Towards the Use of Lichens as a Source of Bioactive Substances for Topical Applications.

The increasing incidence of dermatological diseases prompts the search for new natural methods of treatments, and lichens, with their special symbiotic structure, are a little-known and promising source of biologically active substances. Seven lichen species, Cladonia unicialis (L.) Weber ex F.H. Wigg. (Cladoniaceae), Evernia prunastri (L.) Ach. (Parmeliaceae), Hypogymnia physodes (L.) Nyl. (Parmaliaceae), Parmelia sulcata (Taylor) (Parmeliaceae), Physcia adscendens (Fr.) H. Olivier (Physciaceae), Pseudoevernia furfuracea (L.) Zopf (Parmeliaceae), and Xanthoria parietina (L.) Th. Fr. (Teloschistaceae), were used in our experiment. We identified different metabolites in the acetone extracts of all the lichen species. Based on the high-performance liquid chromatography analysis, the content of lichen substances in the extracts was evaluated. The impact of the individual lichen-specific reference substances, compared to the lichen extracts, on the viability of keratinocytes (HaCaT cell line) and fibroblasts (BJ cell line) and on the activity of selected skin-related enzymes was investigated. Our results revealed that only emodin anthrone at a concentration of 200 mg/L was cytotoxic to keratinocytes and fibroblasts in both cell viability assays. In turn, the C. uncialis extract was only cytotoxic to keratinocytes when used at the same concentration. The other tested treatments showed a positive effect on cell viability and no cytotoxicity or indeterminate cytotoxicity (shown in only one of the tests). Elastase and collagenase activities were inhibited by most of the lichen extracts. In turn, the individual lichen compounds (with the exception of evernic acid) generally had an undesirable stimulatory effect on hyaluronidase and collagenase activity. In addition, almost all the tested compounds and extracts showed anti-inflammatory activity. This suggests that some lichen compounds hold promise as potential ingredients in dermatological and skincare products, but their safety and efficacy require further study. The high cytotoxicity of emodin anthrone highlights its potential use in the treatment of hyperproliferative skin diseases such as psoriasis.

The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense.

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2' site of the SARS-CoV-2 spike protein at two specific sites (815RS816 and 817FI818) as well as NE (813SK814 and 818IE819), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca2+ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion.

Extracellular Vesicle-Associated Neutrophil Elastase Activates Hepatic Stellate Cells and Promotes Liver Fibrogenesis via ERK1/2 Pathway.

Liver fibrosis associated with increased mortality is caused by activation of hepatic stellate cells and excessive production and accumulation of extracellular matrix in response to fibrotic insults. It has been shown that in addition to liver inflammation, systemic inflammation also contributes to liver fibrogenesis. A deeper understanding of mechanisms that control liver fibrotic response to intra- and extra-hepatic inflammation is essential to develop novel clinical strategies against this disease. Extracellular vesicles (EV) have been recognized as immune mediators that facilitate activation of hepatic stellate cells. In inflammatory diseases, activated neutrophils release neutrophil elastase (NE) bound to EV, which has been identified as a significant contributor to inflammation by promoting immune cell activation. Here, we aimed to explore the role of inflammation derived plasma EV-associated NE in liver fibrogenesis and its potential mechanisms. We show EV-associated NE induces activation, proliferation and migration of hepatic stellate cells by promoting activation of the ERK1/2 signaling pathway. This effect did not occur through EV without surface NE, and Sivelestat, a NE inhibitor, inhibited activation of the ERK1/2 signaling pathway mediated by EV-associated NE. Moreover, we found plasma EV-associated NE increases deposition of collagen1 and α-smooth muscle actin in the liver of a mouse model of liver fibrosis (Mdr2-/-). Notably, this effect does not occur in control mice without preexisting liver disease. These data suggest that EV-associated NE is a pro-fibrogenic factor for hepatic stellate cell activation via the ERK1/2 signaling pathway in pre-existing liver injuries. Inhibition of the plasma EV-associated NE in inflammatory conditions may be a therapeutic target for liver fibrosis in patients with inflammatory diseases.

Elastase-targeting biomimic nanoplatform for neurovascular remodeling by inhibiting NETosis mediated AlM2 inflammasome activation in ischemic stroke.

