Neutrophil Activation In Patients Research Articles

Studies of altered phagocytic and bactericidal function of neutrophils in patients with post-COVID syndrome, depending on the degree of lung damage detected by computer tomography in acute period of coronavirus infection

By the beginning of May 2023, the epidemic situation regarding COVID-19 was assessed by WHO as favorable, thus enabling to cancel the international emergency state and declare the end of the pandemic on May 5, 2023. Currently, COVID-19 is becoming a seasonal infection. COVID-19 is often accompanied by imbalanced immune response including decreased number of white blood cells and functional changes of immune cells, e.g., affecting activity of neutrophils, including their phagocytic abilities. About 48% of patients who suffered from COVID-19 developed a post-COVID syndrome which is referred to a number of persistent immune disorders which persist for more than 6-12 months after acute infection. Post-COVID-19 disorders of the cellular component of the immune system can manifest with decreased levels of various lymphocyte subpopulations, e.g., NK cells, T cytotoxic lymphocytes, reduced expression of pan-leukocyte CD46 marker on T lymphocytes, as well as changed number and functionality of neutrophils involved in the antiviral immune response. The neutrophils, as a key population of immune system, play an important role in the response to the infection. So far, however, the mechanisms responsible for their functional changes in the context of the post-COVID syndrome remain poorly understood. The results of the study showed that, in patients with post-COVID syndrome, the neutrophil-lymphocyte ratio (NLR) remains within normal range in patients with lung damage of less than 50%. In patients with lung damage of more than 50%, there is a trend for increase of this index, which may suggest a need for in-depth study of immunity in these groups of patients. Statistically significant differences in the innate immunity indexes were detected with respect to phagocytic and bactericidal activity of neutrophils in patients over post-COVID period who showed different extent of lung damage during the acute phase of coronavirus infection.

Study of the effect of a drug from the macromycetes Ganoderma lucidum on the phagocytic activity of peripheral blood neutrophils in patients with year-round allergic rhinitis

Modern complex therapy of patients with persistent (year-round, chronic) allergic rhinitis (AR), based on the use of antihistamines and intranasal glucocorticosteroids, in many cases is not effective enough, since drug therapy acts only on individual links in the pathogenesis, without preventing the progression of the allergic disease. At the same time, the quantitative and reserve capabilities of neutrophil granulocytes are depleted, which increases the level of antigenic load in patients with AR. The aim of this study is to investigate the phagocytic activity of neutrophils in patients with AR who received basic therapy and patients who received basic therapy and a course of the drug from the macromycetes Ganoderma lucidum "Astmagan". We examined 40 patients of both sexes with AR, the duration of the disease ranged from 6 months to 2 years. Patients were divided into 2 equal groups: The 1st group consisted of patients who received basic therapy (oral Loratadine and intranasal Nasonex). The 2nd group consisted of patients who received basic therapy and a course of "Astmagan". The treatment course lasted 90 days. The control group consisted of 25 healthy individuals. After the treatment, patients in both groups showed a statistically significant positive dynamics of phagocytic system activity: in group 1, the phagocytic index (FI) increased by 30 % (p=0.001), in the second group this indicator reached the normal level. Also, in patients of group 1, the phagocytic number (PN) increased by 35 % (p=0.0001), but did not reach the normal level.
 According to the results of comparative evaluation, a greater efficacy of the treatment complex with the additional use of "Astmagan" was established, which is manifested in the restoration of phagocytic activity of peripheral blood neutrophils, the effect persists for 6 months after treatment.

Salivary levels of azurocidin and soluble azurophilic granules in periodontal disease.

Periodontitis is an infection-driven inflammatory condition of the periodontium. Neutrophils are one of the most important first-line immune cells that protect against pathogen microorganisms in the saliva, but they may also mediate tissue death in inflammatory disorders. The aim of our study was to estimate salivary levels of azurocidin and extracellular azurophilic granules cluster of differentiation (CD63) as biomarkers of neutrophil activation in patients with periodontal diseases and to study the correlation between the levels of these two biomarkers and clinical periodontal parameters. The study included 60 patients with periodontal disease (30 patients with periodontitis and 30 with gingivitis) and 25 healthy controls. The assessed parameters were bleeding on probing, the plaque index, clinical attachment loss, and probing pocket depth. Saliva samples were taken from each study participant, and azurocidin and CD63 levels were measured using ELISA. Azurocidin and CD63 levels were significantly higher in patients with periodontitis and patients with gingivitis than in controls (P < 0.05), and significantly higher in patients with periodontitis than in patients with gingivitis (P < 0.05). Moreover, we found a significant positive correlation between the two biomarkers with clinical attachment loss in the periodontitis group. This study has shown that increased salivary azurocidin and extracellular CD63 levels are associated with enhanced innate response in periodontal disease and can be considered biomarkers of neutrophil activation.

