Neutrophil Enzyme Research Articles

The Interaction of Myeloperoxidase with the Industrial Contaminant 6-PPD: A Potential Pathway for Reactive Metabolites.

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H2O2. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.

Inhibition of myeloperoxidase to treat left ventricular dysfunction in non-ischaemic cardiomyopathy.

Non-ischaemic cardiomyopathy (NICMP), an incurable disease terminating in systolic heart failure (heart failure with reduced ejection fraction [HFrEF]), causes immune activation, however anti-inflammatory treatment strategies so far have failed to alter the course of this disease. Myeloperoxidase (MPO), the principal enzyme in neutrophils, has cytotoxic, pro-fibrotic and nitric oxide oxidizing effects. Whether MPO inhibition ameliorates the phenotype in NICMP remains elusive. Prognostic information from MPO was derived from proteomic data of a large human cardiovascular health cohort (n = 3289). In a murine model of NICMP, we studied the mechanisms of MPO in this disease. In a case series, the MPO inhibitor was also evaluated in NICMP patients. Individuals with increased MPO revealed higher long-term mortality and worsening of heart failure, with impaired prognosis when MPO increased during follow-up. MPO infusion attenuated left ventricular ejection fraction (LVEF) in mice with NICMP, whereas genetic ablation or inhibition of MPO decreased systemic vascular resistance (SVR, 9.4 ± 0.7 mmHg*min/ml in NICMP vs. 6.7 ± 0.8 mmHg*min/ml in NICMP/Mpo-/-mice, n = 8, p = 0.006, data expressed as mean ± standard error of the mean) and improved left ventricular function (LVEF 30.3 ± 2.2% in NICMP vs. 40.7 ± 1.1% in NICMP/Mpo-/- mice, n = 16, p < 0.0001). Four patients diagnosed with NICMP and treated with an MPO inhibitor over 12 weeks showed increase in LVEF, decline in natriuretic peptides and improved 6-min walking distance. MPO inhibitor-related changes in the proteome of NICMP patients predicted reduced mortality when related to the changes in the proteome of the above referenced cardiovascular health cohort. Myeloperoxidase predicts long-term outcome in HFrEF and its inhibition elicits systemic anti-inflammatory and vasodilating effects which translate into improved left ventricular function. MPO inhibition deserves further evaluation as a novel, complementary treatment strategy for HFrEF.

Utilizing Oral Neutrophil Counts as an Indicator of Oral Inflammation Associated With Periodontal Disease: ABlinded Multicentre Study.

Periodontal diseases are chronic inflammatory conditions that require early screening for effective long-term management. Oral neutrophil counts (ONCs) correlate with periodontal inflammation. This study investigates a point-of-care test using a neutrophil enzyme activity (NEA) colorimetric strip for measuring periodontal inflammation. This prospective study had two phases. Phase 1 validated the relationship between ONCs and periodontal inflammation with 90 participants. Phase 2 examined the test's applicability in a real-world setting through a multicentre clinical trial with 375 participants at four sites. ONCs were quantified in oral rinses using laboratory-based methods, and the NEA strip was used for ONC stratification. Clinical measures included bleeding on probing (BoP), probing depth (PD) and clinical attachment loss (CAL). ONCs were significantly elevated in patients with Grade B periodontitis and deep periodontal pockets (PD ≥ 5 mm, CAL ≥ 5 mm). The NEA strip accurately classified patients into high or low ONC categories, showing 80% sensitivity, 82.5% specificity and an AUC of 0.89. It also assessed the effectiveness of periodontal therapy in reducing ONC and inflammation. The test was user-friendly, with no reported discomfort among patients. The NEA strip is a user-friendly and rapid screening tool for detecting high ONCs associated with periodontal inflammation and for evaluating the effectiveness of periodontal therapy.

Notable correlation between serum epidermal growth factor values and inflammatory status in patients with COVID-19.

