Markers Of Leukocyte Activation Research Articles

Markers of systemic leukocyte activation for assessing acute and chronic systemic inflammation

The phenomenon of systemic leukocyte activation is a component of systemic inflammation (SI), known as the systemic inflammatory response (SIR). Currently, there are hundreds of molecular markers that can serve as criteria for systemic activation, including soluble receptor to IL-2 (sIL-2R) or CD25, β2-microglobulin, and eosinophil cationic protein (ECP). The objective of our study was to assess whether markers of systemic leukocyte activation can be used as criteria for acute and chronic systemic inflammation. Data from 121 patients with acute critical infectious conditions – intensive care patients with sepsis-2 (according to the 2001 consensus, without MODS) and sepsis-3 (2016 consensus, with MODS) – and 136 patients with non-infectious genesis (polytrauma with and without MODS) were analyzed to study acute SI. Additionally, data from 148 patients with immune-inflammatory rheumatic diseases were analyzed to study chronic SI. The reactivity level (RL) was calculated using the author’s method to assess the intensity of SIR. The development of SI was verified based on the measured concentrations of IL-6, IL-8, IL-10, TNFα, CRP, D-dimers, cortisol, myoglobin, and troponin I. Criteria for leukocyte activation phenomenon were sIL-2R 700 units/mL, β2-microglobulin 3000 ng/mL, and ECP 10 ng/mL. Based on the study results, TNFα, ECP, and β2-microglobulin were identified as the most stable indices for dividing groups with and without systemic vasculitis in both acute and chronic diseases. These indices indicate the typical character of systemic leukocyte activation and can be used to assess the intensity of the described phenomenon when integrated into the SI score. However, it is doubtful that these indices can be independently applied as criteria for the complex process of SI and even the SIR phenomenon. This is because the registration of threshold values for two criteria simultaneously during the acute process of SI was only observed in 40.2% of cases, and in chronic processes, it was only observed in 23.7%. These values were comparable to the group without acute SI. Additionally, all three additional criteria were only determined in acute SI in 20.5% of cases and in chronic SI in 3.4%.

Abstract LB224: Neoplastic immune mimicry potentiates breast cancer progression

Abstract Dedifferentiation programs are commonly enacted during breast cancer progression where they enable novel cellular phenotypes. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often manifesting as greater resistance to cytotoxic therapies or increased ability to metastasize to distant organs. Here we report that subpopulations of ostensibly stem-like neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins, and have named this unique cellular program ‘immune mimicry’ (IM). We have documented neoplastic cells engaging in IM by examination of histopathological breast tumor specimens and their derived cell lines, public human breast tumor single-cell RNA-seq datasets, and in murine transgenic and cell line-derived mammary cancer models. Immune-mimicked neoplastic cells harbor hallmarks of dedifferentiation and appear enriched for developing aggressive and high-grade tumors. Experimental studies with breast cancer cell lines revealed that neoplastic cells expressing leukocyte surface features are elicited by growth-arresting conditions, such as conventional cytotoxic chemotherapeutic treatments and during metastatic dissemination in mice. In addition, using proof-of-concept studies, we leveraged the canonical leukocyte activation marker CD69, to demonstrate how its expression by neoplastic epithelial cells confers a proliferative advantage under low-density conditions. We conclude that neoplastic breast epithelial cells expressing leukocyte surface receptors may signify a particularly malignant and tumor-initiating cell state. Moving forward, neoplastic IM will be evaluated for prognostic utility in breast cancer to determine whether this unique cell state stratifies patients with increased risks for tumor recurrence and metastasis. The authors acknowledge NIH/NCI (K00 CA212132, T32 CA254888, P30 CA069533), the Collins Medical Trust, the Susan G Komen Foundation, and the National Foundation for Cancer Research for funding. Citation Format: Eric B. Berens, Sokchea Khou, Elaine Huang, Amber Hoffman, Briana Johnson, Zena Werb, Laura M. Heiser, Lisa M. Coussens. Neoplastic immune mimicry potentiates breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB224.

