Vesseg: An Open-Source Tool for Deep Learning-Based Atherosclerotic Plaque Quantification in Histopathology Images-Brief Report.

Objective: Manual plaque segmentation in microscopy images is a time-consuming process in atherosclerosis research and potentially subject to unacceptable user-to-user variability and observer bias. We address this by releasing Vesseg a tool that includes state-of-the-art deep learning models for atherosclerotic plaque segmentation. Approach and Results: Vesseg is a containerized, extensible, open-source, and user-oriented tool. It includes 2 models, trained and tested on 1089 hematoxylin-eosin stained mouse model atherosclerotic brachiocephalic artery sections. The models were compared to 3 human raters. Vesseg can be accessed at https://vesseg .online or downloaded. The models show mean Soerensen-Dice scores of 0.91+/-0.15 for plaque and 0.97+/-0.08 for lumen pixels. The mean accuracy is 0.98+/-0.05. Vesseg is already in active use, generating time savings of >10 minutes per slide. Conclusions: Vesseg brings state-of-the-art deep learning methods to atherosclerosis research, providing drastic time savings, while allowing for continuous improvement of models and the underlying pipeline.

Preoperative neutrophil to lymphocyte ratio and platelet to lymphocyte ratio are associated with major adverse cardiovascular and cerebrovascular events in coronary heart disease patients undergoing non-cardiac surgery.

BACKGROUND: Preoperative risk prediction in patients at elevated cardiovascular risk shows limited accuracy. Platelet to lymphocyte ratio (PLR) and neutrophil to lymphocyte ratio (NLR) indicate systemic inflammation. Both have been investigated for outcome prediction in the field of oncology and cardiovascular medicine, as well as risk prediction of adverse cardiovascular events in non-surgical patients at increased cardiovascular risk. METHODS: For this post-hoc analysis, we included all 38 coronary heart disease patients from the Leukocytes and Cardiovascular Perioperative Events cohort-1 study scheduled for elective non-cardiac surgery. We evaluated preoperative differential blood counts for association with major adverse cardiovascular and cerebrovascular events (MACCE) defined as the composite endpoint of death, myocardial ischemia, myocardial infarction, myocardial injury after non-cardiac surgery, or embolic or thrombotic stroke within 30 days after surgery. We used Youden's index to calculate cut-off values for PLR and NLR. Additive risk-predictive values were assessed using receiver operating characteristic curve and net reclassification (NRI) improvement analyses. RESULTS: Patients with the composite endpoint MACCE had higher PLR and NLR (309 [206; 380] vs. 160 [132; 203], p = 0.001; 4.9 [3.5; 8.1] vs. 2.6 [2.2; 3.4]), p = 0.001). Calculated cut-offs for PLR > 204.4 and NLR > 3.1 were associated with increased risk of 30-day MACCE (OR 7, 95% CI [1.2; 44.7], p = 0.034; OR 36, 95% CI [1.8; 686.6], p = 0.001). Furthermore, NLR improved risk prediction in coronary heart disease patients undergoing non-cardiac surgery when combined with hs-cTnT or NT-proBNP (NRI total = 0.23, p = 0.008, NRI total = 0.26, p = 0.005). CONCLUSIONS: Both PLR and NLR were associated with perioperative cardiovascular adverse events in coronary heart disease patients. NLR proved to be of additional value for preoperative risk stratification. Both PLR and NLR could be used as inexpensive and broadly available tools for perioperative risk assessment. TRIAL REGISTRATION: NCT02874508, August 22, 2016.

Presepsin for pre-operative prediction of major adverse cardiovascular events in coronary heart disease patients undergoing noncardiac surgery: Post hoc analysis of the Leukocytes and Cardiovascular Peri-operative Events-2 (LeukoCAPE-2) Study.

