Therapeutic Anticoagulation with Argatroban and Heparins Reduces Granulocyte Migration: Possible Impact on ECLS-Therapy?

Introduction. Anticoagulants such as argatroban and heparins (low-molecular-weight and unfractionated) play an immense role in preventing thromboembolic complications in clinical practice. Nevertheless, they can also have a negative effect on the immune system. This study is aimed at investigating the influence of these substances on polymorphonuclear neutrophils (PMNs), whose nonspecific defense mechanisms can promote thrombogenesis. Methods. Blood samples from 30 healthy volunteers were investigated, whereby PMNs were isolated by density gradient centrifugation and incubated with 0.8 mu g/mL of argatroban, 1.0 U/mL of low-molecular-weight heparin (LMWH), 1.0 U/mL of unfractionated heparin (UFH), or without drug (control). A collagen-cell mixture was prepared and filled into 3D mu-slide chemotaxis chambers (IBIDI (R) GmbH, Germany). Stimulation was initiated by using a chemokine gradient of n-formyl-methionine-leucyl-phenylalanine (fMLP), and microscopic observation was conducted for 4.5 hours. The cells' track length and track straightness, as well as the number of attracted granulocytes, level of ROS (reactive oxygen species) production, and NET (neutrophil extracellular traps) formation, were analyzed and categorized into migration distances and time periods. Results. All three anticoagulants led to significantly reduced PMN track lengths, with UFH having the biggest impact. The number of tracks observed in the UFH group were significantly reduced compared to the control group. Additionally, the UFH group demonstrated a significantly lower track straightness compared to the control. ROS production and NET formation were unaffected. Conclusion. Our data provide evidence that anticoagulants have an inhibitory effect on the extent of PMN migration and chemotactic migration efficiency, thus indicating their potential immune-modulatory and prothrombotic effects

Model clone detection in practice

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Potential Impact of Local Anesthetics Inducing Granulocyte Arrest and Altering Immune Functions on Perioperative Outcome

Introduction: Local anesthetics (LAs) are frequently used during anesthesia; however, they may influence granulocyte function which in turn could modify immune responses in the perioperative period. Therefore, the aim of this study was to investigate the impact of clinically used doses of bupivacaine and lidocaine on granulocyte function with regard to migration, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETosis) formation, and viability. Methods: A total of 38 granulocyte-enriched samples from healthy subjects were obtained by whole blood lysis. Polymorphonuclear neutrophil (PMN) samples were incubated simultaneously with different concentrations of either bupivacaine (0.03– 3.16 mmol/L) or lidocaine (0.007– 14.21 mmol/L), or without drug (control). Live cell imaging was conducted in order to observe granulocyte chemotaxis, migration, ROS production, and NETosis. Flow cytometry was used to analyze viability and antigen expression. Results: The track length (TL) of PMNs exposed to bupivacaine concentrations of 0.16 mmol/L and above significantly decreased compared to the control. Low concentrations of lidocaine were associated with slight but significant increases in TL, whereas this changed with concentrations above 1.4 mmol/L, showing a significant decrease in TL. PMN incubated with bupivacaine concentrations of 1.58 mmol/L and above or lidocaine concentrations of at least 3.6 mmol/L showed no migration or chemotaxis at all. Time to onset of maximal ROS production and time for half-maximal NETosis decreased in a dose-dependent manner for both substances. Equipotency in NETosis induction was reached by bupivacaine (1.1 mmol/L) at significantly lower concentrations than lidocaine (7.96 mmol/L). Cell viability and oxidative burst were unaffected by LAs. Conclusion: Local anesthetics in clinically used doses ameliorate granulocyte defense mechanisms, thus indicating their potentially decisive effect during the perioperative period

Time course of chemotaxis and chemokinesis of neutrophils following stimulation with IL-8 or FMLP

