within the early post-transplantation period. The postoperative monitoring of LTX patients has gained much importance in recent years. However, reliable plasmatic markers predicting 90-day outcomes are still lacking. METHODS: In the post hoc analysis of a prospective, observational study, butyrylcholinesterase (BChE), mid-regional proadrenomedullin (MR-proADM), as well as conventional inflammatory markers (procalcitonin, C-reactive protein) were evaluated in 93 patients at seven consecutive timepoints within the first 28 days following LTX. RESULTS: Persistently reduced activity of BChE and elevated MR-proADM levels indicated reduced 90-day survival following LTX. Furthermore, reduced BChE and increased MR-proADM activity could indicate early post-transplantation bacterial infections, whereas conventional inflammatory biomarkers showed no diagnostic efficacy within the observation period. CONCLUSION: Concurrent assessment of BChE and MR-proADM activity might serve as a bedside diagnostic tool for early bacterial infections following liver transplantation. Thus, a combined utilization of the two biomarkers may be a useful tool in the risk evaluation of patients following liver transplantation.

A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy.

Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37-4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

Early Use of Methylene Blue in Vasoplegic Syndrome: A 10-Year Propensity Score-Matched Cohort Study.

BACKGROUND: Vasoplegic syndrome is associated with increased morbidity and mortality in patients undergoing cardiac surgery. This retrospective, single-center study aimed to evaluate the effect of early use of methylene blue (MB) on hemodynamics after an intraoperative diagnosis of vasoplegic syndrome (VS). METHODS: Over a 10-year period, all patients diagnosed with intraoperative VS (hypotension despite treatment with norepinephrine ≥0.3 µg/kg/min and vasopressin ≥1 IE/h) while undergoing heart surgery and cardiopulmonary bypass were identified, and their data were examined. The intervention group received MB (2 mg/kg intravenous) within 15 min after the diagnosis of vasoplegia, while the control group received standard therapy. The two groups were matched using propensity scores. RESULTS: Of the 1022 patients identified with VS, 221 received MB intraoperatively, and among them, 60 patients received MB within 15 min after the diagnosis of VS. After early MB application, mean arterial pressure was significantly higher, and vasopressor support was significantly lower within the first hour (p = 0.015) after the diagnosis of vasoplegia, resulting in a lower cumulative amount of norepinephrine (p = 0.018) and vasopressin (p = 0.003). The intraoperative need of fresh frozen plasma in the intervention group was lower compared to the control group (p = 0.015). Additionally, the intervention group had higher creatinine values in the first three postoperative days (p = 0.036) without changes in dialysis incidence. The 90-day survival did not differ significantly (p = 0.270). CONCLUSION: Our results indicate the additive effects of MB use during VS compared to standard vasopressor therapy only. Early MB administration for VS may significantly improve the patients' hemodynamics with minor side effects.

[Sepsis in out-of-hospital emergency medicine]. / Sepsis in der prähospitalen Notfallmedizin: Was können prähospital Tätige aus der Surviving Sepsis Campaign und S3-Leitlinie ziehen?

Background: Sepsis is a challenge in emergency medicine, as this life-threatening organ dysfunction, caused by a dysregulated host response to an infection, presents manifold and therefore is often recognized too late. Objectives: Recently published surviving sepsis campaign and German S3 guidelines provide recommendations for diagnosis and therapy of sepsis in an in-hospital or intensive care setting, but do not particularly address out-of-hospital emergency medical care. We aim to work out the evidence base with regard to the out-of-hospital care of patients with suspected sepsis and to derive treatment recommendations for emergency medical services. Conclusions: Therapy of sepsis and septic shock is summarized in bundles, whereby the first bundle should ideally be completed within the first hour-in analogy to "golden hour" concepts in other emergency medical entities, such as trauma care. In the out-of-hospital setting, therapy focuses on securing vital parameters, according to the ABCDE scheme, with a particular focus on volume therapy. Further procedures within the 1 h bundle, such as lactate measurement, obtaining microbiological samples, and starting an anti-infective therapy, are broadly available in hospital only. The aim is to control the site of infection as soon as possible. Therefore, an appropriate designated hospital should be chosen carefully and informed in advance, in order to initiate and pave the way for further clinical diagnostic and treatment paths. Moreover, structured and target-oriented handovers, as well as regular training, are required.

Gαq modulates the energy metabolism of osteoclasts.

Introduction: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs. Methods: The proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function. Results: PMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid. Discussion: Our study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.

Hyperspectral Imaging for the Evaluation of Microcirculatory Tissue Oxygenation and Perfusion Quality in Haemorrhagic Shock: A Porcine Study.