Neutrophil elastase (NE) is a protease released by activated neutrophils in the brain parenchyma after cerebral ischemia, which plays a pivotal role in the regulation of neutrophil extracellular traps (NETs) formation. The excess NETs could lead to blood-brain barrier (BBB) breakdown, overwhelming neuroinflammation, and neuronal injury. While the potential of targeting neutrophils and inhibiting NE activity to mitigate ischemic stroke (IS) pathology has been recognized, effective strategies that inhibit NETs formation remain under-explored. Herein, a biomimic multifunctional nanoplatform (HM@ST/TeTeLipos) was developed for active NE targeting and IS treatment. The core of the HM@ST/TeTeLipos consisted of sivelestat-loaded ditelluride-containing liposomes with ROS-responsive and NE-inhibiting properties. The outer shell was composed of platelet-neutrophil hybrid membrane vesicles (HMVs), which acted to hijack neutrophils and neutralize proinflammatory cytokines. Our studies revealed that HM@ST/TeTeLipos could effectively inhibit NE activity, thereby suppressing the release of NETs, impeding the activation of the AIM2 inflammasome, and consequently redirecting the immune response away from a pro-inflammatory M1 microglia phenotype. This resulted in enhanced neurovascular remodeling, reduced BBB disruption, and diminished neuroinflammation, ultimately promoting neuron survival. We believe that this innovative approach holds significant potential for improving the treatment of IS and various NE-mediated inflammatory diseases.

Robustaflavone as a novel scaffold for inhibitors of native and auto-proteolysed human neutrophil elastase.

Human neutrophil elastase (HNE) plays a key role in initiating inflammation in the cardiopulmonary and systemic contexts. Pathological auto-proteolysed two-chain (tc) HNE exhibits reduced binding affinity with inhibitors. Using AutoDock Vina v1.2.0, 66 flavonoid inhibitors, sivelestat and alvelestat were docked with single-chain (sc) HNE and tcHNE. Schrodinger PHASE v13.4.132 was used to generate a 3D-QSAR model. Molecular dynamics (MD) simulations were conducted with AMBER v18. The 3D-QSAR model for flavonoids with scHNE showed r2 = 0.95 and q2 = 0.91. High-activity compounds had hydrophobic A/A2 and C/C2 rings in the S1 subsite, with hydrogen bond donors at C5 and C7 positions of the A/A2 ring, and the C4' position of the B/B1 ring. All flavonoids except robustaflavone occupied the S1'-S2' subsites of tcHNE with decreased AutoDock binding affinities. During MD simulations, robustaflavone remained highly stable with both HNE forms. Principal Component Analysis suggested that robustaflavone binding induced structural stability in both HNE forms. Cluster analysis and free energy landscape plots showed that robustaflavone remained within the sc and tcHNE binding site throughout the 100 ns MD simulation. The robustaflavone scaffold likely inhibits both tcHNE and scHNE. It is potentially superior to sivelestat and alvelestat and can aid in developing therapeutics targeting both forms of HNE.

Flagellum-deficient Pseudomonas aeruginosa is more virulent than non-motile but flagellated mutants in a cystic fibrosis mouse model.

Loss of the flagellum marks the pathoadaptation of Pseudomonas aeruginosa to the cystic fibrosis (CF) airway environment during lung disease. Losing the flagellum is advantageous to the bacterium as the flagellum can be recognized by immune cells. The primary purpose of the flagellum is, however, to provide motility to the bacterium. Our goal was to determine whether the loss of flagellar motility or the loss of flagellum expression contributes to P. aeruginosa lung infection in CF. To address this, wild-type and gut-corrected FABP-human cystic fibrosis transmembrane conductance regulator (hCFTR) mice deficient in the murine Cftr gene were infected intratracheally with lethal doses of wild-type or flagellum-deficient P. aeruginosa. While there was no significant difference in the survival of wild-type mice after infection with either of the bacterial strains, a significantly higher mortality was observed in FABP-hCFTR mice infected with flagellum-deficient P. aeruginosa, compared to mice infected with their flagellated counterparts. When FABP-hCFTR mice were infected with isogenic, motility-deficient flagellated mutants, animal survival and lung bacterial titers were similar to those observed in mice infected with the wild-type bacterium. Airway levels of neutrophils and the amount neutrophil elastase were similar in mice infected with either the wild-type bacteria or the flagellum-deficient P. aeruginosa. Our results show that FABP-hCFTR mice have a different response to flagellum loss in P. aeruginosa compared to wild-type animals. The loss of flagellum expression, rather than the loss of motility, is the main driver behind the increased virulence of flagellum-deficient P. aeruginosa in CF. These observations provide new insight into P. aeruginosa virulence in CF.IMPORTANCEPseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis, is known to lose its flagellum during the course of infection in the airways. Here, we show that the loss of flagellum leads to a more enhanced virulence in Cftr-deficient cystic fibrosis mice than in control animals. Loss of flagellum expression, rather than the loss of flagellar swimming motility, represents the main driver behind this increased virulence suggesting that this appendage plays a specific role in P. aeruginosa virulence in cystic fibrosis airways.

