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Background: Postoperative immunosuppression has been recognized as an important driver of surgery-related morbidity and mortality. It is characterized by lymphocyte depression and impaired monocyte capability to present foreign antigens to T-cells via Major Histocompatibility Complex, Class II (MHC-II) molecules. In patients with postoperative abdominal sepsis, we previously detected a persisting differential binding of the CCCTC-Binding Factor (CTCF), a superordinate regulator of transcription, inside the MHC-II region with specific impact on human leucocyte antigen (HLA) gene expression. In this prospective exploratory study, we investigated to which extent major surgery affects the MHC-II region of circulating CD14+-monocytes., Results: In non-immunocompromised patients undergoing elective major abdominal surgery, a postoperative loss of monocyte HLA-DR surface receptor density was accompanied by a decline in the transcription levels of the classical MHC-II genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The surgical event decreased the expression of the transcriptional MHC-II regulators CIITA and CTCF and led to a lower CTCF enrichment at an intergenic sequence within the HLA-DR subregion. During the observation period, we found a slow and only incomplete restoration of monocyte HLA-DR surface receptor density as well as a partial recovery of CIITA, HLA-DRA and HLA-DRB1 expression. In contrast, transcription of HLA-DPA1, HLA-DPB1, CTCF and binding of CTCF within the MHC-II remained altered., Conclusion: In circulating monocytes, major surgery does not globally affect MHC-II transcription but rather induces specific changes in the expression of selected HLA genes, followed by differential recovery patterns and accompanied by a prolonged reduction of CTCF expression and binding within the MHC-II region. Our results hint toward a long-lasting impact of a major surgical intervention on monocyte functionality, possibly mediated by epigenetic changes that endure the life span of the individual cell., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Siegler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Hypercoagulability and thrombo-inflammation are the main reasons for death in COVID-19 patients. It is unclear whether there is a difference between D-dimer levels in patients without or with COVID-19 acute respiratory distress syndrome (ARDS)., Methods: We searched PubMed, EMBASE, and ClinicalTrails.gov databases looking for studies reporting D-dimer levels in patients without or with COVID-19 ARDS. Secondary endpoints included length of hospital stay, and mortality data at the longest follow-up available., Results: We included 12 retrospective and 3 prospective studies with overall 2,828 patients, of whom 1,404 (49.6%) had non-COVID-19 ARDS and 1,424 had COVID-19 ARDS. D-dimer levels were not significantly higher in non-COVID-19 ARDS than in COVID-19 ARDS patients (mean 7.65 mg/L vs. mean 6.20 mg/L MD 0.88 [CI: -0.61 to 2.38] p = 0.25; I² = 85%) while the length of hospital stay was shorter (non-COVID-19 mean 37.4 days vs. COVID-19 mean 48.5 days, MD -10.92 [CI: -16.71 to -5.14] p < 0.001; I² = 44%). No difference in mortality was observed: non-COVID-19 ARDS 418/1167 (35.8%) vs. COVID-19 ARDS 467/1201 (38.8%)., Conclusions: We found no difference in the mean D-dimer levels between non-COVID-19 ARDS and COVID-19 ARDS patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tóth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The COVID-19 pandemic has exerted great pressure on national health systems, which have aimed to ensure comprehensive healthcare at all times. Healthcare professionals working with COVID-19 patients are on the frontline and thereby confronted with enormous demands. Although early reports exist on the psychological impact of the pandemic on frontline medical staff working in Asia, little is known about its impact on healthcare professionals in other countries and across various work sectors. The present cross-sectional, online survey sought to investigate common work stressors among healthcare professionals, their psychological stress as well as coping resources during the pandemic., Methods: A sample of 575 healthcare professionals (57% male) in three different sectors (hospital, prehospital emergency care, and outpatient service) reported their experiences concerning work and private stressors, psychological stress, and coping strategies between April 17, 2020 and June 5, 2020. To capture pandemic-specific answers, most of the items were adapted or newly developed. Exploratory factor analyses (EFA) were conducted to detect underlying latent factors relating to COVID-specific work stressors. In a next step, the effects of these latent stressors across various work sectors on psychological stress (perceived stress, fatigue, and mood) were examined by means of structural equation models (SEM). To add lived experience to the findings, responses to open-ended questions about healthcare professionals' stressors, effective crisis measures and prevention, and individual coping strategies were coded inductively, and emergent themes were identified., Results: The EFA revealed that the examined work stressors can be grouped into four latent factors: "fear of transmission", "interference of workload with private life", "uncertainty/lack of knowledge", and "concerns about the team". The SEM results showed that "interference of workload with private life" represented the pivotal predictor of psychological stress. "Concerns about the team" had stress-reducing effects. The latent stressors had an equal effect on psychological stress across work sectors. On average, psychological stress levels were moderate, yet differed significantly between sectors (all p < .001); the outpatient group experienced reduced calmness and more stress than the other two sectors, while the prehospital group reported lower fatigue than the other two sectors. The prehospital group reported significantly higher concerns about the team than the hospital group (p < .001). In their reports, healthcare professionals highlighted regulations such as social distancing and the use of compulsory masks, training, experience and knowledge exchange, and social support as effective coping strategies during the pandemic. The hospital group mainly mentioned organizational measures such as visiting bans as effective crisis measures, whereas the prehospital sector most frequently named governmental measures such as contact restrictions., Conclusion: The study demonstrated the need for sector-specific crisis measures to effectively address the specific work stressors faced by the outpatient sector in particular. The results on pandemic-specific work stressors reveal that healthcare professionals might benefit from coping strategies that facilitate the utilization of social support. At the workplace, team commitment and knowledge exchange might buffer against adverse psychological stress responses. Schedules during pandemics should give healthcare workers the opportunity to interact with families and friends in ways that facilitate social support outside work. Future studies should investigate cross-sector stressors using a longitudinal design to identify both sector- and time-specific measures. Ultimately, an international comparison of stressors and measures in different sectors of healthcare systems is desirable., Competing Interests: The authors have declared that no competing interests exist.