CD69 is a Promising Immunotherapy and Prognosis Prediction Target in Cancer.

Immunotherapy utilizing T cells that attack tumors is a promising strategy for treatment, but immune suppressive T cell subsets, such as regulatory T cell (Treg), and immune checkpoint molecules, including programmed death-1 (PD-1), can suppress the intensity of a T cell immune reaction and thereby impair tumor clearance. Cluster of differentiation 69 (CD69), known as an early leukocyte activation marker, can be used as a measure or early marker of T cell activation. In recent years, the functions of CD69 in the regulation of Treg/Th17 (T helper cell 17) differentiation and in the tissue retention of T cells have attracted considerable interest. These functions are related to the role of CD69 in immune suppression in tumor environments (TME). In this review, we first summarized current perspectives in the biological function of CD69 and demonstrated that CD69 acts as a regulator of T cell activation, differentiation, retention, and exhaustion. Then, we discussed recent advances in understanding of CD69 deficiency and anti-CD69 antibody administration and shed light on the value of targeting on CD69 for cancer immunotherapy and prognosis prediction.

Oncostatin M for characterizing the inflammatory burden and outcome of V-V ECMO in ARDS patients.

Assessing the outcome of Veno-Venous Extracorporeal Membrane Oxygenation (V-V ECMO) support remains challenging as plasma lactate (pLA), the widely used tool for this purpose, has been shown unreliable. We hypothesized that plasma oncostatin M (pOSM), a sensitive marker of leukocyte activation in infection and inflammation, could address this deficiency. Plasma OSM levels were measured by ELISA in 30 Acute Respiratory Distress Syndrome (ARDS) patients, prior to cannulation (baseline) and decannulation. Based on the absolute pOSM levels at presentation, patients were separated into two groups, A and B. Patients in group A had low pOSM levels (Mean ± SD; Median, 1.1 ± 3.8; 0 pg/mL), whereas group B had high pOSM levels (1548 ± 1999; 767 pg/mL) [t-test: p < 0.01]. The percentage of pOSM levels at decannulation relative to baseline OSM levels was significantly higher in those who died (116.8 ± 68.0; 85.3%) than those who survived (47.6 ± 25.5; 48.9%) [t-test: p = 0.02; Mann-Whitney U Test: p = 0.01]. Conversely, no significant difference was observed in the percentage of pLA levels between those who died (142.9 ± 179.9; 89.8%) and those who survived (79.3 ± 34.3; 81.8%) [t-test: p = 0.31; Mann-Whitney U Test: p = 0.63]. These early findings suggested critical value of absolute and relative pOSM to characterize the inflammatory burden of ARDS patients and the outcome of their V-V ECMO support.

#2668 SUPERIOR BIOCOMPATIBILITY OF REGIONAL CITRATE ANTICOAGULATION COMPARED TO HEPARIN DURING EXPANDED HEMODIALYSIS WITH THERANOVA® MEMBRANE