Despite its crucial role in Epidermal Growth Factor Receptor (EGFR) activation, and the resulting impact on the health-disease process, epidermal growth factor (EGF) is an underexplored molecule in relation to how its serum concentrations relate to other analytes and clinical variables in pathological contexts. To clarify the possible correlation between EGF and clinical and analytical variables in the context of COVID-19. Cross-sectional observational and analytical study, in patients with virological and clinical diagnosis of COVID-19, selected by simple random sampling, admitted between August and September 2021. UMELISA-EGF commercial kits were used. Differences in overall EGF values were observed between groups (566.04 vs. 910.53 pg/ml, p = .0430). In COVID-19 patients, no notable correlations were observed for neutrophil, platelet, triglyceride or liver enzyme values (p > .05). Significant correlations were observed with the neutrophil-lymphocyte indicator (r = 0.4711, p = .0128) as well as with the platelet-lymphocyte index (r = 0.4553, p = .0155). Statistical results of multivariate regression analysis suggest NLR (β = .2232, p = .0353) and PLR (β = .2117, p = .0411) are predictors of inflammation in patients with COVID-19. Serum EGF concentrations in COVID-19 correlate positively with prognostic inflammatory markers of severity and could presumably act as an independent risk factor for the development of inflammation in response to new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Myeloperoxidase-specific antineutrophil cytoplasmic antibody-associated vasculitis.

Myeloperoxidase (MPO)-specific antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-ANCA-associated vasculitis) is one of two major ANCA-associated vasculitis variants characterised by systemic necrotising vasculitis with few or no immune deposits. MPO-ANCA-associated vasculitis predominantly affects small blood vessels and, in contrast to its counterpart proteinase 3-ANCA-associated vasculitis, is generally not associated with granulomatous inflammation. The kidneys and lungs are the most commonly affected organs. The pathogenesis of MPO-ANCA-associated vasculitis is characterised by loss of tolerance to the neutrophil enzyme MPO. This loss of tolerance leads to a chronic immunopathological response where neutrophils become both the target and effector of autoimmunity. MPO-ANCA drives neutrophil activation, leading in turn to tissue and organ damage. Clinical trials have improved the therapeutic approach to MPO-ANCA-associated vasculitis. However, there remains substantial unmet need regarding relapse frequency, toxicity of current treatment, and long-term morbidity. In this Series paper, we present the current state of research regarding pathogenesis, diagnosis, and treatment of MPO-ANCA-associated vasculitis.

Association of neutrophil extracellular trap levels with Raynaud’s phenomenon, glomerulonephritis and disease index score in SLE patients from Brazil

Neutrophil extracellular traps (NETs) are cell-extruded DNA strands coated with neutrophils' nuclear proteins and enzymes from cytotoxic granules, produced by NETosis, a cell death pathway. They perform an important defensive role in innate immunity, but their increased production and/or inefficient degradation expose new antigens, such as DNA or citrullinated histone peptides, triggering autoimmunity. This study aimed to access possible associations between serum NETs levels with epidemiological, clinical, and serological data from a well-characterized SLE Brazilian patients’ cohort. NET levels were evaluated in one hundred seventy serum samples of patients with Systemic Lupus Erythematosus (SLE) using an Immunoassay. Univariate and multivariate binary logistic regression used clinical patients' data as independent variables. Parametric and non-parametric tests compared log10 base serum NET levels transformed between patients' groups. SLE patients were also dichotomized into “High serum NET levels” and “Low serum NET levels” groups. All analyses were performed in R language 4.1.2, and p < 0.05 were considered significant. Increased susceptibility for high serum NET levels was observed in SLE patients with Raynaud's phenomenon (OR = 2.30, 95 % CI = 1.06–5.21 and p = 0.039), independently of any other risk factor. Also, SLE patients with Raynaud's phenomenon presented higher mean NET serum levels (mean = −0.13 vs. −0.51, p = 0.01). In addition, higher mean NET serum levels were associated with glomerulonephritis (mean = -0.45 vs. −0.12, p = 0.03). Ultimately, the SLEDAI index scored higher in the high NETs serum levels group (median = 2.0 vs. 0.0, p = 6 × 10−3). The formation of NETs might be implicated in Raynaud's phenomenon, glomerulonephritis, and disease index score in SLE patients. Our results highlight the importance of serum NET levels as a possible therapeutical target to modulate the clinical course of SLE.