Polymorphonuclear cells (PMNs) attend to inflammatory sites by chemotactic movement, caused by chemoattractants (CAs) like n-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and interleukin-8 (IL-8). However, distinct but applicable assays for investigations of PMNs’ migration limit in vitro examination. We integrated CD15-bead-based isolation of PMNs with analysing their chemotaxis in a novel 3D-µ-Slide migration chamber. The PMNs were exposed to different concentrations of FMLP and IL-8 (1, 10 and 100 nM) and observed for 180 min in cell-physiological environment conditions. Moving PMNs’ percentage (median and interquartile range) decreased from 62% (27%) to 36% (31%) without CA, from 88% (30%) to 22% (26%) for 1 nM IL-8, from 70% (22%) to 28% (13%) for 100 nM IL-8, from 30% (23%) to 18% (46%) for 1 nM FMLP and from 76% (20%) to 28% (13%) for 100 nM FMLP. Centres of cell movement turned towards the CAs (negative values) within a single 30-min observation period: 5.37 µm (16.82 µm) without CA, −181.37 µm (132.18 µm) with 10 nM and −239.34 µm (152.19 µm) with 100 nM IL-8; −116.2 µm (69.07 µm) with 10 nM and −71.59 µm (98.58 µm) with 100 nM FMLP. FMLP and IL-8 ensure chemotaxis without increase of chemokinesis. 3D-µ-Slide chemotaxis chambers facilitate time course analyses of PMNs’ migration in stable conditions over a long time with concise distinction of chemotaxis and chemokinesis

Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn's disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders

Propofol Ameliorates Exaggerated Human Neutrophil Activation in a LPS Sepsis Model

Background: Sepsis is a leading cause of morbidity and mortality worldwide. Many patients suffering from sepsis are treated on intensive care units and many of them require mechanical ventilation under sedation or general anesthesia. Propofol, a drug used for these purposes, is known to interact with polymorphonuclear granulocytes (PMNs). Therefore, the aim of this study was to investigate the influence of propofol on PMN functions after experimental Gram-negative induced sepsis using lipopolysaccharide (LPS) stimulation. Methods: A total of 34 granulocyte-enriched samples were collected from healthy subjects. PMNs were isolated by density gradient centrifugation and incubated simultaneously with either 6 μg/mL or 60 μg/mL propofol, or none (control). Additionally, the experimental sepsis samples were incubated with either 40 pg/mL or 400 pg/mL LPS. Live cell imaging was conducted in order to observe granulocyte chemotactic migration, ROS production, and NETosis. Flow cytometry was used to analyze viability and antigen expression. Results: Propofol led to significantly reduced PMN track length (p < 0.001) and track speed (p < 0.014) after LPS-induced sepsis in a dose-dependent manner. NETosis (p = 0.018) and ROS production (p = 0.039) were accelerated by propofol without LPS incubation, indicating improved immune function. Propofol also ameliorated LPS-induced increased NETosis and ROS-production. Antigen expression for CD11b, CD62l and CD66b was unaffected by propofol. Conclusion: Propofol improves LPS-induced exaggerated PMN activation in an ex vivo model. Beneficial effects due to restored immune function in septic patients might be possible, but needs further investigation

The Image Biomarker Standardization Initiative: Standardized Quantitative Radiomics for High-Throughput Image-based Phenotyping

International audienceBackground Radiomic features may quantify characteristics present in medical imaging. However, the lack of standardized definitions and validated reference values have hampered clinical use. Purpose To standardize a set of 174 radiomic features. Materials and Methods Radiomic features were assessed in three phases. In phase I, 487 features were derived from the basic set of 174 features. Twenty-five research teams with unique radiomics software implementations computed feature values directly from a digital phantom, without any additional image processing. In phase II, 15 teams computed values for 1347 derived features using a CT image of a patient with lung cancer and predefined image processing configurations. In both phases, consensus among the teams on the validity of tentative reference values was measured through the frequency of the modal value and classified as follows: less than three matches, weak; three to five matches, moderate; six to nine matches, strong; 10 or more matches, very strong. In the final phase (phase III), a public data set of multimodality images (CT, fluorine 18 fluorodeoxyglucose PET, and T1-weighted MRI) from 51 patients with soft-tissue sarcoma was used to prospectively assess reproducibility of standardized features. Results Consensus on reference values was initially weak for 232 of 302 features (76.8%) at phase I and 703 of 1075 features (65.4%) at phase II. At the final iteration, weak consensus remained for only two of 487 features (0.4%) at phase I and 19 of 1347 features (1.4%) at phase II. Strong or better consensus was achieved for 463 of 487 features (95.1%) at phase I and 1220 of 1347 features (90.6%) at phase II. Overall, 169 of 174 features were standardized in the first two phases. In the final validation phase (phase III), most of the 169 standardized features could be excellently reproduced (166 with CT; 164 with PET; and 164 with MRI). Conclusion A set of 169 radiomics features was standardized, which enabled verification and calibration of different radiomics software. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Kuhl and Truhn in this issue