Abstract 16734: Patterns of Inflammatory Activation in Cardiovascular Disease: Insights From the Randomized Colchicine PCI Trial

INTRODUCTION: Recent studies demonstrate that neutrophils accumulate in atherosclerotic lesions and that neutrophil counts correlate with disease severity. The randomized double-blind Colchicine-PCI study demonstrated that colchicine attenuated systemic inflammation (IL-6, CRP) after percutaneous coronary intervention compared with placebo. GOALS/AIMS: We characterized neutrophil activity in patients across a spectrum of cardiovascular phenotypes, including severity of coronary artery disease (CAD) and presentation of acute myocardial infarct (AMI). METHODS: The Colchicine-PCI biomarker sub-study enrolled 478 participants with pretreatment assessment of inflammatory biomarkers. White blood cell count and differential were performed using a hematology analyzer. Soluble markers of neutrophil activity, including neutrophil extracellular traps (NETs), and systemic inflammation were assessed using multiplex assays. Neutrophil surface markers of activity and neutrophil platelet aggregates were assessed using an Accuri C6 flow cytometer. RESULTS: We observed neutrophil concentration, adhesion, transmigration, and activation, as well as increased NETs complexes; but no difference in extent of neutrophil platelet aggregation or systemic inflammation in patients with versus without obstructive CAD. Conversely, in patients with versus without AMI, neutrophil markers of adhesion and transmigration did not differ, but systemic markers of inflammation and neutrophil activation were elevated. CONCLUSION: This single-site, prospective study is the largest neutrophil biorepository in patients with cardiovascular risk factors. Inflammatory activation, and particularly neutrophil profiles, differ in patients with obstructive CAD and in the setting of AMI.

Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19

ABSTRACT Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Neutrophil activation in patients treated with endovascular therapy is associated with unfavorable outcomes and mitigated by intravenous thrombolysis

BackgroundAccumulating evidence indicates that neutrophil activation (NA) contributes to microvascular thromboinflammation in acute ischemic stroke (AIS) due to a large vessel occlusion. Preclinical data have suggested that intravenous thrombolysis (IVT)...

Lipocalin-2 and neutrophil activation in pancreatic cancer cachexia.

Cancer cachexia is a multifactorial syndrome characterized by body weight loss and systemic inflammation. The characterization of the inflammatory response in patients with cachexia is still limited. Lipocalin-2, a protein abundant in neutrophils, has recently been implicated in appetite suppression in preclinical models of pancreatic cancer cachexia. We hypothesized that lipocalin-2 levels could be associated with neutrophil activation and nutritional status of pancreatic ductal adenocarcinoma (PDAC) patients. Plasma levels of neutrophil activation markers calprotectin, myeloperoxidase, elastase, and bactericidal/permeability-increasing protein (BPI) were compared between non-cachectic PDAC patients (n=13) and cachectic PDAC patients with high (≥26.9 ng/mL, n=34) or low (<26.9 ng/mL, n=34) circulating lipocalin-2 levels. Patients' nutritional status was assessed by the patient-generated subjective global assessment (PG-SGA) and through body composition analysis using CT-scan slices at the L3 level. Circulating lipocalin-2 levels did not differ between cachectic and non-cachectic PDAC patients (median 26.7 (IQR 19.7-34.8) vs. 24.8 (16.6-29.4) ng/mL, p=0.141). Cachectic patients with high systemic lipocalin-2 levels had higher concentrations of calprotectin, myeloperoxidase, and elastase than non-cachectic patients or cachectic patients with low lipocalin-2 levels (calprotectin: 542.3 (355.8-724.9) vs. 457.5 (213.3-606.9), p=0.448 vs. 366.5 (294.5-478.5) ng/mL, p=0.009; myeloperoxidase: 30.3 (22.1-37.9) vs. 16.3 (12.0-27.5), p=0.021 vs. 20.2 (15.0-29.2) ng/mL, p=0.011; elastase: 137.1 (90.8-253.2) vs. 97.2 (28.8-215.7), p=0.410 vs. 95.0 (72.2-113.6) ng/mL, p=0.006; respectively). The CRP/albumin ratio was also higher in cachectic patients with high lipocalin-2 levels (2.3 (1.3-6.0) as compared to non-cachectic patients (1.0 (0.7-4.2), p=0.041). Lipocalin-2 concentrations correlated with those of calprotectin (rs =0.36, p<0.001), myeloperoxidase (rs =0.48, p<0.001), elastase (rs =0.50, p<0.001), and BPI (rs =0.22, p=0.048). Whereas no significant correlations with weight loss, BMI, or L3 skeletal muscle index were observed, lipocalin-2 concentrations were associated with subcutaneous adipose tissue index (rs =-0.25, p=0.034). Moreover, lipocalin-2 tended to be elevated in severely malnourished patients compared with well-nourished patients (27.2 (20.3-37.2) vs. 19.9 (13.4-26.4) ng/mL, p=0.058). These data suggest that lipocalin-2 levels are associated with neutrophil activation in patients with pancreatic cancer cachexia and that it may contribute to their poor nutritional status.