BACKGROUND: Accurate pre-operative evaluation of cardiovascular risk is vital to identify patients at risk for major adverse cardiovascular and cerebrovascular events (MACCE) after noncardiac surgery. Elevated presepsin (sCD14-ST) is associated with peri-operative MACCE in coronary artery disease (CAD) patients after noncardiac surgery. OBJECTIVES: Validating the prognostic utility of presepsin for MACCE after noncardiac surgery. DESIGN: Prospective patient enrolment and blood sampling, followed by post hoc evaluation of pre-operative presepsin for prediction of MACCE. SETTING: Single university centre. PATIENTS: A total of 222 CAD patients undergoing elective, inpatient noncardiac surgery. INTERVENTION: Pre-operative presepsin measurement. MAIN OUTCOME MEASURES: MACCE (cardiovascular death, myocardial infarction, myocardial ischaemia and stroke) at 30 days postsurgery. RESULTS: MACCE was diagnosed in 23 (10%) patients. MACCE patients presented with increased pre-operative presepsin (median [IQR]; 212 [163 to 358] vs. 156 [102 to 273] pgml, P = 0.023). Presepsin exceeding the previously derived threshold of 184 pg ml was associated with increased 30-day MACCE rate. After adjustment for confounders, presepsin more than 184 pg ml [OR = 2.8 (95% confidence interval 1.1 to 7.3), P = 0.03] remained an independent predictor of peri-operative MACCE. Predictive accuracy of presepsin was moderate [area under the curve (AUC) = 0.65 (0.54 to 0.75), P = 0.023]. While the basic risk model of revised cardiac risk index, high-sensitive cardiac troponin T and N-terminal fragment of pro-brain natriuretic peptide resulted in an AUC = 0.62 (0.48 to 0.75), P = 0.072, addition of presepsin to the model led to an AUC = 0.67 (0.56 to 0.78), P = 0.009 and (ΔAUC = 0.05, P = 0.438). Additive risk predictive value of presepsin was demonstrated by integrated discrimination improvement analysis (integrated discrimination improvement = 0.023, P = 0.022). Net reclassification improvement revealed that the additional strength of presepsin was attributed to the reclassification of no-MACCE patients into a lower risk group. CONCLUSION: Increased pre-operative presepsin independently predicted 30-day MACCE in CAD patients undergoing major noncardiac surgery. Complementing cardiovascular risk prediction by inflammatory biomarkers, such as presepsin, offers potential to improve peri-operative care. However, as prediction accuracy of presepsin was only moderate, further validation studies are needed. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03105427.

Elevated Presepsin Is Associated With Perioperative Major Adverse Cardiovascular and Cerebrovascular Complications in Elevated-Risk Patients Undergoing Noncardiac Surgery: The Leukocytes and Cardiovascular Perioperative Events Study.

BACKGROUND: Perioperative major adverse cardiovascular and cerebrovascular events (MACCEs) are incompletely understood, and risk prediction is imprecise. Atherogenic leukocytes are crucial in cardiovascular events. However, it is unclear if surgical interventions affect leukocyte counts or activation status. Therefore, we investigated whether noncardiac surgery in patients with elevated cardiovascular risk is associated with changes in atherogenic leukocyte subsets and if these changes are related to perioperative MACCEs. METHODS: We enrolled 40 patients in this single-center prospective observational cohort study. Total leukocytes and subpopulations, including classical, intermediate, and nonclassical monocytes and natural killer and regulatory T cells, were quantified before surgery, at 2 and 6 hours after skin incision, and at postoperative days 1 and 2 (POD1+2). The monocyte activation marker presepsin (sCD14-ST) was measured post hoc to determine differentiation of classical to nonclassical monocytes. We evaluated presepsin for prediction of the composite primary end point MACCE (cardiovascular death, myocardial infarction, myocardial ischemia, and stroke) at 30 days. Its additive value to risk assessment based on high-sensitive cardiac troponin T and N-terminal probrain natriuretic peptide (NT-proBNP) was analyzed. RESULTS: We evaluated 38 patients, of whom 5 (13%) reached MACCE. In the entire cohort, classical monocytes continuously increased and peaked at POD1 (0.35 [0.23-0.43] cells per nanoliter blood [nL] vs 0.45 [0.31-0.66] cells·nL, preoperative [pre-OP] vs POD1, P = .002). Intermediate monocytes doubled by POD1 (0.017 [0.013-0.021] vs 0.036 [0.022-0.043] cells·nL, pre-OP versus POD1, P = .0003). Nonclassical monocytes decreased (0.022 [0.012-0.032] vs 0.012 [0.005-0.023] cells·nL, pre-OP vs 6 hours, P = .003). In our patient population, we did not detect changes in any of the other predefined leukocyte subsets investigated. In patients experiencing a MACCE, classical monocyte expansion was reduced (0.081 [-0.16 to 0.081] cells·nL vs 0.179 [0.081 to 0.292] cells·nL, MACCE versus non-MACCE, P = .016). Patients in the event group presented with elevated pre-OP presepsin (1528 [406-1897] pg·mL vs 123 [82.2-174] pg·mL, MACCE versus non-MACCE, P = .0001). Presepsin was associated with MACCE (area under the curve = 0.964, [0.846-0.998], P = .001). Presepsin above the calculated threshold >184 pg·mL was superior to high-sensitive cardiac troponin T for improvement of NT-proBNP-based risk prediction (28 [74%] vs 22 [58%] correctly classified patients, P = .014). CONCLUSIONS: Noncardiac surgery was associated with an increase in atherogenic leukocyte subsets. In a post hoc analysis, elevated pre-OP presepsin was associated with MACCE and improved NT-proBNP-based risk assessment. After validation in an independent data set, a presepsin cutoff of 184 pg·mL might qualify to complement NT-proBNP-based risk prediction, thereby increasing the proportion of correctly identified high-risk patients.

