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Background Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. Methods To follow eosinophil‐virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti‐IL‐5 (mepolizumab), followed by a challenge with rhinovirus‐16 (RV16). Results DiD‐RSV and DiD‐influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus‐induced loss of asthma control. Conclusions This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations., 1,19 – dioctadecyl ‐ 3, 3, 39, 39 – tetramethylindocarbocyanine (DiD)‐respiratory syncytial virus and DiD‐influenza adhered to human eosinophils. Both viruses were internalized and inactivated by eosinophils. The capacity of eosinophils to capture virus was reduced with increasing severity of asthma. IL‐5 Tg: IL‐5 transgenic; RSV: Respiratory syncytial virus; WT: Wild‐type

Multicolour flow cytometry (MFC) is used to measure multiple cellular markers at the single-cell level. Cellular markers may be coloured with different panels of fluorescently-labelled antibodies to enable cell identification or the detection of activated cells in pre-defined, gated’ specific cell subsets. The number of markers that can be used per measurement is technologically limited however, requiring every panel to be analysed in a separate aliquot measurement. The combined analyses of these dedicated panels may enhance the predictive ability of these measurements and could enrich the interpretation of the immunological information. Here we introduce a fusion method for MFC data, based on DAMACY (Discriminant Analysis of Multi-Aspect Cytometry data), which can combine information from complementary panels. This approach leads to both enhanced predictions and clearer interpretations in comparison with the analysis of separate measurements. We illustrate this method using two datasets: the response of neutrophils evoked by a systemic endotoxin challenge and the activated immune status of the innate cells, T cells and B cells in obese versus lean individuals. The data fusion approach was able to detect cells that do not individually show a difference between clinical phenotypes but do play a role in combination with other cells. This research received funding from the Netherlands Organization for Scientific Research (NWO) in the framework of the Technology Area COAST of the Fund New Chemical Innovations. Functions for Matlab are available at http://www.ru.nl/science/analyticalchemistry/research/software/.

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (&lt, 100 cells/μL, p &lt, 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6, 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.

Introduction: Deregulation of polymorphonuclear neutrophils (PMNs) is an essential step in the development of inflammatory complications upon trauma. Different neutrophil subtypes have been identified recently, however, the role of neutrophil subtypes in immunoregulation upon trauma is unclear. We hypothesize that extensive trauma surgery causes instant progressive heterogeneity of the blood neutrophil pool, and increased appearance of young (CD16(dim)/CD62L(bright)) neutrophils in peripheral blood.Material and methods: A standardized extensive thoraco-abdominal porcine trauma surgery model was utilized, and 12 animals were included. Blood was collected at defined timepoints and neutrophil numbers and subtypes were studied by flowcytometry. Neutrophil subtypes were identified by differences in cell surface expression levels of CD16 (Fc gamma RIII) and CD62L (L-selectin). Porcine neutrophil subtypes were further characterized after flow sorting.Results: Eleven animals survived the 3-hour surgical protocol. Neutrophil numbers dropped significantly from a mean of 8,6 +/- 3,5 x 10(6) to 2,4 +/- 1,8 x 10(6) cells/ml during 180 min, (p

The amplitude of the innate immune response reflects the degree of physiological stress imposed by exercise load. An optimal balance of exercise intensity and duration is essential for a balanced immune system and reduces the risk of dysfunction of the immune system. Therefore, it is hypothesized that neutrophils, as key players in the innate immune system, can be used as biomarker in detecting overtraining. The aim was to monitor the state of the innate immune system by phenotyping neutrophils during consecutive bouts of prolonged exercise. Study subjects were recruited from a cohort of walkers participating in a walking event on 3 consecutive days. Participants with immune deficiencies were excluded. Questionnaires to determine the physiological status of the participants were completed. Analysis of neutrophil receptor expression was done by a point‐of‐care fully automated flow cytometer. A total of 45 participants were recruited, of whom 39 participants were included for data analysis. Study participants had a median age of 64 (58‐70) years. The absolute numbers CD16dim/CD62Lbright and CD16bright/CD62Ldim neutrophils were increased after the first 2 days of exercise followed by an adaptation/normalization after the third day. Participants with activated neutrophils (high CD11b expression) had an impaired physical feeling indicated by the participant on a lower visual analog scale compared to participants who did not have activated neutrophils (P = 0.017, P = 0.022). Consecutive days of prolonged exercise results in an initial systemic innate immune response, followed by normalization/adaptation. Increased neutrophil activation was associated with impaired physical feeling measured by a validated VAS score indicated by the participant. Fully automated point‐of‐care flow cytometry analysis of neutrophil phenotypes in a field laboratory might be a useful tool to monitor relevant differences in the systemic innate immune response in response to exercise., Graphical Abstract Fully automated flow cytometry analysis of neutrophils obtained in a field laboratory after repetitive prolonged exercise.

Coronavirus disease 2019 (COVID-19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Critical COVID-19 is thought to be associated with a hyper-inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID-19 patients. Therefore, a prospective study was performed on all patients with suspected COVID-19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point-of-care automated flow cytometry and compared with blood samples collected at later time points. COVID-19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID-19-positive patients and COVID-19-negative patients diagnosed with other bacterial/viral infections, or between COVID-19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation-induced left shift of the neutrophil compartment. In COVID-19 this was associated with disease severity.

The role of eosinophils in allergic inflammation is well recognized. In homeostasis these cells are found in multiple healthy tissues including the lung parenchyma, but the function of these resident eosinophils is unknown. Circulating eosinophils are easily quantifiable and have been used to define “eosinophilic phenotype”, and to select patients who are likely to respond to anti-eosinophil and anti-Th2—directed therapies. However, presence of eosinophils in circulation may not necessarily indicate that the eosinophils are key effector cells for an airway disease such as asthma and this may be reason for not all patients responding well to anti-IL5 therapies despite normalization of blood eosinophils. This pro-con commentary examines the role of enumerating circulating vs luminal (sputum) eosinophils (and their activation status) not only to initiate therapies with monoclonal antibodies, but to monitor their clinical response while on therapy.