Abstract Background and Aims Regional citrate anticoagulation (RCA) is known to improve the biocompatibility of hemodialysis (HD) by reducing complement, leukocyte and platelet activation. As continuous exposure to bio-incompatible HD may accelerate cardiovascular disease and increase mortality, improved biocompatibility may be important for clinical outcomes, including long-term survival of chronic HD patients. We found no reports of RCA during expanded hemodialysis (HDx), except our recent pilot study [1]. The aim of our trial was to compare RCA and full dose unfractionated heparin (UFH) during HDx with respect to biocompatibility parameters. Methods We performed a randomized, cross-over trial in 32 patients on chronic HD. Each had 2 HDx procedures with Theranova® membrane: one with UFH and one with RCA. RCA was achieved with 8% trisodium citrate at a flow rate of 150 mL/min. Anticoagulation with UFH was achieved by a bolus of 30 I.U. per kg of body weight, followed by an infusion of 18 I.U. per kg of body weight per hour. Anticoagulation was monitored by post-filter ionized calcium (RCA) or activated partial thromboplastin time (UFH). The primary outcomes were serum concentrations of C3a (marker of complement activation) and myeloperoxidase (MPO; marker of leukocyte activation) and plasma concentrations of platelet factor 4 (PF4; marker of platelet activation). Blood was drawn prior to, after 15 minutes and just before the end of HDx. The area under the biocompatibility marker concentration-time curve (AUC) was calculated for an individual biocompatibility marker from all three concentrations using the linear trapezoidal rule. As a secondary outcome, adhesion of blood cells and activation of clotting on inner surfaces of dialyzer fibers were visualized using scanning electron microscopy (SEM) in a subset of patients (n = 5). Biocompatibility parameters were compared between the groups using paired t-test or Wilcoxon signed-rank test. Results The trial included 22 males and 10 females. Patients were of mean age 65 ± 12 years with median dialysis vintage of 31 (IQR 17-89) months. Mean duration of HDx was 4.4 ± 0.4 hours (always the same for an individual patient). All patients had arteriovenous fistulas. Analysis of PF4 included only 8 patients, while the rest had concentrations of PF4 above the measuring range, probably due to unspecific platelet degranulation because of preanalytical factors. The concentrations of C3a, MPO and PF4 rose significantly after 15 minutes of HDx with UFH, while we observed no significant rises during HDx with RCA (Table 1). The AUC for biocompatibility markers were significantly smaller during HDx with RCA in comparison to UFH: 1163.0 ± 306.8 vs. 1444.1 ± 385.9 µgh/L for C3a (p&amp;lt;0.001); 78.2 (IQR 39.0-131.2) vs. 275.1 (IQR 136.9-492.9) µgh/L for MPO (p&amp;lt;0.001) and 100.0 (IQR 51.8-196.6) vs. 379.4 (IQR 295.0-553.0) IUh/mL for PF4 (p = 0.04). SEM showed almost no deposition on the membrane with comparable clotting score: 2 (IQR 1-3) for RCA vs. 2 (IQR 1-2.25) for UFH (p = 0.40). Conclusion Given the higher activation of complement, leukocytes and platelets during HDx with UFH, we conclude that RCA offers superior biocompatibility compared to UFH during HDx. Whether improved biocompatibility has clinical relevance remains to be explored.

Hemocompatibility of cellulose phosphate aerogel membranes with potential use in bone tissue engineering.

Cellulose is an appealing material for tissue engineering. In an attempt to overcome some obstacles with cellulose II cell scaffolding materials related to insufficient biomineralization, lack of micron-size porosity, and deficiency in surface charge, respective solutions have been proposed. These included covalent phosphorylation of different cellulose materials targeting relatively low degrees of substitution (DS 0.18-0.23) and processing these cellulose derivatives into scaffolding materials by a dissolution/coagulation approach employing the hitherto rarely used TBAF/DMSO/H2O system for cellulose dissolution. Here, we report bioactivity and preliminary hemocompatibility testing of dual-porous cellulose phosphate aerogels (contrasted with the phosphate-free reference) obtained via coagulation (water/ethanol), solvent exchange and scCO2 drying. Deposition of hydroxyapatite from simulated body fluid (7 days of immersion) revealed good bioactivity (1.5-2.2mg Ca2+ per mg scaffold). Incubation of the scCO2-dried and rehydrated scaffolding materials in heparin anticoagulated human whole blood was conducted to study selected parameters of hemostasis (prothrombin F1+2 fragment, PF4, count of thrombocyte-leukocyte conjugates) and inflammatory response (C5a fragment, leukocyte activation marker CD11b). Adhesion of leukocytes on the surface of the incubated substrates was assessed by scanning electron and fluorescence microscopy (DAPI staining). The results suggest that phosphorylation at low DS does not increase platelet activation. However, a significant increase in platelet activation and thrombin formation was observed after a certain fraction of the negative surface charges had been compensated by Ca2+ ions. The combination of both phosphorylation and calcification turned out to be a potent means for controlling the inflammatory response, which was close to baseline level for some of the studied samples.

Unraveling CD69 signaling pathways, ligands and laterally associated molecules.