Elevation of circulating neutrophil extracellular traps in endometrial cancer: Poor prognostic value of cell-free double-stranded DNA.

OBJECTIVE: Neutrophils produce neutrophil extracellular traps (NETs) by releasing nuclear contents into the extracellular environment. NETs are associated with systemic inflammation and cancer development and progression. We aimed to investigate whether NET markers are associated with the prognosis of endometrial cancer. METHODS: Circulating levels of three NET markers (histone-DNA complex, cell-free double-stranded DNA (dsDNA), and neutrophil elastase) were measured in 98 patients with endometrial cancer who underwent surgery as primary treatment between January 2015 and June 2018 and 45 healthy women. Area under the receiver operating characteristic curve (AUC) analyses were conducted to investigate the diagnostic and prognostic utility of the markers for endometrial cancer. RESULTS: Patients with endometrial cancer showed significantly higher levels of the three NET markers than those in healthy controls. In discriminating endometrial cancer patients from healthy controls, the three NET markers showed AUC values in the following order: cell-free dsDNA (0.832; 95 % CI, 0.760-0.889), histone-DNA complex (0.740; 95 % CI, 0.660-0.809), and neutrophil elastase (0.689; 95 % CI, 0.607-0.764), comparable to those of CA-125 (0.741; 95 % CI, 0.659-0.813). Multivariate analysis adjusting for FIGO stage, histology, and lymphovascular space invasion, and lymph node involvement revealed that cell-free dsDNA level (cutoff: 95.2 ng/mL) was an independent prognostic marker for poor progression-free (adjusted HR, 2.75; 95 % CI, 1.096.92; P = 0.032) and overall survival (adjusted HR, 11.51; 95 % CI, 2.0664.22; P = 0.005) for patients with endometrial cancer. CONCLUSION: High levels of circulating NET markers were observed in patients with endometrial cancer. Cell-free dsDNA levels may play a role as prognostic markers for endometrial cancer.

Exploration of novel human neutrophil elastase inhibitors from natural compounds: Virtual screening, in vitro, molecular dynamics simulation and in vivo study.

BACKGROUND: Human neutrophil elastase (HNE) is an important contributor to lung diseases such as acute lung injury (ALI) or acute respiratory distress syndrome. Therefore, this study aimed to identify natural HNE inhibitors with anti-inflammatory activity through machine learning algorithms, in vitro assays, molecular dynamic simulation, and an in vivo ALI assay. METHODS: Based on the optimized Discovery Studio two-dimensional molecular descriptors, combined with different molecular fingerprints, six machine learning models were established using the Naïve Bayesian (NB) method to identify HNE inhibitors. Subsequently, the optimal model was utilized to screen 6925 drug-like compounds obtained from the Traditional Chinese Medicine Systems Pharmacy Database and Analysis Platform (TCMSP), followed by ADMET analysis. Finally, 10 compounds with reported anti-inflammatory activity were selected to determine their inhibitory activities against HNE in vitro, and the compounds with the best activity were selected for a 100 ns molecular dynamics simulation and its anti-inflammatory effect was evaluated using Poly (I:C)-induced ALI model. RESULTS: The evaluation of the in vitro HNE inhibition efficiency of the 10 selected compounds showed that the flavonoid tricetin had the strongest inhibitory effect on HNE. The molecular dynamics simulation indicated that the binding of tricetin to HNE was relatively stable throughout the simulation. Importantly, in vivo experiments indicated that tricetin treatment substantially improved the Poly (I:C)-induced ALI. CONCLUSION: The proposed NB model was proved valuable for exploring novel HNE inhibitors, and natural tricetin was screened out as a novel HNE inhibitor, which was confirmed by in vitro and in vivo assays for its inhibitory activities.