Neutrophil Activity and Extracellular Matrix Degradation: Drivers of Lung Tissue Destruction in Fatal COVID-19 Cases and Implications for Long COVID.

Pulmonary fibrosis, severe alveolitis, and the inability to restore alveolar epithelial architecture are primary causes of respiratory failure in fatal COVID-19 cases. However, the factors contributing to abnormal fibrosis in critically ill COVID-19 patients remain unclear. This study analyzed the histopathology of lung specimens from eight COVID-19 and six non-COVID-19 postmortems. We assessed the distribution and changes in extracellular matrix (ECM) proteins, including elastin and collagen, in lung alveoli through morphometric analyses. Our findings reveal the significant degradation of elastin fibers along the thin alveolar walls of the lung parenchyma, a process that precedes the onset of interstitial collagen deposition and widespread intra-alveolar fibrosis. Lungs with collapsed alveoli and organized fibrotic regions showed extensive fragmentation of elastin fibers, accompanied by alveolar epithelial cell death. Immunoblotting of lung autopsy tissue extracts confirmed elastin degradation. Importantly, we found that the loss of elastin was strongly correlated with the induction of neutrophil elastase (NE), a potent protease that degrades ECM. This study affirms the critical role of neutrophils and neutrophil enzymes in the pathogenesis of COVID-19. Consistently, we observed increased staining for peptidyl arginine deiminase, a marker for neutrophil extracellular trap release, and myeloperoxidase, an enzyme-generating reactive oxygen radical, indicating active neutrophil involvement in lung pathology. These findings place neutrophils and elastin degradation at the center of impaired alveolar function and argue that elastolysis and alveolitis trigger abnormal ECM repair and fibrosis in fatal COVID-19 cases. Importantly, this study has implications for severe COVID-19 complications, including long COVID and other chronic inflammatory and fibrotic disorders.

Role of neutrophil myeloperoxidase in the development and progression of high-altitude pulmonary edema

BackgroundNeutrophil infiltration and hypoxic pulmonary vasoconstriction induced by hypobaric hypoxic stress are vital in high-altitude pulmonary edema (HAPE). Myeloperoxidase (MPO), an important enzyme in neutrophils, is associated with inflammation and oxidative stress and is also involved in the regulation of nitric oxide synthase (NOS), an enzyme that catalyzes the production of the vasodilatory factor nitric oxide (NO). However, the role of neutrophil MPO in HAPE's progression is still uncertain. Therefore, we hypothesize that MPO is involved in the development of HAPE via NOS. MethodsIn Xining, China (altitude: 2260 m), C57BL/6 N wild-type and mpo−/− mice served as normoxic controls, while a hypobaric chamber simulated 7000 m altitude for hypoxia. L-NAME, a nitric oxide synthase (NOS) inhibitor to inhibit NO production, was the experimental drug, and D-NAME, without NOS inhibitory effects, was the control. After measuring pulmonary artery pressure (PAP), samples were collected and analyzed for blood neutrophils, oxidative stress, inflammation, vasoactive substances, pulmonary alveolar-capillary barrier permeability, and lung tissue morphology. ResultsWild-type mice's lung injury scores, permeability, and neutrophil counts rose at 24 and 48 h of hypoxia exposure. Under hypoxia, PAP increased from 12.89 ± 1.51 mmHg under normoxia to 20.62 ± 3.33 mmHg significantly in wild-type mice and from 13.24 ± 0.79 mmHg to 16.50 ± 2.07 mmHg in mpo−/− mice. Consistent with PAP, inducible NOS activity, lung permeability, lung injury scores, oxidative stress response, and inflammation showed more significant increases in wild-type mice than in mpo−/− mice. Additionally, endothelial NOS activity and NO levels decreased more pronouncedly in wild-type mice than in mpo−/− mice. NOS inhibition during hypoxia led to more significant increases in PAP, permeability, and lung injury scores compared to the drug control group, especially in wild-type mice. ConclusionMPO knockout reduces oxidative stress and inflammation to preserve alveolar-capillary barrier permeability and limits the decline in endothelial NOS activity to reduce PAP elevation during hypoxia. MPO inhibition emerges as a prospective therapeutic strategy for HAPE, offering avenues for precise interventions.