Background: COVID-19 has been reported in over 40million people globally with variable clinical outcomes. In this systematic review and meta-analysis, we assessed demographic, laboratory and clinical indicators as predictors for severe courses of COVID-19., Methods: This systematic review was registered at PROSPERO under CRD42020177154. We systematically searched multiple databases (PubMed, Web of Science Core Collection, MedRvix and bioRvix) for publications from December 2019 to May 31st 2020. Random-effects meta-analyses were used to calculate pooled odds ratios and differences of medians between (1) patients admitted to ICU versus non-ICU patients and (2) patients who died versus those who survived. We adapted an existing Cochrane risk-of-bias assessment tool for outcome studies., Results: Of 6,702 unique citations, we included 88 articles with 69,762 patients. There was concern for bias across all articles included. Age was strongly associated with mortality with a difference of medians (DoM) of 13.15 years (95% confidence interval (CI) 11.37 to 14.94) between those who died and those who survived. We found a clinically relevant difference between non-survivors and survivors for C-reactive protein (CRP; DoM 69.10 mg/L, CI 50.43 to 87.77), lactate dehydrogenase (LDH; DoM 189.49 U/L, CI 155.00 to 223.98), cardiac troponin I (cTnI; DoM 21.88 pg/mL, CI 9.78 to 33.99) and D-Dimer (DoM 1.29mg/L, CI 0.9 to 1.69). Furthermore, cerebrovascular disease was the co-morbidity most strongly associated with mortality (Odds Ratio 3.45, CI 2.42 to 4.91) and ICU admission (Odds Ratio 5.88, CI 2.35 to 14.73)., Discussion: This comprehensive meta-analysis found age, cerebrovascular disease, CRP, LDH and cTnI to be the most important risk-factors that predict severe COVID-19 outcomes and will inform clinical scores to support early decision-making., Competing Interests: I have read the journal’s policy and none of the authors of this manuscript have a competing interest.