Simultaneous In Vivo Imaging of Neutrophil Elastase and Oxidative Stress in Atherosclerotic Plaques Using a Unimolecular Photoacoustic Probe.

Atherosclerosis is a primary global health concern due to its high morbidity and mortality. This disease is characterized by a complex interplay of chronic inflammation, oxidative stress, and proteolytic enzymes. Traditional imaging techniques struggle to capture the dynamic biochemical processes within atherosclerotic plaques. Herein, we have developed a novel unimolecular photoacoustic probe (UMAPP) that combines specific recognition sites for neutrophil elastase (NE) and the redox pair O2•‒/GSH into a cohesive molecular platform, allowing in vivo monitoring of oxidative stress and activated neutrophils within plaques. UMAPP features a boron-dipyrromethene (BODIPY) core linked to a hydrophilic NE-cleavable tetrapeptide, and dual oxidative stress-responsive catechol moieties, enabling NE-mediated modulation of photoinduced electron transfer, affecting the photoacoustic intensity at 685 nm (PA685), while oxidation and reduction of the catechol groups by O2•‒ and GSH lead to reversible, ratiometric changes in the photoacoustic spectrum. Preliminary applications of UMAPP have successfully differentiated between atherosclerotic and healthy mice, assessed the impact of pneumonia on plaque composition, and validated the probe's efficacy in drug-treatment studies, detecting molecular changes prior to observable histopathological alterations. UMAPP's integrated molecular imaging approach holds significant promise for advancing the diagnosis and management of atherosclerosis by enabling earlier and more precise detection of vulnerable plaques.

Altered neutrophil extracellular traps formation among medical residents with sleep deprivation.

Resident physicians on long-term night shifts often face sleep deprivation, affecting the immune response, notably neutrophils, vital to innate defense mechanisms. Sleep-deprived residents exhibit altered neutrophil counts and reduced phagocytosis and NADPH oxidase activity, critical to combating infections. Our study focused on neutrophil extracellular traps (NETs), a defense process against pathogens not previously linked to sleep loss. Results revealed that sleep-deprived residents exhibited a 19.8 % reduction in NET formation compared to hospital workers with regular sleep patterns (P < 0.01). Additionally, key NETs proteins, Neutrophil Elastase and Myeloperoxidase, were less active in sleep-deprived individuals (1.53mU; P < 0.01 and 0.95U; P < 0.001 decrease, accordingly). Interestingly, the ability to form NETs resumed to normal levels three months post-residency among pediatric residents. The causal relationship between reduced NETs due to sleep deprivation and the increased susceptibility to infections, as well as its implications for infection severity, is a critical area for further investigation.

The Potential of Molecular Remission: Tissue Neutrophil Elastase Is Better Than Histological Activity for Predicting Long-Term Relapse in Patients With Ulcerative Colitis in Endoscopic Remission.

BACKGROUND: Growing interest exists in deep remission, beyond clinical and endoscopic remission, to enhance long-term prognosis in patients with ulcerative colitis (UC). Our study aimed to evaluate the risk of relapse according to tissue expression levels of calprotectin and neutrophil elastase (NE) in patients with quiescent UC. METHODS: Rectal biopsies were performed on 218 patients with UC in clinical and endoscopic remission. Histological activity was prospectively scored using the Robarts Histological Index. Tissue calprotectin and NE levels were evaluated using immunohistochemistry. Optimal tissue calprotectin and NE cutoffs for relapse were determined using log-rank analysis. Cox proportional hazard analyses evaluated relapse risk factors. RESULTS: Tissue calprotectin and NE levels were significantly higher in patients with histological activity than in those in histological remission (P < .001). The optimal cutoffs of tissue calprotectin and NE for relapse were 10.61 and 22.08 per mm2, respectively. The 3-year clinical relapse risk was significantly lower in the low-tissue NE group than in the high-tissue NE group (P = .009); however, it did not differ between the low- and high-tissue calprotectin group (P = .094). In multivariate analyses, a low level of tissue NE expression was independently associated with a lower risk of 3-year clinical relapse (adjusted hazard ratio = 0.453, 95% confidence interval = 0.225-0.911, P = .026), unlike histological index and tissue calprotectin. CONCLUSIONS: In patients with UC who have achieved clinical and endoscopic remission, tissue expression of NE is a better predictor of long-term relapse than histological activity.

The tissue expression of neutrophil elastase is a better predictor of long-term relapse than histological activity in patients with ulcerative colitis who achieved clinical and endoscopic remission.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin (PLY), triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.

Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.

Alpha-1 antitrypsin targeted neutrophil elastase protects against sepsis-induced inflammation and coagulation in mice via inhibiting neutrophil extracellular trap formation.

AIMS: Sepsis pathophysiology is complex and identifying effective treatments for sepsis remains challenging. The study aims to identify effective drugs and targets for sepsis through transcriptomic analysis of sepsis patients, repositioning analysis of compounds, and validation by animal models. MAIN METHODS: GSE185263 obtained from the GEO database that includes gene expression profiles of 44 healthy controls and 348 sepsis patients categorized by severity. Bioinformatic algorithms revealed the molecular, function, and immune characteristics of the sepsis, and constructed sepsis-related protein-protein interaction networks. Subsequently, Random Walk with Restart analysis was applied to identify candidate drugs for sepsis, which were tested in animal models for survival, inflammation, coagulation, and multi-organ damage. KEY FINDINGS: Our analysis found 1862 genes linked to sepsis development, enriched in functions like neutrophil extracellular trap formation (NETs) and complement/coagulation cascades. With disease progression, immune activation-associated cells were inhibited, while immune suppression-associated cells were activated. Next, the drug repositioning method identified candidate drugs, such as alpha-1 antitrypsin, that may play a therapeutic role by targeting neutrophil elastase (NE) to inhibit NETs. Animal experiments proved that alpha-1 antitrypsin treatment can improve the survival rate, reduce sepsis score, reduce the levels of inflammation markers in serum, and alleviate muti-organ morphological damage in mice with sepsis. The further results showed that α-1 antitrypsin can inhibit the NETs by suppressing the NE for the treatment of sepsis. SIGNIFICANCE: Alpha-1 antitrypsin acted on the NE to inhibit NETs thereby protecting mice from sepsis-induced inflammation and coagulation.

A novel in vitro cell model of the proteinase/antiproteinase balance observed in alpha-1 antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting from mutations in the alpha-1 antitrypsin (AAT) protein, a major systemic antiproteinase, resulting in reduced/no release of AAT, disrupting the proteinase/antiproteinase balance. A sustained imbalance can cause structural changes to the lung parenchyma, leading to emphysema. Predicting and assessing human responses to potential therapeutic candidates from preclinical animal studies have been challenging. Our aims were to develop a more physiologically relevant in vitro model of the proteinase/antiproteinase balance and assess whether the data generated could better predict the efficacy of pharmacological candidates to inform decisions on clinical trials, together with expected biomarker responses. Methods: We developed an in vitro model assessing the proteinase/antiproteinase balance by the changes in the fibrinogen cleavage products of neutrophil elastase (NE) and proteinase 3 (PR3). This allowed the assessment of physiological and pharmaceutical neutrophil serine proteinase (NSP) inhibitors to determine the putative threshold at which the maximal effect is achieved. Results: AAT significantly reduced NE and PR3 activity footprints, with the maximal reduction achieved at concentrations above 10 µM. The inhibitor MPH966 alone also significantly reduced NE footprint generation in a concentration-dependent manner, leveling out above 100 nM but had no effect on the PR3 footprint. At levels of AAT consistent with AATD, MPH966 had an additive effect, reducing the NE activity footprint more than either inhibitor alone. Conclusion: Our results support an inhibitor threshold above which the activity footprint generation appears resistant to increasing dosage. Our model can support the testing of inhibitors, confirming activity biomarkers as indicators of likely pharmaceutical efficacy, the assessment of NSP activity in the pathophysiology of emphysema, and the likely function of biological or pharmacological inhibitors in disease management.

Identification of lignans as selective cyclooxygenase-2 inhibitors from the extract of Acanthopanacis cortex.

Acanthopanacis cortex (the dried root bark of Acanthopanax gracilistylus W. W. Smith) has been used for the treatment of rheumatic diseases in China for over 2000 years. Four previously undescribed lignans (1-4) and 12 known lignans (5-16) were isolated from Acanthopanacis cortex. In this study, the inhibitory activities of compounds 1-16 against neutrophil elastase (NE), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are reported. The results show that compounds 1-16 exhibit weak inhibitory activities against NE and COX-1. However, compounds 2, 6-8 and 13-16 demonstrate better COX-2 inhibitory effects with IC50 values from 0.75 to 8.17 µΜ. These findings provide useful information for the search for natural selective COX-2 inhibitors.