BACKGROUND Neutrophils and monocytes are key immune effector cells in inflammatory bowel disease (IBD) that is associated with chronic inflammation in the gut. Patients with stable IBD who perform exercise have significantly fewer flare-ups of the disease, but no underlying mechanism has been identified. Therefore, the aim of this study was to compare the responsiveness/refractoriness of these innate immune cells after repeated bouts of prolonged exercise in IBD patients and controls. METHODS Patients with IBD and age- and gender-matched healthy controls were recruited from a cohort of walkers participating in a 4-day walking event. Blood analysis was performed at baseline and after 3 days of walking. Responsiveness to the bacterial/mitochondrial-stimulus N-Formylmethionine-leucyl-phenylalanine (fMLF) was tested in granulocytes and monocytes by measuring the expression of activation markers after adding this stimulus to whole blood. RESULTS In total 38 participants (54 ± 12 years) were included in this study: 19 walkers with and 19 walkers without IBD. After 3 days of prolonged exercise, a significant increase in responsiveness to fMLF was observed in all participants irrespective of disease. However, IBD patients showed significantly less responsiveness in neutrophils and monocytes, compared with non-IBD walkers. CONCLUSIONS Increased responsiveness of neutrophils and monocyte to fMLF was demonstrated after repetitive bouts of prolonged exercise. Interestingly, this exercise was associated with relative refractoriness of both neutrophils and monocytes in IBD patients. These refractory cells might create a lower inflammatory state in the intestine providing a putative mechanism for the decrease in flare-ups in IBD patients after repeated exercise.

Supplemental Digital Content is available in the text., Objectives: The amount of tissue damage and the amplitude of the immune response after trauma are related to the development of infectious complications later on. Changes in the neutrophil compartment can be used as read out of the amplitude of the immune response after trauma. The study aim was to test whether 24/7 point-of-care analysis of neutrophil marker expression by automated flow cytometry can be achieved after trauma. Design: A prospective cohort study was performed. Polytrauma patients who developed infectious complications were compared with polytrauma patients who did not develop infectious complications. Setting: The study was performed in a level 1 trauma center. Patients: All trauma patients presented in the trauma bay were included. Interventions: An extra blood tube was drawn from all patients. Thereafter, a member of the trauma team placed the blood tube in the fully automated flow cytometer, which was located in the corner of the trauma room. Next, a modified and tailored protocol for this study was automatically performed. Main Results: The trauma team was able to successfully start the point-of-care automated flow cytometry analysis in 156 of 164 patients, resulting in a 95% success rate. Polytrauma patients who developed infectious complications had a significantly higher %CD16dim/CD62Lbright neutrophils compared with polytrauma patients who did not develop infectious complications (p = 0.002). Area under the curve value for %CD16dim/CD62Lbright neutrophils is 0.90 (0.83–0.97). Conclusions: This study showed the feasibility of the implementation of a fully automated point-of-care flow cytometry system for the characterization of the cellular innate immune response in trauma patients. This study supports the concept that the assessment of CD16dim/CD62Lbright neutrophils can be used for early detection of patients at risk for infectious complications. Furthermore, this can be used as first step toward immuno-based precision medicine of polytrauma patients at the ICU.

Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PI-negative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness.

Deregulation of polymorphonuclear neutrophils (PMNs) is an essential step in the development of inflammatory complications upon trauma. Different neutrophil subtypes have been identified recently, however, the role of neutrophil subtypes in immunoregulation upon trauma is unclear. We hypothesize that extensive trauma surgery causes instant progressive heterogeneity of the blood neutrophil pool, and increased appearance of young (CD16A standardized extensive thoraco-abdominal porcine trauma surgery model was utilized, and 12 animals were included. Blood was collected at defined timepoints and neutrophil numbers and subtypes were studied by flowcytometry. Neutrophil subtypes were identified by differences in cell surface expression levels of CD16 (FcγRIII) and CD62L (L-selectin). Porcine neutrophil subtypes were further characterized after flow sorting.Eleven animals survived the 3-hour surgical protocol. Neutrophil numbers dropped significantly from a mean of 8,6 ± 3,5 × 10Standardized extensive trauma surgery was associated with instant progressive neutropenia and increased heterogeneity of the blood neutrophil pool. Furthermore, three different neutrophil subsets in peripheral porcine blood were identified over the course of surgery. Further studies should clarify their precise role in the development of early organ failure upon extensive trauma surgery. This for the first time exemplifies experimentally the time constraints and impact of damage control surgery after severe trauma.

Nowadays, more trauma patients develop chronic critical illness (CCI), a state characterized by prolonged intensive care. Some of these CCI patients have disproportional difficulties to recover and suffer from recurrent infections, a syndrome described as the persistent inflammation, immunosuppression and catabolism syndrome (PICS). A total of 78 trauma patients with an ICU stay of &ge, 14 days (CCI patients) between 2007 and 2017 were retrospectively included. Within this group, PICS patients were identified through two ways: (1) their clinical course (&ge, 3 infectious complications) and (2) by laboratory markers suggested in the literature (C-reactive protein (CRP) and lymphocytes), both in combination with evidence of increased catabolism. The incidence of PICS was 4.7 per 1000 multitrauma patients. The sensitivity and specificity of the laboratory markers was 44% and 73%, respectively. PICS patients had a longer hospital stay (median 83 vs. 40, p &lt, 0.001) and required significantly more surgical interventions (median 13 vs. 3, p = 0.003) than other CCI patients. Thirteen PICS patients developed sepsis (72%) and 12 (67%) were readmitted at least once due to an infection. In conclusion, patients who develop PICS experience recurrent infectious complications that lead to prolonged hospitalization, many surgical procedures and frequent readmissions. Therefore, PICS forms a substantial burden on the patient and the hospital, despite its low incidence.