Modulation of Peripheral CD4+CD25+Foxp3+ Regulatory T Cells Ameliorates Surgical Stress-Induced Atherosclerotic Plaque Progression in ApoE-Deficient Mice.

Systemic inflammation associated with major surgery rapidly accelerates atherosclerotic plaque progression in mice. Regulatory T cells (Tregs) have emerged as important modulators of atherogenesis. In coronary artery disease patients, low frequency of Tregs constitutes an independent risk factor for cardiovascular complications after non-cardiac surgery. In this exploratory analysis, we investigate whether preoperative Treg levels affect surgery-induced atherosclerotic lesion destabilization in a murine model of perioperative stress. After 9 weeks of high-cholesterol diet, atherosclerotic apolipoprotein E-deficient mice with modulated Treg levels were subjected to a 30-minute surgical procedure consisting of general isoflurane anesthesia, laparotomy and moderate blood loss. Controls underwent general anesthesia only. Brachiocephalic arteries were harvested 3 days after the intervention for histomorphological analyses of atherosclerotic plaques. Tregs were depleted by a single dose of anti-CD25 monoclonal antibody (mAb) administered 6 days prior to the intervention. Expansion of Tregs was induced by daily injections of IL-2/anti-IL-2 complex (IL-2C) on three consecutive days starting 3 days before surgery. Isotype-matched antibodies and PBS served as controls. Antibody-mediated modulation was Treg-specific. IL-2C treatment resulted in an eight-fold elevation of peripheral CD4+CD25+Foxp3+ Tregs compared to mice administered with anti-CD25 mAb. In mice treated with PBS and anti-CD25 mAb, surgical stress response caused a significant increase of atherosclerotic plaque necrosis (PBS: p < 0.001; anti-CD25 mAb: p = 0.037). Preoperative Treg expansion abrogated perioperative necrotic core formation (p = 0.556) and significantly enhanced postoperative atherosclerotic plaque stability compared to PBS-treated mice (p = 0.036). Postoperative plaque volume (p = 0.960), stenosis (p = 0.693), lesional collagen (p = 0.258), as well as the relative macrophage (p = 0.625) and smooth muscle cell content (p = 0.178) remained largely unaffected by preoperative Treg levels. In atherosclerotic mice, therapeutic expansion of Tregs prior to major surgery mitigates rapid effects on perioperative stress-driven atherosclerotic plaque destabilization. Future studies will show, whether short-term interventions modulating perioperative inflammation qualify for prevention of cardiovascular events associated with major non-cardiac surgery.

Presepsin as a biomarker in perioperative medicine.