Biomarkers of Neutrophil Activation in Patients with Symptomatic Chronic Peripheral Artery Disease Predict Worse Cardiovascular Outcome

Neutrophils play a role in cardiovascular (CV) disease. However, relatively scant evidence exists in the setting of peripheral artery disease (PAD). The aims of this study were to measure biomarkers of neutrophil activation in patients with symptomatic chronic PAD compared with healthy controls, to assess their association with PAD severity, and to evaluate their prognostic value in patients with PAD. The following circulating markers of neutrophil degranulation were tested: polymorphonuclear neutrophil (PMN) elastase, neutrophil gelatinase-associated lipocalin (NGAL), and myeloperoxidase (MPO). Neutrophil extracellular traps (NETs) were quantified by measuring circulating MPO-DNA complexes. Patients with PAD underwent a comprehensive series of vascular tests. The occurrence of 6-month major adverse CV (MACE) and limb events (MALE) was assessed. Overall, 110 participants were included, 66 of which had PAD. After adjustment for conventional CV risk factors, PMN-elastase (adjusted odds ratio [OR]: 1.008; 95% confidence interval [CI]: 1.002-1.015; p = 0.006), NGAL (adjusted OR: 1.045; 95%CI: 1.024-1.066; p < 0.001), and MPO (adjusted OR: 1.013; 95%CI: 1.001-1.024; p = 0.028) were significantly associated with PAD presence. PMN-elastase (adjusted hazard ratio [HR]: 1.010; 95%CI: 1.000-1.020; p = 0.040) and MPO (adjusted HR: 1.027; 95%CI: 1.004-1.051; p = 0.019) were predictive of 6-month MACE and/or MALE. MPO displayed fair prognostic performance on receiver operating characteristic (ROC) curve analyses, with an area under the curve (AUC) of 0.74 (95%CI: 0.56-0.91) and a sensitivity and specificity of 0.80 and 0.65, respectively, for a cut-off of 108.37 ng/mL. MPO-DNA showed a weak inverse correlation with transcutaneous oximetry (TcPO2) on proximal foot (adjusted ρ -0.287; p = 0.032). In conclusion, in patients with symptomatic chronic PAD, enhanced neutrophil activity may be associated with an increased risk of acute CV events, rather than correlate with disease severity. Further research is needed to clarify the role of neutrophils in PAD natural history.

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.

Analysis of neutrophil functional activity in patients with chronic polyposis rhinosinusitis after treatment with interferon gamma.