We investigate the feasibility of molecular-level sample classification of sepsis using microarray gene expression data merged by in silico meta-analysis. Publicly available data series were extracted from NCBI Gene Expression Omnibus and EMBL-EBI ArrayExpress to create a comprehensive meta-analysis microarray expression set (meta-expression set). Measurements had to be obtained via microarray-technique from whole blood samples of adult or pediatric patients with sepsis diagnosed based on international consensus definition immediately after admission to the intensive care unit. We aggregate trauma patients, systemic inflammatory response syndrome (SIRS) patients, and healthy controls in a non-septic entity. Differential expression (DE) analysis is compared with machine-learning-based solutions like decision tree (DT), random forest (RF), support vector machine (SVM), and deep-learning neural networks (DNNs). We evaluated classifier training and discrimination performance in 100 independent iterations. To test diagnostic resilience, we gradually degraded expression data in multiple levels. Clustering of expression values based on DE genes results in partial identification of sepsis samples. In contrast, RF, SVM, and DNN provide excellent diagnostic performance measured in terms of accuracy and area under the curve (>0.96 and >0.99, respectively). We prove DNNs as the most resilient methodology, virtually unaffected by targeted removal of DE genes. By surpassing most other published solutions, the presented approach substantially augments current diagnostic capability in intensive care medicine., Competing Interests: The authors have declared that no competing interests exist.

Background: Postoperative abdominal infections belong to the most common triggers of sepsis and septic shock in intensive care units worldwide. While monocytes play a central role in mediating the initial host response to infections, sepsis-induced immune dysregulation is characterized by a defective antigen presentation to T-cells via loss of Major Histocompatibility Complex Class II DR (HLA-DR) surface expression. Here, we hypothesized a sepsis-induced differential occupancy of the CCCTC-Binding Factor (CTCF), an architectural protein and superordinate regulator of transcription, inside the Major Histocompatibility Complex Class II (MHC-II) region in patients with postoperative sepsis, contributing to an altered monocytic transcriptional response during critical illness., Results: Compared to a matched surgical control cohort, postoperative sepsis was associated with selective and enduring increase in CTCF binding within the MHC-II. In detail, increased CTCF binding was detected at four sites adjacent to classical HLA class II genes coding for proteins expressed on monocyte surface. Gene expression analysis revealed a sepsis-associated decreased transcription of (i) the classical HLA genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1 and (ii) the gene of the MHC-II master regulator, CIITA (Class II Major Histocompatibility Complex Transactivator). Increased CTCF binding persisted in all sepsis patients, while transcriptional recovery CIITA was exclusively found in long-term survivors., Conclusion: Our experiments demonstrate differential and persisting alterations of CTCF occupancy within the MHC-II, accompanied by selective changes in the expression of spatially related HLA class II genes, indicating an important role of CTCF in modulating the transcriptional response of immunocompromised human monocytes during critical illness., Competing Interests: The authors have declared that no competing interests exist.

Background: With more than 18 million annual new cases, cancer belongs to the major challenges of modern healthcare. Surgical resection of solid tumours under general anaesthesia is the prime therapy. Different aspects of anaesthesia are under discussion to independently influence the long-term outcome of cancer patients. Most recently, the commonly used volatile anaesthetics like sevoflurane have entered the spotlight, as retrospective studies suggest a detrimental outcome in certain cancer aetiologies with sparse mechanistic understanding. Our objective was to investigate this concept in a murine melanoma model, herein comparing the consequence of inhalative and injection anesthesia on tumour composition and growth., Methods: We used a murine model of malignant melanoma in male, adult C57BL/6 mice (n = 92), induced by the subcutaneous injection of B16-F10 cells. We either exposed the melanoma cells to sevoflurane before implantation or subjected the animals to single or double anaesthesia with either volatile or injection drugs. After a maximum follow-up of 4 weeks, leucocytes within the tumour microenvironment (TME) were comprehensively analysed by flow cytometry with focus on tumor-associated macrophages (TAM)., Results: We found that exposure of melanoma cells to sevoflurane before implantation induced long-lasting transcriptome changes and aggravated tumour growth, without extensive changes of the TME. Contrastingly, both a single and double anaesthesia with sevoflurane led to a significant reduction of TAMs (injection vs. sevoflurane: 2,0 vs. 0.3% and 1.2 vs. 0.6%, respectively), whilst increasing PD-L1 expression on the remaining cells (mean fluorescent intensity injection vs. sevoflurane: 3,804 vs. 7,143 and 9,090 vs. 32,228, respectively). No changes in tumour growth were observed in these groups., Conclusion: In sharp contrast to the detrimental impact of sevoflurane on patients' outcome reported in retrospective clinical studies, we propose here that sevoflurane might actually exert a beneficial effect by decreasing TAMs within the TME, rendering the tumour again susceptible for cytotoxic T cells and immunotherapies. Further research is warranted to delineate, how these results translate into the clinic., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To test the feasibility of a randomized controlled study design comparing epidural analgesia (EDA) with continuous wound infiltration (CWI) in respect to postoperative complications and mobility to design a future multicentre randomized controlled trial., Design, Setting, Participants: CWI has been developed to address drawbacks of EDA. Previous studies have established the equivalent analgesic potential of CWI compared to EDA. This is a single centre, non-blinded pilot randomized controlled trial at a tertiary surgical centre. Patients undergoing elective non-colorectal surgery via a midline laparotomy were randomized to EDA or CWI. Endpoints included recruitment, feasibility of assessing postoperative mobility with a pedometer and morbidity. No primary endpoint was defined and all analyses were explorative., Interventions: CWI with local anaesthetics (experimental group) vs. thoracic EDA (control)., Results: Of 846 patients screened within 14 months, 71 were randomized and 62 (31 per group) included in the intention-to-treat analysis. Mobility was assessed in 44 of 62 patients and revealed no differences within the first 3 postoperative days. Overall morbidity did not differ between the two groups (measured via the comprehensive complication index). Median pain scores at rest were comparable between the two groups, while EDA was superior in pain treatment during movement on the first, but not on the second and third postoperative day. Duration of preoperative induction of anaesthesia was shorter with CWI than with EDA. Of 17 serious adverse events, 3 were potentially related to EDA, while none was related to CWI., Conclusion: This trial confirmed the feasibility of a randomized trial design to compare CWI and EDA regarding morbidity. Improvements in the education and training of team members are necessary to improve recruitment., Trial Registration: DRKS00008023., Competing Interests: The authors have declared that no competing interests exist.