CD69 is an early leukocyte activation marker involved in the regulation of the immune response. Initial in vitro studies evaluated its function using monoclonal antibodies until knock-out mice were developed. Subsequently, four ligands for CD69 have been identified, namely galectin-1, S100A8/S100A9 complex, myosin light chains 9 and 12, and oxidized low-density lipoproteins. In addition, several molecules are laterally associated with and regulated by CD69, including calreticulin and two transmembrane receptors, sphingosine-1-phosphate receptor (S1P1) and the heterodimeric amino acid transporter complex SLC7A5-SLC3A2 (LAT1-CD98). Recently, CD69 engagement has been shown to induce the expression of the immunoregulatory receptor programmed cell death-1 (PD-1) in T cells. The molecular signaling induced by CD69 has been explored in different scenarios and cell types. This review provides a perspective on the molecular pathways, ligands and cellular functions known to be regulated by CD69.

The Decrease in Serum sRAGE Levels Upon Smoking is Associated with Activated Neutrophils

The serum level of the soluble Receptor for Advanced Glycation End-products (sRAGE) is a promising blood biomarker for the development, severity, and progression of chronic obstructive pulmonary disease (COPD). However, cigarette smoking causes a nearly instant drop in circulating sRAGE levels, strongly impacting on the variability in sRAGE levels. In the current study, we investigated the possible mechanism behind the sudden drop in sRAGE upon smoking. We showed that the number of activated neutrophils in blood significantly increases within two hours upon smoking three cigarettes within one hour. Furthermore, an increased expression of the leukocyte activation marker CD11b, which is a known ligand for RAGE, was observed upon smoking. Additionally, the in vitro activation of neutrophils increased their capacity to bind sRAGE. Together, these data indicate that smoking activates neutrophils in the circulation with concomitant upregulation of the RAGE ligand CD11b, leading to reduced levels of sRAGE in serum.

Circulating T Cell Activation and Exhaustion Markers Are Associated With Radiation Pneumonitis and Poor Survival in Non-Small-Cell Lung Cancer.

IntroductionPersistent inflammation and immune activation in the lungs are associated with adverse outcomes such as radiation pneumonitis (RP) and poor survival in non-small-cell lung cancer (NSCLC) patients. However, it is unknown how this is reflected by leukocyte activation markers in serum.ObjectiveThe aim was to evaluate the serum levels of activation of different leukocyte subsets and to examine those in relation to the pathogenesis of RP and survival in NSCLC.MethodsWe analyzed the serum levels of MPO, sCD25, sTIM-3, sPD-L1, sCD14, sCD163, CCL19 and CCL21 in 66 inoperable NSCLC patients with stage IA-IIIA disease. The patients were treated with stereotactic body radiation therapy (SBRT) or concurrent chemoradiation therapy (CCRT), followed by regular blood sampling for 12 months after treatment and for 5 years for survival.ResultsNineteen (29%) patients developed RP, which occurred more frequently and earlier in patients receiving CCRT than in those receiving SBRT. Increases in sCD25, sTIM-3 and CCL21 levels were observed at the last 6 months of follow-up in patients who had RP after SBRT. Patients who had RP after CCRT had higher sTIM-3 levels during the first 3 months of follow-up. Baseline sCD25 was independently associated with both 2- and 5-year mortality outcomes, while baseline sTIM-3 was independently associated with 2-year mortality.ConclusionWe showed that T cell activation and exhaustion markers such as sCD25 and sTIM-3 are enhanced in patients developing RP and are associated with poor survival in NSCLC.

Cellular immunophenotype of major spine surgery in adults.