Fusobacterium nucleatum elicits subspecies-specific responses in human neutrophils.

Fusobacterium nucleatum as a Gram-negative anaerobe plays a key bridging role in oral biofilms. It is involved in periodontal and extraoral diseases, the most prominent being colorectal cancer. Five subspecies are recognised: animalis, fusiforme, nucleatum, polymorphum and vincentii. Subspecies interact with neutrophils constantly patrolling tissues to remove microbial intruders. Neutrophil antimicrobial activities include generation of reactive oxygen species (ROS), formation of neutrophil extracellular traps (NETs) and release of cytokines and neutrophil enzymes. Subspecies-specific differences in immunogenicity have previously been observed in a neutrophil-like cell line but were not investigated in human neutrophils. Additionally, neutrophil responses to planktonic and biofilm-grown F. nucleatum have not been studied to date. The aims of this study were to compare the immunogenicity of planktonic and biofilm-grown F. nucleatum and to investigate potential differences in human neutrophil responses when stimulated with individual F. nucleatum subspecies. Human neutrophils isolated from peripheral blood were stimulated with planktonic and biofilm-grown F. nucleatum subspecies. Generation of ROS and NET formation were quantified by luminescence and fluorescence assays, respectively. Secretion of cytokines (IL-1β, TNF-α, IL-6, IL-8), neutrophil elastase and matrix metalloproteinase-9 was quantified by enzyme-linked immunosorbent assay (ELISA). Neutrophil responses showed biofilm-grown bacteria induced a significantly higher total and intracellular ROS response, as well as shorter time to total ROS release. Biofilm-grown F. nucleatum led to significantly lower IL-1β release. We found significant differences among individual subspecies in terms of total, intracellular ROS and extracellular superoxide. Subspecies polymorphum stimulated the highest mean amount of NET release. Amounts of cytokines released differed significantly among subspecies, while no differences were found in lysosomal enzyme release. Immunogenicity of F. nucleatum in human neutrophils is highly subspecies-specific in vitro with regard to ROS release and cytokine production. Understanding subspecies-specific immunogenicity of F. nucleatum may facilitate the discovery of novel therapeutic targets in F. nucleatum-mediated diseases.

Features of the respiratory burst state of neutrophils and the activity of NAD(P)-dependent dehydrogenases in patients with widespread purulent peritonitis in the prognosis of the development of sepsis

Aim: Studying of the respiratory burst state and NAD(P)-dependent dehydrogenases activities in blood neutrophils features in the forecast of the development of abdominal sepsis in patients with widespread purulent peritonitis (WPP).&#x0D; Materials and methods. The study involved 50 patients with WPP in the preoperative period. Abdominal sepsis was developed by 35 patients (70.0%) from 5 to 10 days postoperative period, 15 patients (30.0%) hadn’t complications. The respiratory burst condition of blood neutrophils was examined using a chemiluminescent assay. Intracellular activity of the NAD- and NADP-dependent dehydrogenases was researched with using bioluminescent methods.&#x0D; Results. It was found that patients with WPP whose dynamics of the preoperative period will develop sepsis the chemiluminescent activity of blood neutrophils was characterized by a reduced level of spontaneous synthesis of the primary reactive oxygen species (ROS) and elevated levels of spontaneous synthesis of secondary ROS relative of the indicators identified in patients without subsequent complications. The feature of neutrophil metabolism in WPP patients without subsequent development of sepsis was high activity of the anaerobic lactate dehydrogenase reaction and decrease in activity of the NADP-dependent decarboxylating malate dehydrogenase. In patients with WPP and the subsequent development of sepsis was found high level of NAD-dependent substrates outflow citric acid cycle in the reaction of amino acid metabolism via glutamate dehydrogenase that may affect the activity of aerobic respiration in the neutrophils. Using correlation analysis was found that the intensity of the neutrophils respiratory burst in patients with no subsequent complications depends on the activity of anaerobic glycolysis.&#x0D; Conclusion. The established differences in the state of the respiratory burst and the activity of enzymes in neutrophils in patients with WPP, in depending on the subsequent development of the sepsis, determine the possibility of developing the method of forecasting complications and developing immunoactive therapy in the postoperative period of WPP.