Chronic polyposis rhinosinusitis (CPRS) is an inflammatory disease of the nose and paranasal sinuses, accompanied by the formation and recurrent growth of polyps. PDRS is an urgent medical problem, because it is difficult to treat and is accompanied by constant exacerbations. The important role of neutrophil granulocytes in the pathogenesis of CPRS has been proved, as they are the first line of defense in response to tissue damage and active participants in the pathological process. There is evidence of successful use of immunocorrectors in the treatment of patients with CPPS, but they are often prescribed without regard to possible pathogenetic mechanisms of the disease. One of the promising immunomodulators of local use is a preparation of human recombinant interferon gamma. It is known that interferon gamma is able to activate neutrophils due to the receptors to this cytokine, which are located on their surface. The aim of the study was to investigate the functional activity of neutrophils in patients with CPPS and the effect of human recombinant interferon gamma on these indicators. Thirty-five patients with CHRS were examined before and after therapy with intranasal interferon gamma. The control group included 30 healthy subjects. Functional activity of neutrophils was studied in whole blood by chemiluminescent method using double stimulation. Patients with CPRS before treatment revealed increased indexes of neutrophils stimulated activity, maximal intensity of cells luminescence, activation coefficient and decreased time of neutrophils output at maximal luminescence. After treatment with intranasal preparation of interferon gamma there was significant decrease of spontaneous and stimulated activity of neutrophils and maximum intensity of cell luminescence. As a result, after the treatment, in patients with CHRS the values of stimulated production of neutrophils and maximum intensity of cell luminescence were reduced to the level of the control group, and the spontaneous activity of neutrophils was even lower than in healthy subjects, while the neutrophils activation factor remained elevated as in patients before therapy. The results obtained testify to normalization of the main indexes of neutrophil functional activity in CHPS patients after treatment with human recombinant interferon gamma.

Immune complex-mediated neutrophil activation in patients with polymyalgia rheumatica.

Neutrophils are important in host defence. However, neutrophils are also linked to inflammation and organ damage. The purpose of this study was to assess whether markers of neutrophil activation are increased in PMR. Levels of immune complexes (IC), calprotectin and neutrophil extracellular traps (NETs) were measured in plasma of healthy individuals (n = 30) and patients with PMR (n = 60), at flare and upon treatment with glucocorticoids using ELISA. Plasma-mediated neutrophil activation was assessed in presence of an FcγRIIA inhibitory antibody (IV.3). Plasma levels of calprotectin and NETs were elevated in PMR (P < 0.001). Mechanistically, neutrophil activation was driven by ICs, present in plasma, able to up-regulate neutrophil activation markers CD66b and CD11b (P < 0.0001) in an FcγRIIA-dependent manner (P < 0.01). Of note, circulating levels of IC correlated with plasma induced CD66b and CD11b (r = 0.51, P = 0.004, and r = 0.46, P = 0.01, respectively) and decreased after glucocorticoid therapy. In contrast to NETs, calprotectin significantly decreased after glucocorticoid therapy (P < 0.001) and was higher in PMR without overlapping GCA compared with patients with overlapping disease (P = 0.014). Interestingly, musculoskeletal involvement was associated with elevated levels of calprotectin before initiation of glucocorticoid therapy (P = 0.036). Neutrophil activation, including NET formation, is increased in PMR, through IC-mediated engagement of FcγRIIA. Clinically, neutrophil activation is associated with musculoskeletal involvement, with calprotectin, but not NETs, being a biomarker of treatment response in PMR patients. In all, IC-mediated neutrophil activation is a central process in PMR pathogenesis identifying potential novel therapeutic targets (FcγRIIA), as well as soluble markers for disease monitoring (calprotectin).

A Role of Immunothrombosis in Acquired Antibody-Mediated Thrombophilia's: Classical Complement and Neutrophil Activation in APS and HIT