Presepsin is the soluble fragment of CD14, a multifunctional glycoprotein expressed on the surface of innate immune cells. In healthy individuals, presepsin is present in very low concentrations with reference values ranging from 60 to 382 pg/mL. Several studies have shown that presepsin is a valuable biomarker for sepsis diagnosis in adults. Only lately, presepsin has been evaluated for prediction and early detection of neonatal sepsis and septic shock. Elevated plasma presepsin concentration has also been reported in patients undergoing cardiac and non-cardiac surgery and it has further been evaluated as a potential independent predictor of perioperative cardiovascular complications and mortality. Combined cardiac and inflammatory biomarker evaluation may offer additive predictive information, but further investigations in large populations are required to determine presepsin diagnostic and prognostic value, in order to personalize therapy and reduce surgical patients' morbidity and mortality.

Frontline Science: Low regulatory T cells predict perioperative major adverse cardiovascular and cerebrovascular events after noncardiac surgery.

Immune cells drive atherosclerotic lesion progression and plaque destabilization. Coronary heart disease patients undergoing noncardiac surgery are at risk for perioperative major adverse cardiac and cerebrovascular events (MACCE). It is unclear whether differential leukocyte subpopulations contribute to perioperative MACCE and thereby could aid identification of patients prone to perioperative cardiovascular events. First, we performed a hypothesis-generating post hoc analysis of the LeukoCAPE-1 study (n = 38). We analyzed preoperative counts of 6 leukocyte subpopulations in coronary heart disease patients for association with MACCE (composite of cardiac death, myocardial infarction, myocardial ischemia, myocardial injury after noncardiac surgery, thromboembolic stroke) within 30 d after surgery. Regulatory T cells (Tregs) were the only leukocyte subgroup associated with MACCE. We found reduced Tregs in patients experiencing MACCE versus no-MACCE (0.02 [0.01; 0.03] vs. 0.04 [0.03; 0.05] Tregs nl-1 , P = 0.002). Using Youden index, we derived the optimal threshold value for association with MACCE to be 0.027 Tregs nl-1 . Subsequently, we recruited 233 coronary heart disease patients for the prospective, observational LeukoCAPE-2 study and independently validated this Treg cutoff for prediction of MACCE within 30 d after noncardiac surgery. After multivariate logistic regression, Tregs < 0.027 cells nl-1 remained an independent predictor for MACCE (OR = 2.54 [1.22; 5.23], P = 0.012). Tregs improved risk discrimination of the revised cardiac risk index based on ΔAUC (area under the curve; ΔAUC = 0.09, P = 0.02), NRI (0.26), and IDI (0.06). Preoperative Treg levels below 0.027 cells nl-1 predicted perioperative MACCE and can be measured to increase accuracy of established preoperative cardiac risk stratification in coronary heart disease patients undergoing noncardiac surgery.

Successive bacterial colonisation of pork and its implications for forensic investigations.

AIMS: Bacteria are considered one of the major driving forces of the mammalian decomposition process and have only recently been recognised as forensic tools. At this point, little is known about their potential use as 'post-mortem clocks'. This study aimed to establish the proof of concept for using bacterial identification as post-mortem interval (PMI) indicators, using a multi-omics approach. METHODS AND RESULTS: Pieces of pork were placed in the University's outdoor facility and surface swabs were taken at regular intervals up to 60 days. Terminal restriction fragment length polymorphism (T-RFLP) of the 16S rDNA was used to identify bacterial taxa. It succeeded in detecting two out of three key contributors involved in decomposition and represents the first study to reveal Vibrionaceae as abundant on decomposing pork. However, a high fraction of present bacterial taxa could not be identified by T-RFLP. Proteomic analyses were also performed at selected time points, and they partially succeeded in the identification of precise strains, subspecies and species of bacteria that colonized the body after different PMIs. CONCLUSION: T-RFLP is incapable of reliably and fully identifying bacterial taxa, whereas proteomics could help in the identification of specific strains of bacteria. Nevertheless, microbial identification by next generation sequencing might be used as PMI clock in future investigations and in conjunction with information provided by forensic entomologists. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this work represents the first attempt to find a cheaper and easily accessible, culture-independent alternative to high-throughput techniques to establish a 'microbial clock', in combination with proteomic strategies to address this issue.