Eucalyptus-derived essential oils alleviate microbes and modulate inflammation by suppressing superoxide and elastase release

The Eucalyptus tree, belonging to the myrtle family, grows all over the world for its pharmaceutical and industrial benefits. In this article, we present a comparative analysis of the chemical composition of the hydrodistilled oils obtained from three different Eucalyptus species growing in Egypt viz. E. citriodora, E. camaldulensis, and E. ficifolia. Gas Chromatography-Mass Spectrometric guided analysis resulted in the identification of a total of 20 metabolites in E. citriodora oil with citronellal (54.9%) and citronellol (25.4%) being the most dominant components. β-cymene (12.7%) and 1,8-cineole (11.7%) were the major volatile constituents identified in E. camaldulensis oil, while trans-β-ocimene (22.4%), 1,8-cineole (13.5%), and L-trans-pinocarveol (12.5%) were the dominating components in the oil of E. ficifolia. The essential oils of the studied species were evaluated for their in vitro anti-inflammatory, antiviral including anti-SARS-CoV-2 (severe acute respiratory syndrome corona virus 2), antibacterial, and antifungal activities. E. citriodora oil displayed the highest inhibitory activity on the release of the superoxide radical (32%) and elastase enzyme (31%) in human neutrophils, while E. ficifolia oil had enhancing effects on elastase. The latter showed significant antiviral effects against hepatitis A, herpes simplex, and coxsackie viruses with IC50 values at 2.1, 2.5, and 5.6 μg/mL, respectively. Moderate antibacterial and antifungal activities were observed for Eucalyptus oils with Staphylococcus aureus being the most susceptible bacterial strain. E. ficifolia oil, similarly, displayed the best antibacterial activity with minimum inhibitory concentration (MIC) value at ca. 25 μg/mL (for S. aureus). On the contrary, E. camaldulensis oil was the most active against Candida albicans with an MIC value at 45 μg/mL. In silico studies were performed with a number of macromolecular drug targets for confirming the biological activities of the identified compounds and for interpreting their ADME (absorption-distribution-metabolism-elimination) parameters.

Neutrophils extracellular traps formation may serve as a biomarker for disease activity in oligoarticular juvenile idiopathic arthritis: a pilot study

BackgroundJuvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children, causing significant morbidity. Despite the dramatic improvement in treatment, many patients do not achieve complete remission, and biomarkers for subclinical disease, flares, and response to treatment are lacking. Neutrophils and neutrophil extracellular traps (NETs) play key roles in the pathogenesis of autoimmune and inflammatory conditions. In this study, we characterized neutrophil enzyme activity and NETs formation in oligoarticular and polyarticular JIA and explored their association with disease activity.MethodsNeutrophils from 6 healthy controls and 7 patients with oligoarticular and polyarticular JIA were freshly isolated at time of diagnosis and after glucocorticoid intra-articular injection. Enzymatic activity of neutrophil granular enzymes was monitored by colorimetry and PMA-activated NETs formation was assessed using fluorescent microscopy.ResultsIn this pilot and feasibility study, we revealed that NETs were significantly increased in oligoarticular JIA patients at time of diagnosis compared to healthy controls. Anti-inflammatory treatment using intra-articular steroid injection normalized NETs formation in these patients. Correlation between NETs formation and clinical Juvenile Activity Disease Activity Score-10 (cJADAS-10) was linear and significant (P = 0.007) in oligo but not in poly JIA patients.ConclusionsThis is the first study exploring the link of NETs formation with oligo and poly JIA activity. We demonstrated a statistically significant linear correlation between cJADAS-10 and NETs formation in oligo but not in poly JIA patients. Hence, we suggest that NETs may reflect clinical disease activity in JIA, and may serve as a putative biomarker. Further work is needed to validate these initial results and determine the dynamics of NETs formation in JIA.