Introduction: Heparin induced thrombocytopenia (HIT) and APS (antiphospholipid syndrome) are acquired thrombophilia's characterized by both arterial and venous thromboembolic events (TE). As a common feature APS and HIT share the presence of pathological antibodies, which play a crucial role in disease pathogenesis. In HIT, antibodies are directed to platelet factor (PF) 4 and heparin, respectively, and in APS, antibodies target anti-phospholipids (anti-cardiolipin antibody, anti-Beta2- glycoprotein I and lupus anticoagulant). There is growing evidence that complement- and neutrophil activation in the form of neutrophil extracellular traps (NETs) play an important role in the pathogenesis of thrombosis, the so-called "immunothrombosis". Insufficient control with subsequent unrestrained complement activation results in thrombosis as seen in patients with paroxysmal nocturnal hemoglobinuria (PNH). Therapeutic inhibition of complement activation abrogates thrombosis in these patients, emphasizing the important role of complement activation in the pathogenesis of thrombosis. It has been demonstrated, that complement activation products efficiently induce neutrophil activation, and therapeutic complement inhibition in sepsis and PNH prevents neutrophil activation. Here, we investigate the role of complement activation and neutrophil activation in patients with APS and HIT, respectively. Methods To assess the association of complement activation and NET Formation, we measured complement activation products (C4bc, C3bc) and marks for neutrophil activation, such as nucleosomes and human neutrophil elastase-α1- antitrypsin complexes (HNE) by ELISA in plasma of 34 primary APS- and 26 HIT- patients and 31 healthy controls. Groups have been compared using the Mann- Whitney-Rank Test. Values are indicated as median and range, p<0.05 is considered statistically significant. Results Patients with APS and HIT showed sign. higher levels of complement activation products as compared to healthy controls. Complement activation mainly of the classical (C4b/c) and alternate pathway (C3b/c) in APS (11 nM and 263 nM) and in HIT (33 nM and 324 nM) were sign. higher (p<0.0001) as compared to the healthy controls (7 nM and 19 nM). C4bc levels were sign. higher in HIT as compared to APS patients (p<0001), but there was no difference in C3b/c levels between these two groups (see figure 1). Patients with HIT had a sig. (p<0.0001) higher nucleosomes (440 U/ml) and HNE (121 ng/ml) as compared to healthy controls (12 U/ml and 26 ng/ml). HNE levels were sign. (p<0.001) higher in APS patients (37 ng/ml) as compared to healthy controls (26 ng/ml), however no difference was observed in nucleosome levels (12 U/ml vs 14 U/ml) (see figure 2). Based on the number of positive antibodies, eleven APS patients have been stratified as high-risk and 24 APS patients as low- to intermediate risk. C4bc levels were sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in C3b/c levels could be observed between the 2 groups. Similarly, HNE complexes sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in nucleosome levels could be observed between the 2 groups. Six HIT patients (22%) had a diagnosed TE event whereas 17 patients (63%) had no diagnosed TE. In four patients (15%) no clinical data were available, and they have been excluded from subgroup analysis. Interestingly, the measured markers for complement - and neutrophil activation in HIT patient with and without thrombosis were not different. Conclusions We demonstrate complement activation in patients with HIT and APS. Based on the simultaneous raise in nucleosomes and HNE, there is evidence of NET formation in HIT, whereas only weak neutrophil activation can be found in APS. In APS classical complement activation and neutrophil activation correlates with APS risk stratification. Our results demonstrate classical complement pathway- and neutrophil activation in HIT and APS patients. These findings may form the base for new therapeutic strategies targeting complement and neutrophil activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phagocytic and chemiluminescent activity of blood neutrophils in patients with bladder cancer

BACKGROUND: Tumor microenvironment modulates (including with the help of metabolites) the functional activity of the neutrophils that contribute to the reprogramming of the antitumor activity into a protumor one.&#x0D; AIMS: To study the phagocytic and chemiluminescent activity of neutrophils in patients with bladder cancer (BC) under the influence of metabolites of the tumor microenvironment in vitro.&#x0D; MATERIALS AND METHODS: We examined 37 patients with superficial BC (T1,а,isN0M0) and considered 32 healthy individuals as a control group. Neutrophils isolated from their blood were incubated in vitro with lactate, ADP, and glutamate. Phagocytic activity was examined using flow cytometry, and the intensity of the respiratory burst of neutrophils was evaluated via chemiluminescent analysis.&#x0D; RESULTS: In patients with BC, the phagocytic index (PhI) values are reduced compared to the control sample (without in vitro metabolite exposure) and when exposed to glutamate, while the effect of lactate on cells causes an increase in the phagocytic number and PhI. Moreover, under the influence of lactate in vitro, the activity of spontaneous and zymosan-induced chemiluminescence of neutrophils decreases. ADP causes a decrease in spontaneous chemiluminescence only. Finally, under the influence of glutamate, the indicators of spontaneous and induced chemiluminescence decrease.&#x0D; CONCLUSIONS: Under the influence of lactate and ADP (products of tumor cells), the phagocytic activity of a population of immature neutrophils is stimulated, which leads to myeloid suppressor cells that inhibit antitumor immunity. Thus, metabolites of the tumor microenvironment modulate the activity of the respiratory burst of neutrophils in patients with BC.