Neutrophil antibacterial capacity is measured in animal models and in vitro as an important indicator of neutrophil function. To be able to extrapolate their conclusions, in vitro experiments should mimic the in vivo situation. In vivo, antibacterial capacity depends on multiple steps of bacterial sensing, priming, chemotaxis, phagocytosis and intracellular killing. Therefore, we developed a simply executed assay that involves multiple steps in one assay. The neutrophils were incorporated into a three-dimensional matrix of fibrin fibers, in which they could freely migrate. The fibrin matrix provided a more physiological representation of tissue structure than a shaken suspension and extended ex vivo survival of neutrophils. Staphylococci endogenously producing GFP (Green Fluorescent Protein) provided a real-time quantification of the bacterial load without the need for lysing the fibrin matrix or counting of colony forming units on agar plates. The delay in bacterial outgrowth serves as a measure for the relative antibacterial capacity of the neutrophils. Additionally, neutrophil capacity could easily be measured high-throughput in a 96-wells format. In this new assay we study neutrophil behavior in a physiologically relevant setting and explore many functions of the neutrophil in a single test. The functional capacity of neutrophils from different in vitro treatments or different donors can directly be compared.

IL‐5 is an important cytokine for priming and survival of mature eosinophils and for proliferation and maturation of their progenitors. Hence, IL‐5(Rα) targeting will be increasingly used in diseases where eosinophils are the key immune effector cells such as eosinophilic asthma (EA), hypereosinophilic syndrome (HES), eosinophilic esophagitis (EE), and eosinophilic granulomatosis with polyangiitis (EGPA). Therefore, several neutralizing monoclonal antibodies directed against IL‐5 (mepolizumab and reslizumab) and its receptor IL‐5Rα (benralizumab) have found or will find their way to the clinic. While the clinical effect of these drugs has been extensively investigated and reviewed, the understanding of the underlying immunological and hematological mechanisms remains less clear. This review will discuss the translational outcomes of treatment with these monoclonal antibodies in humans to shed light on the mechanisms underlying the main immunological and hematological findings from these clinical trials in humans.

Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis. ECLIPSE combines dimensionality reduction by Simultaneous Component Analysis with Kernel Density Estimates. A Difference between Densities (DbD) is used to eliminate cells in responder samples that overlap in marker expression with cells of controls. Thereby, subsequent data analyses focus on the immune response-specific cells, leading to more informative and focused models. To prove the power of ECLIPSE, we applied the method to study two distinct datasets: the in vivo neutrophil response induced by systemic endotoxin challenge and in studying the heterogeneous immune-response of asthmatics. ECLIPSE described the well-characterized common response in the LPS challenge insightfully, while identifying slight differences between responders. Also, ECLIPSE enabled characterization of the immune response associated to asthma, where the co-expressions between all markers were used to stratify patients according to disease-specific cell profiles.

Upon activation granulocytes upregulate several adhesion molecules (CD11b) and granule proteins (CD35, CD66b) and shed surface l-selectin (CD62L). These changes in expression, as assessed by flow cytometry, can be used as markers for activation. Whereas these markers are usually studied in fresh blood samples, a new method is required when samples are collected at a field site with no direct access to a flow cytometer. Therefore, we developed and tested a field-applicable method in which fixed leukocytes were cryopreserved. Using this method, the intensity of granulocyte activation markers was compared to samples that were either stained fresh, or fixed prior to staining but not cryopreserved. In addition, the response to an in vitro stimulation with fMLF was determined. While we observed differences in marker intensities when comparing fresh and fixed granulocytes, similar intensities were found between fixed cells that had been cryopreserved and fixed cells that did not undergo cryopreservation. Although fixation using FACS lysing solution might lead to membrane permeabilization, activation markers, and the responsiveness to fMLF or eotaxin could still be clearly measured. This method will, therefore, enable future studies of granulocyte activation in settings with limited resources and will allow simultaneous analysis of samples collected at different time points. © 2018 International Society for Advancement of Cytometry.

Summary Mepolizumab (anti-IL-5) is a successful biological for treatment of T2/eosinophilic asthma by blocking the IL-5-eosinophil axis. The kinetics of human eosinophils in blood and sputum was determined to better understand the underlying mechanism(s). Pulse-chase labeling was performed with 6,6-2H2-glucose in patients with asthma after short term (4 days) and long term (84 days) treatment with mepolizumab (n = 10) or placebo (n = 10). The retention time of eosinophils in sputum was longer than in blood. Treatment with mepolizumab induced a fast and long-lasting eosinopenia with no reduction of eosinophil progenitors. The retention time of eosinophils in blood was delayed only after short-term treatment. This leads to the hypothesis that IL-5 increases the number of IL-5-responsive progenitors and potentiates homing to the tissues, leading to reactive eosinophilia. Long-term treatment is associated with low numbers of IL-5-independent eosinophils in blood and tissues. Therefore, long-term treatment with mepolizumab restores the kinetics of eosinophils as normally found in homeostasis., Graphical abstract, Highlights • Anti-IL-5 (mepolizumab) treatment leads to inhibition of reactive eosinophilia • Reactive blood eosinophils have a high retention time in the absence of IL-5 • Eosinophils are long lived in the sputum of eosinophil asthmatics • Anti-IL-5 reduces proliferating progenitors rather than inhibiting differentiation, Health sciences; Immunology; Respiratory medicine; Clinical medicine; Drugs