Evaluation of Salivary Biomarkers and Spirometry for Diagnosing COPD in Non-Smokers and Smokers of Polish Origin.

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory condition with global implications. Accurate and timely diagnosis is critical; however, traditional diagnostic methods (based on spirometry) show limitations, prompting the search for predictive biomarkers and modern diagnostic techniques. This study explored the validation of COPD-related biomarkers (C-reactive protein, procalcitonin, neutrophil elastase, and alpha-1 antitrypsin) in saliva. A diverse cohort, including healthy non-smokers, healthy smokers, and COPD patients of Polish origin, underwent spirometry and marker analysis. The data correlated with clinical factors, revealing noteworthy relations. Firstly, salivary biomarker levels were compared with serum concentrations, demonstrating notable positive or negative correlations, depending on the factor. Further analysis within healthy individuals revealed associations between biomarker levels, spirometry, and clinical characteristics such as age, sex, and BMI. Next, COPD patients exhibited an enhanced concentration of biomarkers compared to healthy groups. Finally, the study introduced a breathing assessment survey, unveiling significant associations between self-perceived breathing and spirometric and tested parameters. Outcomes emphasized the relevance of subjective experiences in COPD research. In conclusion, this research underscored the potential of salivary biomarkers as diagnostic tools for COPD, offering a non-invasive and accessible alternative to traditional methods. The findings paved the way for improved modern diagnostic approaches.

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients.

Objectives: Elevated circulating DNA (cirDNA) concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release. We carried out the first prospective, multicenter study of the dynamics of cirDNA and neutrophil extracellular trap (NETs) markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients. Methods: We examined the plasma levels of two NETs protein markers [myeloperoxidase (MPO) and neutrophil elastase (NE)], as well as levels of cirDNA of nuclear (cir-nDNA) and mitochondrial (cir-mtDNA) origin in 29 colon, prostate, and breast cancer patients and in 114 healthy individuals (HI). Results: The synergistic analytical information provided by these markers revealed that: (i) NETs formation contributes to post-surgery conditions; (ii) post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer [r = 0.60 (P < 0.001) and r = 0.53 (P < 0.01), respectively], but not in prostate and breast cancer; (iii) each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics, but overall the pre- and post-surgery median values of cir-nDNA, NE, and MPO were significantly higher in cancer patients than in HI. Conclusion: Taken as a whole, our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period, depending on surgical procedure or cancer type. By contrast, cir-mtDNA is poorly associated with NETs formation in the studied perioperative period, which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

Unveiling the metabolites underlying the skin anti-ageing properties of Cytinus hypocistis (L.) L. through a biochemometric approach.

BACKGROUND: The genus Cytinus, recognised as one of the most enigmatic in the plant kingdom, has garnered attention for its bioactive potential, particularly its skin anti-ageing properties. Despite this recognition, much remains to be accomplished regarding deciphering and isolating its most active compounds. HYPOTHESIS: This study aimed to identify the compounds responsible for C. hypocistis skin anti-ageing potential. METHODS: Using multivariate analysis, a biochemometric approach was applied to identify the discriminant metabolites by integrating extracts' chemical profile (Liquid Chromatography-High-Resolution Mass Spectrometry, LCHRMS) and bioactive properties. The identified bioactive metabolite was structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR). RESULTS: Among the studied bioactivities, the anti-elastase results exhibited a significant variation among the samples from different years. After the biochemometric analysis, the compound 2,3:4,6-bis(hexahydroxydiphenoyl)glucose, with a molecular mass of 784.075 Da, was structurally elucidated as the discriminant feature responsible for the outstanding human neutrophil elastase inhibition. Remarkably, the subfraction containing this compound exhibited a tenfold improvement in neutrophil elastase inhibition efficacy compared to the crude extract; its effectiveness fell within the same range as SPCK, a potent irreversible neutrophil elastase inhibitor. Moreover, this subfraction displayed no cytotoxicity or phototoxicity and excellent efficacy for the tested anti-ageing properties. CONCLUSIONS: Hydrolysable tannins were confirmed as the metabolites behind C. hypocistis skin anti-ageing properties, effectively mitigating critical molecular mechanisms that influence the phenotypically distinct ageing clinical manifestations. Pedunculagin was particularly effective in inhibiting neutrophil elastase, considered one of the most destructive enzymes in skin ageing.