ASD reconstructions are a major, sterile traumatic insult, likely causing perturbations to the immune systems. The immune response to surgery is associated with outcomes. The purpose of this study was to examine for a detectable immune signature associated with ASD surgery. Consecutive patients undergoing ASD surgery were approached and enrolled. Peripheral blood was drawn before incision, 4h after, and 24h after incision. Blood was stabilized and comprehensive flow cytometric immunophenotyping performed. Leukocyte population frequency, absolute number and activation marker expression were defined. Immunologic features were defined and analyzed by hierarchical clustering and principal component analysis (PCA). Changes over time were evaluated by repeated measures ANOVA (RMANOVA) and were corrected for a 1% false discovery rate. Post hoc testing was by Dunn's test. p values of < = 0.05 were considered significant. Thirteen patients were enrolled; 11(85%) F, 65.4years (± 7.5), surgical duration 418 ± 83min, EBL 1928 ± 1253mL. Hierarchical clustering and PCA found consistent time from incision-dependent changes. HLA-DR and activating co-stimulatory molecule CD86 were depressed at 4h and furthermore at 24h on monocyte surfaces. CD4 + HLA-DR + T cells, but not CD8 +, increased over time with increased expression of PD-1 at 4 and 24h. Despite surgery and patient heterogeneity, we identified an immune signature associated with the sterile trauma of ASD surgery. Circulating leukocyte populations change in composition and signaling protein expression after incision and persisting to 24h after incision, suggesting an immunocompromised state. Further work may determine relationships between this state and poor outcomes after surgery.

The cellular and molecular basis of CD69 function in anti-tumor immunity.

Cancer immunotherapy utilizes our immune system to attack cancer cells and is an extremely promising strategy for cancer treatment. Although immune-checkpoint blockade, such as anti-PD-1 (programmed cell death 1) antibody, has demonstrated significant enhancement of anti-tumor immunity and has induced notable clinical outcomes, its response rates remain low, and adverse effects are always a matter of concern; therefore, new targets for cancer immunotherapy are always desired. In this situation, new concepts are needed to fuel the investigation of new target molecules for cancer immunotherapy. We propose that CD69 is one such target molecule. CD69 is known to be an activation marker of leukocytes and is also considered a crucial regulator of various immune responses through its interacting proteins. CD69 promotes T-cell retention in lymphoid tissues via sphingosine-1-phosphate receptor 1 (S1P1) internalization and also plays roles in the pathogenesis of inflammatory disorders through interacting with its functional ligands Myl9/12 (myosin light chains 9, 12a and 12b). In anti-tumor immunity, CD69 is known to be expressed on T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TDLNs). We revealed that CD69 negatively regulates the effector function of intratumoral T cells and importantly controls the 'exhaustion' of CD8 T cells. In addition, we and others showed that either CD69 deficiency or the administration of anti-CD69 monoclonal antibody enhances anti-tumor immunity. Thus, CD69 is an attractive target for cancer immunotherapy.

N-Oleoyl dopamine induces IL-10 via central nervous system TRPV1 and improves endotoxemia and sepsis outcomes

BackgroundThe transient receptor potential vanilloid 1 (TRPV1) participates in thermosensation and inflammatory pain, but its immunomodulatory mechanisms remain enigmatic. N-Oleoyl dopamine (OLDA), an endovanilloid and endocannabinoid, is a TRPV1 agonist that is produced in the central nervous system and the peripheral nervous system. We studied the anti-inflammatory effects and TRPV1-dependent mechanisms of OLDA in models of inflammation and sepsis.MethodsMice were challenged intratracheally or intravenously with LPS, or intratracheally with S. aureus to induce pneumonia and sepsis, and then were treated intravenously with OLDA. Endpoints included plasma cytokines, leukocyte activation marker expression, mouse sepsis scores, lung histopathology, and bacterial counts. The role of TRPV1 in the effects of OLDA was determined using Trpv1−/− mice, and mice with TRPV1 knockdown pan-neuronally, in peripheral nervous system neurons, or in myeloid cells. Circulating monocytes/macrophages were depleted using clodronate to determine their role in the anti-inflammatory effects of OLDA in endotoxemic mice. Levels of exogenous OLDA, and of endovanilloids and endocannabinoids, at baseline and in endotoxemic mice, were determined by LC–MS/MS.ResultsOLDA administration caused an early anti-inflammatory response in endotoxemic and septic mice with high serum levels of IL-10 and decreased levels of pro-inflammatory cytokines. OLDA also reduced lung injury and improved mouse sepsis scores. Blood and lung bacterial counts were comparable between OLDA- and carrier-treated mice with S. aureus pneumonia. OLDA’s effects were reversed in mice with pan-neuronal TRPV1 knockdown, but not with TRPV1 knockdown in peripheral nervous system neurons or myeloid cells. Depletion of monocytes/macrophages reversed the IL-10 upregulation by OLDA in endotoxemic mice. Brain and blood levels of endovanilloids and endocannabinoids were increased in endotoxemic mice.ConclusionsOLDA has strong anti-inflammatory actions in mice with endotoxemia or S. aureus pneumonia. Prior studies focused on the role of peripheral nervous system TRPV1 in modulating inflammation and pneumonia. Our results suggest that TRPV1-expressing central nervous system neurons also regulate inflammatory responses to endotoxemia and infection. Our study reveals a neuro-immune reflex that during acute inflammation is engaged proximally by OLDA acting on neuronal TRPV1, and through a multicellular network that requires circulating monocytes/macrophages, leads to the systemic production of IL-10.