Background: In the search for specific phenotypes of chronic obstructive pulmonary disease (COPD) computed tomography (CT) derived Parametric Response Mapping (PRM) has been introduced. This study evaluates the association between PRM and currently available biomarkers of disease severity in COPD.Methods: Smokers with and without COPD were characterized based on questionnaires, pulmonary function tests, body plethysmography, and low-dose chest CT scanning. PRM was used to calculate the amount of emphysema (PRMEmph) and non-emphysematous air trapping (i.e. functional small airway disease, PRMfSAD). PRM was first compared with other biomarkers for emphysema (Perc15) and air trapping (E/I-ratio(MLD)). Consequently, linear regression models were utilized to study associations of PRM measurements with clinical parameters.Results: 166 participants were included with a mean +/- SD age of 50.5 +/- 17.7 years. Both PRMEmph and PRMfSAD were more strongly correlated with lung function parameters as compared to Perc15 and E/I-ratio(MLD). PRMEmph and PRMfSAD were higher in COPD participants than non-COPD participants (14.0% vs. 1.1%, and 31.6% vs. 8.2%, respectively, both p Conclusions: PRM strongly associates with the presence and severity of COPD. PRM therefore appears to be a valuable tool in differentiating COPD phenotypes. (C) 2016 Elsevier Ltd. All rights reserved.

Background Septic shock is a severe complication in polytrauma patients. Early identification of patients at risk can guide future prevention strategies. Platelets (PLTs) and leucocytes presumably play an important role in the post-injury inflammatory response. The role of early changes in PLT and leucocyte counts was investigated in search for the aetiology of the development of septic complications. Methods Polytrauma patients (aged 16-80 years) admitted to the intensive care unit with an expected stay of at least 3 days were included. PLT and leucocyte counts were measured on a daily basis for 14 days. Results A total of 41 patients were included, of whom nine (22%) developed septic shock. There was no difference in (New) Injury Severity Score or Acute Physiology and Chronic Health Evaluation II scores between patients who developed septic shock and patients who did not. Three patients died, one of them in septic shock. Patients who developed septic shock during hospital stay had lower PLTs and a slower recovery to normal PLT counts than patients without septic shock. Patients who developed either a decrease in both PLTs and leucocytes or an increase in PLTs and leucocytes in the first 24 h after trauma were more likely to develop septic shock. This correlation was not found in patients who did not develop septic shock. Conclusion A similar change in PLT and leucocyte counts in the first 24 h after trauma is associated with the development of septic shock after a week. This indicates an early interaction between PLTs and leucocytes, which needs further investigation to gain more insight in the aetiology of post-injury septic complications.

Introduction Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated “load & go” flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. Methods Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® “load & go” flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. Results The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×–2.2×) p = .058, CD11b: 2.13× (1.6×–2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. Conclusion This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15–20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. Fund This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Introduction: Cystic fibrosis (CF) is the most common monogenic disease that is eventually lethal. It is characterized by chronic bacterial infection of the lung and affecting additional organs including pancreas and liver. Neutrophils form the largest number of inflammatory cells in the airways of cystic fibrosis patients. Previous studies have shown that the killing function of neutrophils in CF patients is preserved, but maybe influenced by Pseudomonas aeruginosa (PA) colonization during the disease progression. Objective: To evaluate the phagocytosis and bacterial killing of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) by neutrophils in CF patients in comparison with cells of healthy controls. Materials and Methods: Our study was conducted in 10 CF patients (confirmed by clinical examination, sweat test and mutational analysis) in the Masih Daneshvari hospital (Tehran, Iran) and 10 age- and gender-matched healthy control subjects in 2017. Neutrophils were isolated from the peripheral blood of patients with CF patients and control subjects. The cells were incubated with SA and PA -labeled with GFP in a FLUOstar OPTIMA Microplate Fluorescent Reader. The fluorescence was measured over time as a measure of bacterial counts. Results: Neutrophils of CF patients were able to contain SA for long periods of time (72±5 h) that were significantly longer than by cells from healthy control (25±6 h) P≤0.05). Whereas, no differences in releasing of PA was observed. Our data support the hypothesis that neutrophils of CF patients exhibit a primed phenotype that enables them to contain SA longer than unprimed cells isolated from control individuals.

The aim of this study is to investigate whether the change in (sub)maximal heart rate after intensified training is associated with the change in performance. Thirty subjects were recruited who performed cardiopulmonary exercise tests to exhaustion 2 weeks before (pre), 1 week after (post) and 5 weeks after (follow-up) an 8-day non-competitive amateur cycling event (TFL). The exercise volume during the TFL was 7.7 fold the volume during the preparation period. Heart rate and cardiopulmonary parameters were obtained at standardised absolute submaximal workloads (low, medium and high intensity) and at peak level each test. Subjects were classified as functionally overreached (FOR) or acute fatigued (AF) based on the change in performance. No differences between FOR and AF were observed for heart rate (P =.51). On total group level (AF + FOR), post-TFL heart rate decreased significantly at low (−4.4 beats·min −1 , 95% CI [−8.7, −0.1]) and medium (−5.5 beats·min −1 [−8.5, −2.4]), but not at high intensity. Peak heart rate decreased −3.4 beats·min −1 [−6.1, −0.7]. O 2 pulse was on average 0.49 ml O 2 ·beat −1 [0.09, 0.89] higher at all intensities after intensified training. No changes in ⩒O 2 (P =.44) or the ventilatory threshold (P =.21) were observed. Pearson’s correlation coefficients revealed negative associations between heart rate and O 2 pulse at low (r = −.56, P 2 or any other submaximal parameter. (Sub)maximal heart rate decreased after the TFL. However, this decrease is unrelated to the change in performance. Therefore, heart rate seems inadequate to prescribe and monitor intensified training.