BACKGROUND: The ultimate goal of haemodynamic therapy is to improve microcirculatory tissue and organ perfusion. Hyperspectral imaging (HSI) has the potential to enable noninvasive microcirculatory monitoring at bedside. METHODS: HSI (Tivita® Tissue System) measurements of tissue oxygenation, haemoglobin, and water content in the skin (ear) and kidney were evaluated in a double-hit porcine model of major abdominal surgery and haemorrhagic shock. Animals of the control group (n = 7) did not receive any resuscitation regime. The interventional groups were treated exclusively with either crystalloid (n = 8) or continuous norepinephrine infusion (n = 7). RESULTS: Haemorrhagic shock led to a drop in tissue oxygenation parameters in all groups. These correlated with established indirect markers of tissue oxygenation. Fluid therapy restored tissue oxygenation parameters. Skin and kidney measurements correlated well. High dose norepinephrine therapy deteriorated tissue oxygenation. Tissue water content increased both in the skin and the kidney in response to fluid therapy. CONCLUSIONS: HSI detected dynamic changes in tissue oxygenation and perfusion quality during shock and was able to indicate resuscitation effectivity. The observed correlation between HSI skin and kidney measurements may offer an estimation of organ oxygenation impairment from skin monitoring. HSI microcirculatory monitoring could open up new opportunities for the guidance of haemodynamic management.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Hyperspectral imaging for perioperative monitoring of microcirculatory tissue oxygenation and tissue water content in pancreatic surgery - an observational clinical pilot study.

BACKGROUND: Hyperspectral imaging (HSI) could provide extended haemodynamic monitoring of perioperative tissue oxygenation and tissue water content to visualize effects of haemodynamic therapy and surgical trauma. The objective of this study was to assess the capacity of HSI to monitor skin microcirculation and possible relations to perioperative organ dysfunction in patients undergoing pancreatic surgery. METHODS: The hyperspectral imaging TIVITA® Tissue System was used to evaluate superficial tissue oxygenation (StO2), deeper layer tissue oxygenation (near-infrared perfusion index (NPI)), haemoglobin distribution (tissue haemoglobin index (THI)) and tissue water content (tissue water index (TWI)) in 25 patients undergoing pancreatic surgery. HSI parameters were measured before induction of anaesthesia (t1), after induction of anaesthesia (t2), postoperatively before anaesthesia emergence (t3), 6 h after emergence of anaesthesia (t4) and three times daily (08:00, 14:00, 20:00 ± 1 h) at the palm and the fingertips until the second postoperative day (t5-t10). Primary outcome was the correlation of HSI with perioperative organ dysfunction assessed with the perioperative change of SOFA score. RESULTS: Two hundred and fifty HSI measurements were performed in 25 patients. Anaesthetic induction led to a significant increase of tissue oxygenation parameters StO2 and NPI (t1-t2). StO2 and NPI decreased significantly from t2 until the end of surgery (t3). THI of the palm showed a strong correlation with haemoglobin levels preoperatively (t2: r = 0.83, p < 0.001) and 6 h postoperatively (t4: r = 0.71, p = 0.001) but not before anaesthesia emergence (t3: r = 0.35, p = 0.10). TWI of the palm and the fingertip rose significantly between pre- and postoperative measurements (t2-t3). Higher blood loss, syndecan level and duration of surgery were associated with a higher increase of TWI. The perioperative change of HSI parameters (∆t1-t3) did not correlate with the perioperative change of the SOFA score. CONCLUSION: This is the first study using HSI skin measurements to visualize tissue oxygenation and tissue water content in patients undergoing pancreatic surgery. HSI was able to measure short-term changes of tissue oxygenation during anaesthetic induction and pre- to postoperatively. TWI indicated a perioperative increase of tissue water content. Perioperative use of HSI could be a useful extension of haemodynamic monitoring to assess the microcirculatory response during haemodynamic therapy and major surgery. TRIAL REGISTRATION: German Clinical Trial Register, DRKS00017313 on 5 June 2019.

Antiproliferative effect of GTS-21 in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant brain tumour in adults. The poor prognosis and short median overall survival of patients with GBM is associated with resistance to therapy after surgical and adjuvant treatment. The expression of various acetylcholine receptors (AChR) in GBM has been widely reported. The present study aimed to investigate the expression of cholinergic system-related genes in primary GBM and to explore the antiproliferative effect of 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) in GBM cell lines. Therefore, the expression of 28 genes associated with the cholinergic system was detected using a customized RT2 Profiler PCR Array in 44 GBM and 5 healthy control brain tissue samples. In addition, the activity of GTS-21, an alpha 7 subunit nicotinic AChR (α7 nAChR) agonist, and that of α-bungarotoxin (α-BTX), an α7 nAChR antagonist, was determined in primary and established GBM cells. Therefore, the A172, U87 and G28 cell lines and primary GBM cells were treated with GTS-21, ACh or nicotine. Cell viability was evaluated using MTT assay at 24, 48 and 72 h following cell treatment with the corresponding compounds. The results revealed that the expression of cholinergic system-related components was notably downregulated, except that of cholinergic receptor nicotinic alpha 7 subunit (CHRNA7), in primary GBM and U87 cells. However, the dominant-negative duplicate form of CHRNA7 was also downregulated. Furthermore, A172 and G28 cells exhibited a heterogeneous gene expression pattern. Additionally, GTS-21 inhibited the proliferation of GBM cells in a dose- and time-dependent manner. Interestingly, treatment with α-BTX restored the proliferation of U87 cells, but not that of A172 and G28 cells. Collectively, the findings of the present study suggested that GTS-21 may inhibit the proliferation of GBM cells and may therefore serve as a novel therapeutic approach to the treatment of GBM, which warrants further investigation.