Oncostatin M: a Potential Biomarker to Predict Infection in Patients with Left Ventricular Assist Devices.

Infection is a serious adverse event limiting left ventricular assist device (LVAD) therapy in advanced heart failure patients, but a reliable means to identify patients at increased risk of infection is still lacking. We hypothesized that preoperative elevated levels of plasma Oncostatin M (OSM), a cytokine marker of leukocyte activation and inflammation, would be predictive of subsequent infection. We measured plasma OSM in 41 LVAD patients one day before LVAD implantation and postoperatively over two months. Preoperative plasma OSM levels were normal in 27 patients (group A, 4.9 ± 3.2 pg/ml) but elevated in 14 patients (group B, 1649.0 ± 458.9 pg/ml) ( p = 0.003). Early postoperative levels rose in both groups and declined rapidly in group A, with group B declining slowly over two months. Significantly more infections developed in group B than group A patients over two months postimplantation ( p = 0.004). No other routine clinical assessment or laboratory testing afforded this differentiation. These findings suggest that preoperative plasma OSM levels may assist in identifying patients at increased risk of infections after LVAD implantation.

Fibroblasts Regulate Mast Cell Activation in Nasal Polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory sinus disease associated with mast cell (MC) expansion and activation. We find that MCs in the subepithelium of nasal polyps exhibit elevated CD69 expression, a known leukocyte activation marker. However, it is unclear which factors within the polyp drive this activation. As subepithelial MCs colocalize with fibroblasts, cells known to influence MC development and mediator release, we hypothesized that nasal polyp fibroblasts could directly drive MC activation.

Intrinsic and Extrinsic Cell Apoptotic Pathways in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis: A Systematic Review.

We aimed to verify the influence of intrinsic and extrinsic cell apoptotic pathways on the inhibition of cellular apoptosis in patients with tropical spastic paralysis/myelopathy related to human T cell lymphotropic virus type 1. The databases accessed were PubMed, Scopus, Science Direct, and Web of Science. Neither the time of publishing nor the language of the articles was limited. The descriptors used for this systematic literature review were: Tropical Paraparesis, Proto-Oncogenic Protein C, Bcl-2, Bcl-X Protein, Bax protein, Fas ligand (FasL) protein, Fas receptor, TNF-related apoptosis-inducing ligand and Fas-associated protein with death domain (FADD)-like apoptosis regulating. The search resulted in 546 articles from which 9 articles were selected for analysis; ranging from serum levels of Bcl-2, Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) measured by enzyme-linked immunosorbent assay and the levels of cellular expression of Bcl-2 and Bcl-xL the TCD4+ lymphocytes accessed by western blot. Most studies accessed either gene expression or polymorphism of Fas, FasL, and TRAIL in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas one study used flow cytometry and fluorescence to determine Fas expression. Increased Bcl-xL expression inhibited T lymphocyte apoptosis, whereas Bcl-2, serum levels, and cellular expression did not influence T lymphocyte apoptosis and serum levels of Fas were significantly higher and associated with markers of leukocyte activation in patients with HAM/TSP. In addition, Fas polymorphism (FAS-670AA) was associated with higher proviral load. There is a need for additional research on this issue since the number of patients was small and the studies presented higher heterogeneity.