Flow Cytometry is an analytical technology to simultaneously measure multiple markers per single cell. Ten thousands to millions of single cells can be measured per sample and each sample may contain a different number of cells. All samples may be bundled together, leading to a ‘multi-set’ structure. Many multivariate methods have been developed for Flow Cytometry data but none of them considers this structure in their quantitative handling of the data. The standard pre-processing used by existing multivariate methods provides models mainly influenced by the samples with more cells, while such a model should provide a balanced view of the biomedical information within all measurements. We propose an alternative ‘multi-set’ preprocessing that corrects for the difference in number of cells measured, balancing the relative importance of each multi-cell sample in the data while using all data collected from these expensive analyses. Moreover, one case example shows how multi-set pre-processing may benefit removal of undesired measurement-to-measurement variability and another where class-based multi-set pre-processing enhances the studied response upon comparison to the control reference samples. Our results show that adjusting data analysis algorithms to consider this multi-set structure may greatly benefit immunological insight and classification performance of Flow Cytometry data.

Item does not contain fulltext BACKGROUND: Eosinophils are a prominent cell type in the host response to helminths, and some evidence suggests that neutrophils might also play a role. However, little is known about the activation status of these granulocytes during helminth infection. METHODS: We analyzed the expression of eosinophil and neutrophil activation markers in peripheral blood by flow cytometry and measured serum levels of eosinophil granule proteins in 300 subjects residing in an area endemic for soil-transmitted helminths (STH). The data generated are on samples before and after 1 year of 3-monthly albendazole treatment. RESULTS: Anthelmintic treatment significantly reduced the prevalence of STH. While eosinophil numbers were significantly higher in STH-infected compared to uninfected subjects and significantly decreased following albendazole treatment, there was no effect exerted by the helminths on either eosinophil nor neutrophil activation. Although at baseline eosinophil granule protein levels were not different between STH-infected and uninfected subjects, treatment significantly reduced the levels of eosinophil-derived neurotoxin (EDN) in those infected at baseline. CONCLUSIONS: These results show that besides decreasing eosinophil numbers, anthelmintic treatment does not significantly change the activation status of eosinophils, nor of neutrophils, and the only effect seen was a reduction in circulating levels of EDN. CLINICAL TRIALS REGISTRATION: http://www.isrctn.com/ISRCTN75636394.

Recurrent severe bacterial and fungal infections are characteristic features of the rare genetic immunodeficiency disorder chronic granulomatous disease (CGD). The disease usually manifests within the first years of life with an incidence of 1 in approximately 200,000 live births. The incidence is higher in Iran and Morocco where it reaches 1.5 per 100,000 live births. Mutations have been described in the 5 subunits of NADPH oxidase, mostly in gp91phox and p47phox, with fewer mutations reported in p67phox, p22phox, and p40phox. These mutations cause loss of superoxide production in phagocytic cells. CYBB, the gene encoding the large gp91phox subunit of the transmembrane component cytochrome b558 of the NADPH oxidase complex, is localized on the X-chromosome. Genetic defects in CYBB are responsible for the disease in the majority of male CGD patients. CGD is associated with the development of granulomatous reactions in the skin, lungs, bones, and lymph nodes, and chronic infections may be seen in the liver, gastrointestinal tract, brain, and eyes. There is usually a history of repeated infections, including inflammation of the lymph glands, skin infections, and pneumonia. There may also be a persistent runny nose, inflammation of the skin, and inflammation of the mucous membranes of the mouth. Gastrointestinal problems can also occur, including diarrhea, abdominal pain, and perianal abscesses. Infection of the bones, brain abscesses, obstruction of the genitourinary tract and/or gastrointestinal tract due to the formation of granulomatous tissue, and delayed growth are also symptomatic of CGD. The prevention of infectious complications in patients with CGD involves targeted prophylaxis against opportunistic microorganisms such as Staphylococcus aureus, Klebsiella spp., Salmonella spp. and Aspergillus spp. In this review, we provide an update on organ involvement and the association with specific isolated microorganisms in CGD patients.

Background Trauma leads to a complex inflammatory cascade that induces both immune activation and a refractory immune state in parallel. Although both components are deemed necessary for recovery, the balance is tight and easily lost. Losing the balance can lead to life-threatening infectious complications as well as long-term immunosuppression with recurrent infections. Neutrophils are known to play a key role in these processes. Therefore, this review focuses on neutrophil characteristics and function after trauma and how these features can be used to identify trauma patients at risk for infectious complications. Results Distinct neutrophil subtypes exist that play their own role in the recovery and/or development of infectious complications after trauma. Furthermore, the refractory immune state is related to the risk of infectious complications. These findings change the initial concepts of the immune response after trauma and give rise to new biomarkers for monitoring and predicting inflammatory complications in severely injured patients. Conclusion For early recognition of patients at risk, the immune system should be monitored. Several neutrophil biomarkers show promising results and analysis of these markers has become accessible to such extent that they can be used for point-of-care decision making after trauma.