Enhanced neutrophil activity, but not neutrophil extracellular trap markers, is found in patients with symptomatic chronic peripheral arterial disease

Abstract Background/Introduction Neutrophil degranulation and neutrophil extracellular traps (NETs) are suggested to play a role in cardiovascular (CV) disease development [1]. Human studies showed that markers of NETs, are increased in patients with severe coronary atherosclerosis and predict risk of cardiac events [2]. The role of NETs in peripheral artery disease (PAD) has not been investigated. Purpose To measure circulating markers of neutrophil activity in patients with symptomatic PAD compared with matched healthy controls, evaluate their association with disease severity, and assess their prognostic value. Methods Multicenter, prospective case-control study. Circulating levels of polymorphonuclear neutrophil (PMN)-elastase, neutrophil gelatinase-associated lipocalin (NGAL), myeloperoxidase (MPO), and NETs-associated MPO-DNA complexes were measured in patients with symptomatic PAD and heathy controls. The former underwent comprehensive vascular evaluation including ankle-brachial index (ABI) calculation, transcutaneous oximetry measurement, constant-load treadmill test, 6-minute walking test, pulse wave velocity, and flow-mediated dilatation of the brachial artery. Six-month outcomes were: occurrence of major adverse CV (MACE) and limb events (MALE), ABI reduction ≥0.15, and 6-minute walking distance reduction ≥20 m. Results Overall, 110 participants were included, 66 had symptomatic PAD. Levels of all but one biomarker (MPO-DNA) were significantly higher in patients with PAD compared with healthy controls (Fig. 1). No significant correlation was found between biomarkers and vascular tests in patients with PAD. Baseline PMN-elastase (adjusted hazard ratio [HR]: 1.010; 95% CI: 1.000–1.020; p=0.040) and MPO (adjusted HR: 1.027; 95% CI: 1.004–1.051; p=0.019) were predictive of MACE and/or MALE at 6-month follow-up in patients with symptomatic PAD (Table 1). Conclusions Present data show enhanced neutrophil activity, but not NETs levels, in patients with symptomatic PAD compared to controls. Baseline PMN-elastase and MPO levels were associated with worst CV outcome in patients with chronic PAD. These data suggest a role of neutrophils in PAD development and severity that needs to be further elucidated. Funding Acknowledgement Type of funding sources: None.

The frequencies of infgamma-producing t-helpers 17 and Lp(a) concentration in blood in patients with atherosclerosis

Background and Aims : Chronic inflammation accompanied by the innate and adaptive immune response as well as lipid disorders are key players in atherogenesis. We analyzed the content of minor T-cell populations with pro- and ant-inflammatory activity, the concentration of lipoprotein(a) [Lp(a)] and markers of neutrophil activation in patients with atherosclerosis.

Neutrophil activation in patients with anti-neutrophil cytoplasmic autoantibody-associated vasculitis and large-vessel vasculitis

ObjectiveTo assess markers of neutrophil activation such as calprotectin and N-formyl methionine (fMET) in anti-neutrophil cytoplasmic autoantibody-associated vasculitis (AAV) and large-vessel vasculitis (LVV).MethodsLevels of fMET, and calprotectin, were measured in the plasma of healthy controls (n=30) and patients with AAV (granulomatosis with polyangiitis (GPA, n=123), microscopic polyangiitis (MPA, n=61)), and LVV (Takayasu’s arteritis (TAK, n=58), giant cell arteritis (GCA, n=68)), at times of remission or flare. Disease activity was assessed by physician global assessment. In vitro neutrophil activation assays were performed in the presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H.ResultsLevels of calprotectin, and fMET were elevated in patients with vasculitis as compared to healthy individuals. Levels of fMET correlated with markers of systemic inflammation: C-reactive protein (r=0.82, p<0.0001), and erythrocyte sedimentation rate (r=0.235, p<0.0001). The neutrophil activation marker, calprotectin was not associated with disease activity. Circulating levels of fMET were associated with neutrophil activation (p<0.01) and were able to induce de novo neutrophil activation via FPR1-mediated signaling.ConclusionCirculating fMET appears to propagate neutrophil activation in AAV and LVV. Inhibition of fMET-mediated FPR1 signaling could be a novel therapeutic intervention for systemic vasculitides.