The relationship between specialized pro-resolving lipid mediators, morbid obesity and weight loss after bariatric surgery

Obesity and diabetes are associated with chronic inflammation. Specialized pro-resolving lipid mediators (SPMs)—resolvins (Rv), protectins (PD) and maresins (MaR)—actively resolve inflammation. Bariatric surgery achieves remission of diabetes, but mechanisms are unclear. We measured SPMs and proinflammatory eicosanoid levels using liquid chromatography-tandem mass spectrometry in 29 morbidly obese subjects (13 with diabetes) and 15 nondiabetic, mildly obese subjects. Compared to the mildly obese, the morbidly obese had higher levels of SPMs—RvD3, RvD4 and PD1—and white blood cells (WBC) and platelets. Post-surgery, SPM and platelet levels decreased in morbidly obese nondiabetic subjects but not in diabetic subjects, suggesting continued inflammation. Despite similar weight reductions 1 year after surgery (44.6% vs. 46.6%), 8 diabetes remitters had significant reductions in WBC and platelet counts whereas five non-remitters did not. Remitters had a 58.2% decrease (p = 0.03) in 14-HDHA, a maresin pathway marker; non-remitters had an 875.7% increase in 14-HDHA but a 36.9% decrease in MaR1 to a median of 0. In conclusion, higher levels of RvD3, PD1 and their pathway marker, 17-HDHA, are markers of leukocyte activation and inflammation in morbid obesity and diabetes and diminish with weight loss in nondiabetic but not diabetic subjects, possibly representing sustained inflammation in the latter. Lack of diabetes remission after surgically-induced weight loss may be associated with reduced ability to produce MaR1 and sustained inflammation.

Distinct patterns of soluble leukocyte activation markers are associated with etiology and outcomes in precapillary pulmonary hypertension

Activation of inflammatory processes has been identified as a major driver of pulmonary vascular remodeling that contributes to the development of precapillary pulmonary hypertension (PH). We hypothesized that circulating markers of leukocyte activation, reflecting monocytes/macrophages (sCD163, sCD14), T-cells (sCD25) and neutrophils (myeloperoxidase [MPO], neutrophil gelatinase-associated lipocalin [NGAL]) activity, could give prognostic information in precapillary PH. Circulating markers of leucocyte activation, sCD163, sCD14, sCD25, MPO and NGAL were measured by enzyme immunoassays in plasma from patients with idiopathic PAH (IPAH; n = 30); patients with PAH related to associated conditions (APAH; n = 44) and patients with chronic thromboembolic PH (CTEPH) (n = 32), and compared with 23 healthy controls. Markers of leucocyte activation were elevated in precapillary PH with particularly high levels in APAH. The elevated levels of monocyte/macrophage marker sCD163 was independently associated with poor long-term prognosis in the group as a whole, and elevated levels of sCD25 was associated with poor prognosis in APAH, while elevated levels of sCD163 and NGAL was associated with poor prognosis in IPAH and CTEPH. Our data show leucocyte activation in precapillary PH with different profiles and impact on prognosis according to etiology. The association of sCD163 with poor outcome in fully adjusted model may be of particular interest.

Leukocyte Activation and Antioxidative Defense Are Interrelated and Moderately Modified by n-3 Polyunsaturated Fatty Acid-Enriched Eggs Consumption-Double-Blind Controlled Randomized Clinical Study.