Acute inflammation recruits neutrophils with a band-shaped nucleus to the circulation. This neutrophil population was recently shown to have superior antibacterial capacity. Early recruitment of banded neutrophils to an infection site will likely improve the outcome of the immune response, yet it critically depends on efficient migration. However, the current dogma states that the segmentation of the mature neutrophil nucleus has evolved to favor migration through narrow pores as found between endothelial cells and in the interstitium. Therefore, we hypothesized that banded neutrophils migrate less efficiently than neutrophils with segmented nuclei, whereas recently described neutrophils with hypersegmented nuclei would in turn migrate more efficiently. Acute inflammation was evoked in a human model of experimental endotoxemia to recruit neutrophil subsets with different nuclear segmentation to the circulation. To simulate migration toward an infection site, migration of the subsets was studied in in vitro models of transendothelial migration or interstitial chemokinesis and chemotaxis. In both models, nuclear segmentation did not increase migration speed. In dense collagen matrices, the speed of the hypersegmented neutrophils was even reduced compared with the banded neutrophils. Fluorescence microscopy suggested that the hypersegmented neutrophils displayed reduced rear release and deposited more membrane vesicles. Vice versa, migration through narrow pores did not induce nuclear segmentation in the neutrophils. In conclusion, like neutrophils with a segmented nucleus, the banded subset exhibited efficient migration through narrow pores. These findings suggest that the nucleus does not preclude the banded subset from reaching an infection site.

The role of inflammatory cells in bone regeneration remains unclear. We hypothesize that leukocytes contribute to fracture healing by rapidly synthesizing an "emergency extracellular matrix (ECM)" before stromal cells infiltrate the fracture hematoma (FH) and synthesize the eventual collagenous bone tissue. 53 human FHs were isolated at different time points after injury, ranging from day 0 until day 23 after trauma and stained using (immuno)histochemistry. FHs isolated within 48 h after injury contained fibronectin(+) ECM, which increased over time. Neutrophils within the early FHs stained positive for cellular fibronectin and neutrophil derived particles were visible within the fibronectin(+) ECM. Stromal cells appeared at day 5 after injury or later and collagen type I birefringent fibrils could be identified during the second week after injury. Our study suggests that neutrophils contribute to bone regeneration by synthesizing an "emergency ECM" before stromal cells infiltrate the FH and synthesize the eventual bone tissue.

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Endurance exercise is associated with a transient increase in neutrophil counts in the peripheral blood. Here we investigate the impact of intensified endurance exercise on the neutrophil compartment. We hypothesized that intensified endurance exercise leads to mobilization of neutrophil subsets, which are normally absent in the blood. Furthermore, we followed the potential build-up of neutrophil activation and the impact on overnight recovery of the neutrophil compartment during a seven-day cycling tour. The neutrophil compartment was studied in 28 healthy amateur cyclists participating in an eight-day strenuous cycling tour. Blood samples were taken at baseline, after 4 days and after 7 days of cycling. The neutrophil compartment was analyzed in terms of numbers and its phenotype by deep phenotyping of flow cytometry data with the multi-dimensional analysis method FLOOD. Repeated endurance exercise led to a gradual increase in total neutrophil counts over the days leading to a 1.26 fold-increase (95%CI 1.01-1.51 p = 0.0431) in the morning of day 8. Flow cytometric measurements revealed the appearance of 2 additional neutrophil subsets: CD16brightCD62Ldim and CD16dimCD62Lbright. A complex change in neutrophil phenotypes was present characterized by decreased expression of both CD11b and CD62L and marked increased expression of LAIR-1, VLA-4 and CBRM1/5. The changes in expression were found on all neutrophils present in the blood. Strikingly, in strong contrast to our findings during acute inflammation evoked by LPS challenge, these neutrophils did not upregulate classical degranulation markers. In fact, our FLOOD analysis revealed that the exercise induced neutrophil phenotype did not overlap with the neutrophil subsets arising upon acute inflammation. In conclusion, during multiple days of endurance exercise the neutrophil compartment does not regain homeostasis overnight. Thereby our study supports the concept of a build-up of inflammatory cues during repeated endurance exercise training, causing a prolonged change of the systemic neutrophil compartment.

Neutrophils are one of the most important effector cells of the innate immune response (1). They are traditionally seen as a homogenous population of short‐lived cells mainly involved in the defence against extracellular microorganisms by phagocytosis and intracellular killing (1,2). The cells contain a large armamentarium that aids in this function and ranges from the production of reactive oxygen species by a membrane‐bound NADPH oxidase to cytotoxic proteins and peptides residing in the different granules present in the cytoplasm (3). Recently, the view of neutrophils belonging to a homogenous population of cells has been challenged, and several neutrophil phenotypes have been described that exhibit specialized functions, such as involvement in tissue repair, tumour killing and immune regulation (4). It is not clear whether these cells belong to separate parallel lineages originating from the bone marrow or that neutrophils become instructed in the distant tissues, thus changing their phenotypes. In addition, functional heterogeneity in a phenotypically homogenous population of neutrophils adds to the complexity of neutrophil phenotypes(5). This article will review the current literature describing the heterogeneity within the neutrophil compartment with respect to both phenotype and function in health and disease.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Background A neuroinflammatory response is suggested to play an important role in delirium, a common complication in older hospitalized patients. We examined whether hip fracture patients who develop postoperative delirium have a different proteome in cerebrospinal fluid (CSF) prior to surgery. Methods Patients (≥ 75 years) were admitted for hip fracture surgery. CSF was collected during spinal anaesthesia; proteins were separated using gel electrophoresis and identified with mass spectrometry. We compared the proteome of patients with and without postoperative delirium. Findings were validated in an independent, comparable cohort using immuno-assays. Results In the derivation cohort 53 patients were included, 35.8% developed postoperative delirium. We identified differences in levels of eight CSF proteins between patients with and without subsequent delirium: complement factor C3, contactin-1, fibulin-1 and I-beta-1,3-N-acetylglucosaminyltransferase were significantly lower in patients with postoperative delirium, while neural cell adhesion molecule-2, fibrinogen, zinc-α-2-glycoprotein and haptoglobin levels were significantly higher. In the validation cohort 21.2% of 52 patients developed postoperative delirium. Immuno-assays confirmed contactin-1 results although not statistically significant. Complement factor C3 was significantly higher in patients with postoperative delirium. Conclusion Our results show the complexity of pathophysiological mechanisms involved in delirium and emphasizes the need of independent validation of findings. General significance This study highlights the challenges and inconsistent findings in studies of delirium, a serious complication in older patients. We analysed proteins in CSF, the most proximal fluid to the brain. All patients were free from delirium at the time of sampling., Highlights • Patients prone to delirium have different CSF protein levels. • Pathophysiological mechanisms involved in delirium are complex. • There is a need of independent validation of findings in research on delirium. • This study highlights the challenges and inconsistent findings in delirium studies.