Pediatric systemic lupus erythematosus: phagocytic defect and oxidase activity of neutrophils.

There are limited data on neutrophil function in pediatric-onset systemic lupus erythematosus (pSLE) patients. This study aimed to evaluate phagocytosis and oxidase activity of neutrophils in patients with pSLE. Eighty-seven patients with pSLE and 44 controls were enrolled. Phagocytic activity was assayed using pHrodoTMRed E. coli BioParticles Phagocytosis Kit by flow cytometry. Determination of NADPH oxidase activity was carried out by Dihyrdrorhodamine-123 (DHR-123) flow cytometry assay. Phagocytic activity of patients' neutrophils (mean 76.59%) was lower than that in controls (91.30%) (p < 0.001). Median delta median fluorescence intensity (ΔMFI) and stimulation index (SI) in patients (ΔMFI: 0.09; SI: 2.79) were also decreased compared to controls (ΔMFI: 0.18; SI: 5.00) (p < 0.002; p < 0.001 respectively). Disease activity showed an inverse correlation with phagocytic activity. Oxidase activity was also significantly low (SI DHR < 40) in 16% of patients. No significant correlation was found between oxidative burst and disease activity. Neutrophil function is impaired in patients with pSLE, as evidenced by the markedly reduced phagocytic activity. Phagocytic activity is also inversely correlated with disease activity. The oxidative activity was also reduced but not significantly. Neutrophil phagocytic function is impaired in pediatric-onset systemic lupus erythematosus (pSLE). There is an inverse correlation between disease activity in pSLE and phagocytic activity. NADPH oxidase activity in patients with pSLE did not show significant correlation with disease activity.

The state of functional activity of neutrophils in patients with various variants of carbohydrate metabolism disorders who have had SARS-COV-2S infection

According to the Federal State Statistics Service of the Russian Federation, there has been a significant increase in the prevalence of prediabetes and type 2 diabetes mellitus (DM). Type 2 diabetes is characterized by frequent disability due to the progression of complications of diabetes and high mortality. Domestic and foreign authors have shown that neutrophils play an important role in the development of diabetic micro- and macroangiopathies. It has been shown that in DM, changes in the functional activity of neutrophils are observed, manifested in a decrease in chemotactic activity, changes in phagocytic activity, leukotriene production, and secretion of lysosomal enzymes. According to the results of previous studies, ambiguous data were obtained on the effect of high hyperglycemia in DM on the phagocytic activity of neutrophils. Thus, foreign authors have shown that in DM, when the target levels of glycemic control are not reached, the phagocytic activity of neutrophils is inhibited. Other researchers have found that with DM, activation of the phagocytic function of neutrophils is detected, but at the same time, the reaction of neutrophils to additional stimulation with latex (according to the results of the HCT test), a decrease in the expression of adhesion molecules on their surface and the ability to form extracellular traps occurs. Neutrophils, being the main cells of innate immunity, play an important role in the antiviral response. Patients with impaired carbohydrate metabolism are at high risk of severe course and unfavorable outcome of SARS-CoV-2 infection. The course and outcome of an infectious disease depends on how the effector functions of neutrophils are realized. The aim of the study was to study the features of the functional activity of neutrophils in patients with various variants of carbohydrate metabolism disorders who had SARS-CoV-2 infection. The study included 20 patients with impaired carbohydrate metabolism who had suffered SARS-CoV-2 infection, the control group included 15 clinically healthy volunteers. In all groups, the phagocytic activity of latex particles with a diameter of 1.7 microns was evaluated by neutrophils (phagocytosis activity, phagocytosis intensity, phagocytic number); spontaneous and induced NBT activity of neutrophils by morphological method. A significant increase in the absorption capacity of peripheral blood neutrophils, an increase in the phagocytic activity of leukocytes in the group of patients with impaired carbohydrate metabolism was revealed. A simultaneous increase in the indicators of spontaneous and induced HCT tests was found in the group of patients with impaired glucose tolerance and in the group of patients with type 2 diabetes mellitus. The observed disorders in the phagocytic link are an important factor in reducing the overall immunoreactivity of patients, which determines the long rehabilitation period. Identifying the features of innate immunity in patients with various variants of carbohydrate metabolism disorders who have suffered from SARS-CoV-2 infection will further help develop an individual strategy for the prevention and treatment of these diseases.