Novel inhibitory effect of galectin-3 on the respiratory burst induced by Staphylococcus aureus in human neutrophils.

Among the responders to microbial invasion, neutrophils represent the earliest and perhaps the most important immune cells that contribute to host defense with the primary role to kill invading microbes using a plethora of stored anti-microbial molecules. One such process is the production of reactive oxygen species (ROS) by the neutrophil enzyme complex NADPH-oxidase, which can be assembled and active either extracellularly or intracellularly in phagosomes (during phagocytosis) and/or granules (in the absence of phagocytosis). One soluble factor modulating the interplay between immune cells and microbes is galectin-3 (gal-3), a carbohydrate-binding protein that regulates a wide variety of neutrophil functions. Gal-3 has been shown to potentiate neutrophil interaction with bacteria, including Staphylococcus aureus, and is also a potent activator of the neutrophil respiratory burst, inducing large amounts of granule-localized ROS in primed cells. Herein, the role of gal-3 in regulating S. aureus phagocytosis and S. aureus-induced intracellular ROS was analyzed by imaging flow cytometry and luminol-based chemiluminescence, respectively. Although gal-3 did not interfere with S. aureus phagocytosis per se, it potently inhibited phagocytosis-induced intracellular ROS production. Using the gal-3 inhibitor GB0139 (TD139) and carbohydrate recognition domain of gal-3 (gal-3C), we found that the gal-3-induced inhibitory effect on ROS production was dependent on the carbohydrate recognition domain of the lectin. In summary, this is the first report of an inhibitory role of gal-3 in regulating phagocytosis-induced ROS production.

Role of oxidase and antioxidant enzymes in neutrophils and blood circulation in patients with acute coronary syndrome

Acute coronary syndrome (ACS), a subcategory of cardiovascular diseases, has become a major cause of mortality and morbidity worldwide. Oxidative stress resulting from increased production of reactive oxygen species and decreased antioxidants plays a major role in the pathophysiology of ACS. This study evaluated the activities of certain oxidase and antioxidant enzymes in circulation and neutrophils to determine their roles in increased oxidative stress in patients with ACS. A total of 52 patients with ACS admitted in the coronary care unit of two tertiary hospitals and 52 healthy controls were enrolled. Blood samples were collected from all subjects, and various oxidase and antioxidant enzymes in neutrophils and circulation were assayed. The patients had significantly higher white blood cell and neutrophil counts than the controls. In patients, the mean (±SD) serum activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (12.83±6.6 U/L) and superoxidase dismutase (4.34±1.41 U/mL) were significantly higher than in the control group (4.88±3.03 U/L and 3.02±1.7 U/mL, respectively) while the catalase had significantly lower activities (33.36±13.16 U/mL vs. 63.98±31.86 U/mL). In neutrophils, the activities of myeloperoxidase, NADPH oxidase and catalase were significantly higher in ACS patients, while superoxidase dismutase was significantly lower. Further, significant positive correlations were found between activities of myeloperoxidase and catalase, and NADPH oxidase and superoxidase dismutase in neutrophils of ACS patients. These findings revealed that higher activities of myeloperoxidase and NADPH oxidase, both in serum and neutrophils, lead to increased oxidative stress and form the inflammatory basis of ACS, and the antioxidant enzymes combat the events.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Do circulating neutrophil extracellular traps predict recurrence in early breast cancer?