This placebo-controlled, double-blind, randomized, interventional study investigated the effects of low/intermediate doses of n-3 polyunsaturated fatty acids (PUFAs) on the endothelial function, markers of leukocyte activation, and oxidative status following dietary intake of n-3 PUFA-enriched hen eggs in young healthy individuals. Twenty young healthy adults of both sexes who consumed n-3 PUFA-enriched hen eggs (two eggs per day, for three weeks, total of approximately 407 mg/day n-3 PUFAs) or regular eggs (two eggs per day for three weeks, total of approximately 75 mg/day n-3 PUFAs) participated in this study. Skin microvascular endothelium-independent and endothelium-dependent vasodilation were assessed by laser Doppler flowmetry. Serum lipid profile and content of free fatty acids, markers of leukocyte activation, biochemical parameters of oxidative stress, as well as antioxidative enzymes serum activity were measured before and after respective dietary protocol. The results of this study revealed significant differences in the markers of leukocyte activation (such as CD11a/LFA-1) and antioxidative defense, which are related to increased intake of n-3 PUFAs, providing the evidence that consumption of nutritionally enriched hen eggs may affect physiological processes related to oxidative balance. The absence of significant changes in microvascular reactivity following supplementation with a low-intermediate dose of n-3 PUFAs, unlike in our previous studies where functional eggs contained ~1 g of n-3 PUFA, suggests the existence of a dose-dependent effect.

The effect of hyperoxia on inflammation and platelet responses in an ex vivo extracorporeal membrane oxygenation circuit.

Use of extracorporeal membrane oxygenation (ECMO) is expanding, however, it is still associated with significant morbidity and mortality. Activation of inflammatory and innate immune responses and hemostatic alterations contribute to complications. Hyperoxia may play a role in exacerbating these responses. Nine ex vivo ECMO circuits were tested using fresh healthy human whole blood, with two oxygen levels: 21% inspired fraction of oxygen (FiO2 ; mild hyperoxia; n=5) and 100% FiO2 (severe hyperoxia; n=4). Serial blood samples were taken for analysis of platelet aggregometry, leukocyte activation, inflammatory, and oxidative stress markers. ECMO resulted in reduced adenosine diphosphate- (P<.05) and thrombin receptor activating peptide-induced (P<.05) platelet aggregation, as well as increasing levels of the neutrophil activation marker, neutrophil elastase (P=.013). Additionally, levels of the inflammatory chemokine interleukin-8 were elevated (P<.05) and the activity of superoxide dismutase, a marker of oxidative stress, was increased (P=.002). Hyperoxia did not augment these responses, with no significant differences detected between mild and severe hyperoxia. Our ex vivo model of ECMO revealed that the circuit itself triggers a pro-inflammatory and oxidative stress response, however, exposure to supra-physiologic oxygen does not amplify that response. Extended-duration studies and inclusion of an endothelial component could be beneficial in characterizing longer term changes.

Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition.

There is evidence for the effects of platelet inhibition on innate immune activation. Circulating microRNAs (miRNAs) have been implicated as markers of platelet and leukocyte activation. In the present study, we assessed the effects of P2Y12 inhibitors on platelet and leukocyte miRNAs during endotoxemia. Healthy volunteers were randomly assigned to receive oral ticagrelor (n = 10), clopidogrel (n = 8) or no drug (n = 8) for one week, followed by an intravenous bolus of 2 ng/kg endotoxin. Serum was collected at baseline, after one week of antiplatelet treatment and 6 and 24 h after endotoxin administration. MiRNAs were screened using LNA-based qPCR, followed by TaqMan-qPCR validation of candidates. Clinical validation was performed in 41 sepsis patients. Platelet-enriched miR-197, miR-223 and miR-223* were decreased in volunteers following antiplatelet therapy. Endotoxin increased platelet miRNAs, whilst the opposite effect was seen for leukocyte-enriched miR-150. Neither of these endotoxin-mediated effects were altered by P2Y12 inhibitors. Sepsis patients with fatal outcomes (n = 12) had reduced miR-150 levels compared with survivors (n = 29). In conclusion, we show that miR-150 is downregulated in experimental endotoxemia and can predict survival in sepsis but is unaffected by P2Y12 inhibition. While P2Y12 inhibition reduces platelet-associated miRNAs in healthy volunteers, it fails to attenuate the response of platelet miRNAs to endotoxemia.