Plasma Exchange Therapy Using Solvent Detergent-Treated Plasma: An Observational Pilot Study on Complement, Neutrophil and Endothelial Cell Activation in a Case Series of Patients Suffering from Atypical Hemolytic Uremic Syndrome.

Plasma exchange therapy (PEX) was standard treatment for thrombotic microangiopathy before eculizumab was available and is still widely applied. However, most PEX patients still ultimately progress to end-stage renal disease (ESRD). It has been suggested that infusion of plasma that contains active complement may induce additional complement activation with subsequent activation of neutrophils and endothelial cells, leading to exacerbation of organ damage and deterioration of renal function. This observational pilot study examines the effect of hemodialysis, eculizumab and PEX before and after treatment in plasma of aHUS patients on complement-, neutrophil and endothelial cell activation. Eleven patients were included in this pilot study. Six patients were treated with hemodialysis, 2 patients received regular infusions of eculizumab, and 3 patients were on a regular schedule for PEX. Patients were followed during 3 consecutive treatments. Blood samples were taken before and after patients received their treatment. Complement activation products increased in plasma of patients after PEX, as opposed to patients treated with hemodialysis or eculizumab. Increased levels of complement activation products were detected in omniplasma used for PEX. Additionally, activation of neutrophils and endothelial cells was observed in patients after hemodialysis and PEX, but not in patients receiving eculizumab treatment. In this pilot study we observed that PEX induced complement and neutrophil activation, and that omniplasma contains significant amounts of complement activation products. Additionally, we demonstrate that hemodialysis induces activation of neutrophils and endothelial cells. Complement activation with subsequent neutrophil activation may contribute to the deterioration of organ function and may result in ESRD. Further randomized controlled studies are warranted to investigate the effect of PEX on complement- and neutrophil activation in patients with thrombotic microangiopathy.

Mitochondrial N-formyl methionine peptides associate with disease activity as well as contribute to neutrophil activation in patients with rheumatoid arthritis

ObjectivesLiterature suggests that neutrophils of patients with rheumatoid arthritis (RA) are primed to respond to N-formyl methionine group (formylated peptides). Animal models indicate that formylated peptides contribute to joint damage via neutrophil recruitment and inflammation in joints. Non-steroidal anti-inflammatory drugs are also known to inhibit formyl peptide-induced neutrophil activation. The predominant source of formylated peptides in sterile inflammatory conditions like RA is mitochondria, organelles with prokaryotic molecular signatures. However, there is no direct evidence of mitochondrial formyl peptides (mtNFPs) in the circulation of patients with RA and their potential role in neutrophil-mediated inflammation in RA, including their clinical significance. MethodsLevels of mtNFPs (total fMet, MT-ND6) were analyzed using ELISA in plasma and serum obtained from patients in 3 cross-sectional RA cohorts (n = 275), a longitudinal inception cohort (n = 192) followed for a median of 8 years, and age/gender-matched healthy controls (total n = 134). Neutrophil activation assays were done in the absence or presence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H. ResultsElevated levels of total fMet were observed in the circulation of patients with RA as compared to healthy controls (p < 0.0001) associating with disease activity and could distinguish patients with the active disease from patients with inactive disease or patients in remission. Baseline levels of total fMet correlated with current and future joint involvement, respectively and predicted the development of rheumatoid nodules (OR = 1.2, p = 0.04). Further, total fMet levels improved the prognostic ability of ACPA in predicting erosive disease (OR of 7.9, p = 0.001). Total fMet levels correlated with markers of inflammation and neutrophil activation. Circulating mtNFPs induced neutrophil activation in vitro through FPR1-dependent mechanisms. ConclusionsCirculating mtNFPs could be novel biomarkers of disease monitoring and prognosis for RA and in investigating neutrophil-mediated inflammation in RA. We propose, FPR1 as a novel therapeutic target for RA.