Neutrophil extracellular traps (NETs), three-dimensional structures formed by neutrophil enzymes such as neutrophil elastase (NE) and nuclear components (DNA), have been associated with progression and metastasis in breast cancer (BC). Thus, the aim of this study was to evaluate the association of circulating NETs with clinicopathological characteristics and outcomes in early BC. A prospective cohort included women with newly diagnosed early BC. NETs were defined as the presence of NE-DNA complexes in plasma, measured by optical density. Levels of NETs were dichotomized according to the median, as low and high levels of circulating NETs. Fisher's exact test was used to evaluate associations between NETs and clinicopathological characteristics and outcomes. Survival was assessed using the Kaplan Meier method and log-rank test. Forty patients were included, 23 (57.5%) patients with low and 17 (42.5%) with high levels of circulating NETs. No associations were found between clinicopathological characteristics and circulating NETs levels. Recurrence (p = 0.99) and site of recurrence (p = 0.99) were not statistically associated with plasma NETs levels. Overall, recurrence-free survival was not statistically different between circulating levels of NETs. With a short follow-up and low number of events, our results suggest that circulating levels of NETs at diagnosis of early BC are not associated with more aggressive clinicopathological characteristics, recurrence, or site of recurrence.

A CASE OF MICROSCOPIC POLYANGIITIS (MPA) FOLLOWING SARS-COV-2 INFECTION

A CASE OF MICROSCOPIC POLYANGIITIS (MPA) FOLLOWING SARS-COV-2 INFECTION

Cholecystoduodenostomy for biliary obstruction treatment in a white‐collared lemur (Eulemur cinereiceps)

AbstractA 16‐year‐old male, white‐collared lemur (Eulemur cinereiceps) was presented for lethargy, anorexia and nausea. General examination showed dehydration and icterus. Blood analysis revealed neutrophilic leucocytosis and elevated hepatic enzyme concentrations. Abdominal CT scan revealed two biliary calculi and a dilatation of the pancreatic and common bile ducts, indicating biliary obstruction. Due to lack of response to supportive treatment (intravenous fluid therapy, long‐acting amoxicillin, tramadol, omeprazole, methionine, silybin), cholecystoduodenostomy was performed, and a hepatic granuloma and gallstone were removed. The animal recovered from the surgery within a week and blood parameters were within normal range 3 weeks later. Microbiological evaluation of the calculus revealed an Enterococcus casseiflavus biliary infection, and hepatic cysticercosis by Taenia martis was diagnosed by histology and PCR. Cholelithiasis has been described in primates, but successful surgical treatment is rare. To the authors’ knowledge, this is the first description of an obstructive cholelithiasis treated successfully by cholecystoduodenostomy in a primate.

Bilirubin as a Predictor of Complicated Appendicitis in a District General Hospital: A Retrospective Analysis.

AimsThe objectives of the study were to establish the function of bilirubin as a novel diagnostic tool for predicting complex appendicitis and to compare the impact of other variables such as white blood cell count (WCC), C-reactive protein (CRP), and neutrophil.MethodsThis retrospective, single-center cohort analysis included all patients admitted to Torbay General Hospital in Torquay, United Kingdom, between January 2020 and December 2020 with a clinical diagnosis of acute appendicitis. In addition to serum CRP, WCC, and neutrophil, serum bilirubin and other liver enzymes were obtained from the patients’ blood on admission.ResultsThe quantitative analysis included 174 patients from the group that remained. The large majority of the sample consisted of adults and males (77% and 51.7%, respectively). Overall, 49.4% of participants in the study were diagnosed with complicated appendicitis; 74.7% of adults had complicated appendicitis, with 58.6% being male. In 68.6% of cases, perforated appendicitis was the most prevalent form of complicated appendicitis. Patients with complicated appendicitis had significantly higher WCC, neutrophil, and CRP levels than those with uncomplicated appendicitis (14.15 vs. 12.88, p = 0.016; 11.63 vs. 10.19, p = 0.007; and (89.28 vs. 40.65, p = 0.0001, respectively).. The significantly greater alkaline phosphatase and total serum bilirubin discrepancies were observed in patients with complicated appendicitis. There were statistically significant differences in the means of the patients: (18.46 vs. 10.98, p = 0.0001 and 110.64 vs. 102.24, p = 0.033).ConclusionSerum bilirubin is a crucial diagnostic aid for determining the existence of complicated appendicitis.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm−1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.