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b-/- mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b-/- mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b-/- mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b-/- mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b-/- neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.

To the editor: Monocytes originate from the bone marrow (BM), are distributed in the bloodstream, and can differentiate in the tissue into skin macrophages or intestinal dendritic cells (DCs).[1][1] They play an essential role in the defense against pathogens[2][2] and are implicated in a range of

Objective Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to investigate the role of neutrophils in systemic-onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation. Methods Fifty patients with systemic-onset JIA who were receiving treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence-activated cell-sorted neutrophils from 3 patients with active systemic-onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic-onset JIA and 15 healthy controls. Results Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL-1Ra therapy. RNA-sequencing analysis revealed a substantial up-regulation of inflammatory processes in neutrophils from patients with active systemic-onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic-onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL-1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL-1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL-1Ra. In vitro, rIL-1Ra antagonized the priming effect of IL-1β on neutrophils from healthy subjects. Conclusion These results strongly support the notion that neutrophils play an important role in systemic-onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic-onset JIA are both susceptible to IL-1 blockade.

Purpose: Reaction time has been proposed as a training monitoring tool, but to date, results are equivocal. Therefore, it was investigated whether reaction time can be used as a monitoring tool to establish overreaching. Methods: The study included 30 subjects (11 females and 19 males, age: 40.8 [10.8] years, VO2max: 51.8 [6.3] mL/kg/min) who participated in an 8-day cycling event. The external exercise load increased approximately 900% compared with the preparation period. Performance was measured before and after the event using a maximal incremental cycling test. Subjects with decreased performance after the event were classified as functionally overreached (FOR) and others as acutely fatigued (AF). A choice reaction time test was performed 2 weeks before (pre), 1 week after (post), and 5 weeks after (follow-up), as well as at the start and end of the event. Results: A total of 14 subjects were classified as AF and 14 as FOR (2 subjects were excluded). During the event, reaction time at the end was 68 ms (95% confidence interval, 46-89) faster than at the start. Reaction time post event was 41 ms (95% confidence interval, 12-71) faster than pre event and follow-up was 55 ms faster (95% confidence interval, 26-83). The time by class interaction was not significant during (P = .26) and after (P = .43) the event. Correlations between physical performance and reaction time were not significant (all Ps < .30). Conclusions: No differences in choice reaction time between AF and FOR subjects were observed. It is suggested that choice reaction time is not valid for early detection of overreaching in the field.

Parametric response mapping (PRM) of paired CT lung images has been shown to improve the phenotyping of COPD by allowing for the visualization and quantification of non-emphysematous air trapping component, referred to as functional small airways disease (fSAD). Although promising, large variability in the standard method for analyzing PRMfSAD has been observed. We postulate that representing the 3D PRMfSAD data as a single scalar quantity (relative volume of PRMfSAD) oversimplifies the original 3D data, limiting its potential to detect the subtle progression of COPD as well as varying subtypes. In this study, we propose a new approach to analyze PRM. Based on topological techniques, we generate 3D maps of local topological features from 3D PRMfSAD classification maps. We found that the surface area of fSAD (SfSAD) was the most robust and significant independent indicator of clinically meaningful measures of COPD. We also confirmed by micro-CT of human lung specimens that structural differences are associated with unique SfSAD patterns, and demonstrated longitudinal feature alterations occurred with worsening pulmonary function independent of an increase in disease extent. These findings suggest that our technique captures additional COPD characteristics, which may provide important opportunities for improved diagnosis of COPD patients.

Contains fulltext : 177455.pdf (Publisher’s version ) (Open Access) BACKGROUND: COPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD. METHOD: Our aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients. RESULTS: We identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). CONCLUSION: This illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00807469 registered December 11th 2008.

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, up-regulation of host defense proteins through Toll-like receptors (TLRs) and nuclear factor B requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4–Myd88 (myeloid differentiation primary response gene 88)–dependent and abl tyrosine kinase–dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an in vivo model of bacteremia, neutrophils crawled to and rapidly phagocytosed Escherichia coli sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.

Background Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common s-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. Objective We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. Methods Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation, which was then compared to IgG-mediated degranulation of freshly isolated (unstimulated) airway eosinophils activated in vivo by segmental allergen challenge. Results When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMs2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. Conclusions We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMs2-mediated eosinophil degranulation. This article is protected by copyright. All rights reserved.

Purpose:To investigate whether monitoring of easily measurable stressors and symptoms can be used to distinguish early between acute fatigue (AF) and functional overreaching (FOR).Methods:The study included 30 subjects (11 female, 19 male; age 40.8 ± 10.8 y, VO2max 51.8 ± 6.3 mL · kg–1 · min–1) who participated in an 8-d cycling event over 1300 km with 18,500 climbing meters. Performance was measured before and after the event using a maximal incremental test. Subjects with decreased performance after the event were classified as FOR, others as AF. Mental and physical well-being, internal training load, resting heart rate, temperature, and mood were measured daily during the event. Differences between AF and FOR were analyzed using mixed-model ANOVAs. Logistic regression was used to determine the best predictors of FOR after 3 and 6 d of cycling.Results:Fifteen subjects were classified as FOR and 14 as AF (1 excluded). Although total group changes were observed during the event, no differences between AF and FOR were found for individual monitoring parameters. The combination of questionnaire-based changes in fatigue and readiness to train after 3 d cycling correctly predicted 78% of the subjects as AF or FOR (sensitivity = 79%, specificity = 77%).Conclusions:Monitoring changes in fatigue and readiness to train, using simple visual analog scales, can be used to identify subjects likely to become FOR after only 3 d of cycling. Hence, we encourage athlete support staff to monitor not only fatigue but also the subjective integrated mental and physical readiness to perform.