Background: Antigen presentation on monocyte surface to T-cells by Major Histocompatibility Complex, Class II (MHC-II) molecules is fundamental for pathogen recognition and efficient host response. Accordingly, loss of Major Histocompatibility Complex, Class II, DR (HLA-DR) surface expression indicates impaired monocyte functionality in patients suffering from sepsis-induced immunosuppression. Besides the impact of Class II Major Histocompatibility Complex Transactivator (CIITA) on MHC-II gene expression, X box-like (XL) sequences have been proposed as further regulatory elements. These elements are bound by the DNA-binding protein CCCTC-Binding Factor (CTCF), a superordinate modulator of gene transcription. Here, we hypothesized a differential interaction of CTCF with the MHC-II locus contributing to an altered monocyte response in immunocompromised septic patients., Methods: We collected blood from six patients diagnosed with sepsis and six healthy controls. Flow cytometric analysis was used to identify sepsis-induced immune suppression, while inflammatory cytokine levels in blood were determined via ELISA. Isolation of CD14++ CD16-monocytes was followed by (i) RNA extraction for gene expression analysis and (ii) chromatin immunoprecipitation to assess the distribution of CTCF and chromatin modifications in selected MHC-II regions., Results: Compared to healthy controls, CD14++ CD16-monocytes from septic patients with immune suppression displayed an increased binding of CTCF within the MHC-II locus combined with decreased transcription of CIITA gene. In detail, enhanced CTCF enrichment was detected on the intergenic sequence XL9 separating two subregions coding for MHC-II genes. Depending on the relative localisation to XL9, gene expression of both regions was differentially affected in patients with sepsis., Conclusion: Our experiments demonstrate for the first time that differential CTCF binding at XL9 is accompanied by uncoupled MHC-II expression as well as transcriptional and epigenetic alterations of the MHC-II regulator CIITA in septic patients. Overall, our findings indicate a sepsis-induced enhancer blockade mediated by variation of CTCF at the intergenic sequence XL9 in altered monocytes during immunosuppression., Competing Interests: The authors have declared that no competing interests exist.