Low-Density Granulocyte Contamination From Peripheral Blood Mononuclear Cells of Patients With Sepsis and How to Remove It - A Technical Report.

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15+ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15+ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

The Optimization and Biological Significance of a 29-Host-Immune-mRNA Panel for the Diagnosis of Acute Infections and Sepsis.

In response to the unmet need for timely accurate diagnosis and prognosis of acute infections and sepsis, host-immune-response-based tests are being developed to help clinicians make more informed decisions including prescribing antimicrobials, ordering additional diagnostics, and assigning level of care. One such test (InSep™, Inflammatix, Inc.) uses a 29-mRNA panel to determine the likelihood of bacterial infection, the separate likelihood of viral infection, and the risk of physiologic decompensation (severity of illness). The test, being implemented in a rapid point-of-care platform with a turnaround time of 30 min, enables accurate and rapid diagnostic use at the point of impact. In this report, we provide details on how the 29-biomarker signature was chosen and optimized, together with its molecular, immunological, and medical significance to better understand the pathophysiological relevance of altered gene expression in disease. We synthesize key results obtained from gene-level functional annotations, geneset-level enrichment analysis, pathway-level analysis, and gene-network-level upstream regulator analysis. Emerging findings are summarized as hallmarks on immune cell interaction, inflammatory mediators, cellular metabolism and homeostasis, immune receptors, intracellular signaling and antiviral response; and converging themes on neutrophil degranulation and activation involved in immune response, interferon, and other signaling pathways.

The COVID-19 puzzle: deciphering pathophysiology and phenotypes of a new disease entity.

The zoonotic SARS-CoV-2 virus that causes COVID-19 continues to spread worldwide, with devastating consequences. While the medical community has gained insight into the epidemiology of COVID-19, important questions remain about the clinical complexities and underlying mechanisms of disease phenotypes. Severe COVID-19 most commonly involves respiratory manifestations, although other systems are also affected, and acute disease is often followed by protracted complications. Such complex manifestations suggest that SARS-CoV-2 dysregulates the host response, triggering wide-ranging immuno-inflammatory, thrombotic, and parenchymal derangements. We review the intricacies of COVID-19 pathophysiology, its various phenotypes, and the anti-SARS-CoV-2 host response at the humoral and cellular levels. Some similarities exist between COVID-19 and respiratory failure of other origins, but evidence for many distinctive mechanistic features indicates that COVID-19 constitutes a new disease entity, with emerging data suggesting involvement of an endotheliopathy-centred pathophysiology. Further research, combining basic and clinical studies, is needed to advance understanding of pathophysiological mechanisms and to characterise immuno-inflammatory derangements across the range of phenotypes to enable optimum care for patients with COVID-19.

Perioperative changes in the plasma metabolome of patients receiving general anesthesia for pancreatic cancer surgery.

BACKGROUND: Modern anesthesia strives to offer personalized concepts to meet the patient's individual needs in sight of clinical outcome. Still, little is known about the impact of anesthesia on the plasma metabolome, although many metabolites have been shown to modulate the function of various immune cells, making it particularly interesting in the context of oncological surgery. In this study longitudinal dynamics in the plasma metabolome during general anesthesia in patients undergoing pancreatic surgery were analyzed. MATERIALS AND METHODS: Prospective, observational study with 10 patients diagnosed with pancreatic (pre-) malignancy and subjected to elective resection surgery under general anesthesia. Plasma metabolites (n = 630) were quantified at eight consecutive perioperative timepoints using mass spectrometry-based targeted metabolomics. RESULTS: 39 metabolites significantly changed during the perioperative period. Tryptophan concentrations decreased by 45% with the maximum decrease after anesthesia induction (p = 6.24E-07), while taurine synthesis increased (p = 1.46E-04). Triacylglycerides and lysophosphatidylcholines were significantly reduced with increased liberation of free monounsaturated fatty acids (p = 0.03). Carnitine levels decreased significantly (p = 9.30E-04). CONCLUSIONS: The major finding of this study was perioperative tryptophan depletion and increased taurine synthesis. Both are essential for immune cell function and are therefore of significant interest for perioperative management. Further studies are needed to identify influencing anesthetic factors.