Background The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. Objectives To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. Methods Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). Results The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl‐methionyl‐leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). Conclusions The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Summary Improvements in the control of haemorrhage after trauma have resulted in the survival of many people who would otherwise have died from the initial loss of blood. However, the danger is not over once bleeding has been arrested and blood pressure restored. Two-thirds of patients who die following major trauma now do so as a result of causes other than exsanguination. Trauma evokes a systemic reaction that includes an acute, non-specific, immune response associated, paradoxically, with reduced resistance to infection. The result is damage to multiple organs caused by the initial cascade of inflammation aggravated by subsequent sepsis to which the body has become susceptible. This Series examines the biological mechanisms and clinical implications of the cascade of events caused by large-scale trauma that leads to multiorgan failure and death, despite the stemming of blood loss. Furthermore, the stark and robust epidemiological finding—namely, that age has a profound influence on the chances of surviving trauma irrespective of the nature and severity of the injury—will be explored. Advances in our understanding of the inflammatory response to trauma, the impact of ageing on this response, and how this information has led to new and emerging treatments aimed at combating immune dysregulation and reduced immunity after injury will also be discussed.

Background. Cigarette smoking is the main cause of chronic obstructive pulmonary disease (COPD) inducing oxidative stress and local tissue injury, resulting in pulmonary inflammation. Advanced glycation end products (AGEs) are produced by glycation and oxidation processes and their formation is accelerated in inflammatory conditions. In this study we assessed whether AGE accumulation in the skin is elevated in COPD and associates with disease severity.Methods. 202 mild-to-very-severe COPD patients and 83 old (40-75 years) and 110 young (18-40 years) healthy smokers and never-smokers were included. AGEs were measured by skin autofluorescence (SAF). Demographic variables, smoking habits, co-morbidities and lung function values were obtained.Results. COPD patients (FEV1 = 55% predicted) had significantly higher SAF values than old and young healthy controls: 2.5 vs. 1.8 and 1.2 (arbitrary units, p Conclusions. SAF is increased in mild-to-very severe COPD patients compared with healthy controls. Interestingly, SAF was not associated with disease severity as values were comparable between different GOLD stages (stage I-IV) of COPD. This may suggest that AGEs play a role in the induction phase of COPD in susceptible smokers. Future studies should further investigate the mechanisms underlying AGEs formation and accumulation in COPD. (C) 2014 Elsevier Inc. All rights reserved.

Introduction Trauma is one of the major causes of morbidity and mortality. Thoracic injuries are associated with inflammatory complications such as ARDS. The pathogenesis of this complication after pulmonary injury is incompletely understood, but neutrophils are thought to play a pivotal role. The aim of this project was to gain more insight in the role of thoracic injuries in the pathophysiological processes that link systemic neutrophil activation with inflammatory complications after trauma. Methods In this prospective cohort study fifty-five patients with isolated penetrating thoracic injury were included at a level one Trauma Unit. Blood samples were analysed for neutrophil phenotype with the use of flowcytometry within 3h of trauma and repeated six and 24h after injury. The presence of inflammatory complications (e.g. ARDS or sepsis/septic shock) was assessed during admission, and this was related to the neutrophil phenotpe. Results The clinical follow-up of fifty-three patients was uneventful. Only two patients developed an inflammatory complication. Within 3h after trauma, neutrophils showed a decreased expression of FcγRII ( p =0.007) and FcγRIII ( p =0.001) compared to healthy individuals. After 6h, expression of active FcγRII ( p =0.017), C5aR ( p =0.004) and CAECAM8 ( p =0.043) increased, whereas L-selectin ( p =0.002) decreased. After 24h also CXCR-2 (CD182) expression increased compared to healthy individuals ( p =0.001). Conclusions Penetrating thoracic trauma leads to a distinct primed activation status of circulating neutrophils within hours. In addition to activation of cells, both young and reverse migrated neutrophils are released into the circulation. This degree of systemic inflammation does not exceed a threshold of inflammation that is needed for the development of inflammatory complications like ARDS.

Neutrophils are recognized as an essential part of the innate immune response, but an active debate still exists regarding the life cycle of these cells. Neutrophils first differentiate in the bone marrow through progenitor intermediaries before entering the blood, in a process that gauges the extramedullary pool size. Once believed to be directly eliminated in the marrow, liver, and spleen, neutrophils, after circulating for less than 1 day, are now known to redistribute into multiple tissues with poorly understood kinetics. In this review, we provide an update on the dynamic distribution of neutrophils across tissues in health and disease, and emphasize differences between humans and model organisms. We further highlight issues to be addressed to exploit the unique features of neutrophils in the clinic. The work in the authors laboratories is funded by (ERC) Medical Research Council, Wellcome Trust, GlaxoSmithKline, MedImmune, the NIHR Cambridge Biomedical Research Centre, British Heart Foundation, National Institute for Health, Cambridge NIHR Biomedical Research Centre, the MCIU (Ministerio de Ciencia, Innovación y Universidades), the Pro-CNIC Foundation, the Dutch Science Agenda (NWA) and the Netherlands Organization for Scientific Research (NWO). The CNIC is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Sí