Background: Sepsis is defined as a life-threatening condition, resulting from a dysregulated and harmful response of the hosts' immune system to infection. Apart from this, the (over-)compensating mechanisms counterbalancing the inflammatory response have been proven to render the host susceptible to further infections and increase delayed mortality. Our study aimed to unravel the heterogeneity of immune response in early sepsis and to explain the biology behind it., Methods: A systematic search of public repositories yielded 949 microarray samples from patients with sepsis of different infectious origin and early after clinical manifestation. These were merged into a meta-expression set, and after applying sequential conservative bioinformatics filtering, an in-deep analysis of transcriptional heterogeneity, as well as a comparison to samples of healthy controls was performed., Results: We can identify two distinct clusters of patients (cluster 1: 655 subjects, cluster 2: 294 subjects) according to their global blood transcriptome. While both clusters exhibit only moderate differences in direct comparison, a comparison of both clusters individually to healthy controls yielded strong expression changes of genes involved in immune responses. Both comparisons found similar regulated genes, with a stronger dysregulation occurring in the larger patient cluster and implicating a loss of monocyte and T cell function, co-occurring with an activation of neutrophil granulocytes., Conclusion: We propose a consistent-but in its extent varying-presence of immunosuppression, occurring as early in sepsis as its clinical manifestation and irrespective of the infectious origin. While certain cell types possess contradictory activation states, our finding underlines the urgent need for an early host-directed therapy of sepsis side-by-side with antibiotics., Competing Interests: The authors have declared that no competing interests exist.

Background: Ciliary beating by respiratory epithelial cells continuously purges pathogens from the lower airways. Here we investigated the effect of the fungal cell wall polysaccharides Galactomannan (GM) and Zymosan (Zym) on the adrenergic activated particle transport velocity (PTV) of tracheal epithelium., Methods: Experiments were performed using tracheae isolated from male C57BL/6J mice. Transport velocity of the cilia bearing epithelial cells was measured by analysing recorded image sequences. Generation of reactive oxygen species (ROS) were determined using Amplex Red reagents. PCR experiments were performed on isolated tracheal epithelium to identify adrenergic receptor mRNA., Results: The adrenergic receptors α1D, α2A, β1 and β2 have been identified in isolated tracheal epithelium. We found epinephrine responsible for an increase in PTV, which could only be reduced by selective β-receptor-inhibition. In addition, either GM or Zym prevented the epinephrine induced PTV increase. Furthermore, we observed a strong ROS generation evoked by GM or Zym. However, epinephrine induced increase in PTV recovered in the presence of GM and Zym after application of ROS scavengers., Conclusion: Both GM or Zym trigger reversible ROS generation in tracheal tissue leading to inhibition of the β-adrenergic increase in PTV.

Background: Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host's systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown., Methods and Findings: In a pilot approach, we performed combined chromatin immunoprecipitation followed by high-throughput sequencing to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 and 27 trimethylation and lysine 9 acetylation in monocytes isolated from healthy donors (n = 4) and patients with sepsis (n = 2). Despite different underlying causes for sepsis, a comparison over promoter regions shows a high correlation between the patients for all chromatin marks. These findings hold true also when comparing patients to healthy controls. Despite the global similarity, differential analysis reveals a set of distinct promoters with significant enrichment or depletion of histone marks. Further analysis of overrepresented GO terms show an enrichment of genes involved in immune function. To the most prominent ones belong different members of the HLA family located within the MHC cluster together with the gene coding for the major regulator of this locus-CIITA., Conclusions: We are able to show for the first time that sepsis in humans induces selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes, shedding light on basal regulatory mechanisms that might be contributing to the functional changes occurring in monocytes.

Unlabelled: Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner., Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

The tracheal epithelium prevents via its highly effective clearance mechanism the contamination of the lower airways by pathogens. This mechanism is driven by ciliary bearing cells which are not only in contact with the gas phase; in addition they are also influenced by inflammatory mediators. These mediators can alter the protective function of the epithelium. Since the pro-inflammatoric cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role within the inflammatory cascade, we investigated its effect onto the tracheal epithelium measured by its ciliary beat frequency and the particle transport velocity. In organ explant experiments the ciliary beat frequency and the particle transport velocity were measured under the application of TNF-α using tracheae from male C57BL6J mice. We observed a dose dependent TNF-α induced increase of both particle transport velocity and ciliary beat frequency. Knock out mice experiments made evident that the increase was depended on the expression of tumor necrosis factor receptor 1 (TNF-R1). The increases in ciliary beat frequency as well as the accelerated particle transport velocity were either inhibited by the unspecific serotonin antagonist methysergide or by cyproheptadine a specific 5-HT2 receptor antagonist. Thus, acetylcholine antagonists or nitric oxide synthase (NOS) inhibitors failed to inhibit the TNF-α induced activation. In conclusion, TNF-α may play a pivotal role in the protection of lower airways by inducing ciliary activity and increase in particle transport velocity via TNF-R1 and 5-HT2 receptor.