Elastase Research Articles

Streptozotocin-induced morphological changes in rat lungs

ABSTRACT Streptozotocin (STZ) is a commonly used compound for the induction of type 2 diabetes (T2D) in animal models, but its effects on non-pancreatic tissues like the lungs are not well understood. This study aimed to examine the histopathological impact of STZ on the lungs using male Sprague-Dawley rats. The rats were divided into two groups: a control group on a normal diet and an STZ-treated group receiving a high-fat diet and 10% sucrose water for 8 weeks, followed by an STZ injection (30 mg/kg body weight). All rats were terminated 9 days after STZ administration, and lung samples were collected for light microscopy, transmission electron microscopy (TEM), and confocal laser scanning microscopy. Light microscopy revealed thickening of alveolar septa, narrowing of alveoli, and inflammatory infiltrates in the STZ group. TEM showed mitochondrial damage in type 2 pneumocytes, including membrane fragmentation, cristae loss, and formation of mitochondrial-derived vesicles. Confocal microscopy revealed significantly higher expression of myeloperoxidase, neutrophil elastase, and citrullinated histone 3 in the STZ group compared to controls. These findings suggest that STZ induces considerable lung damage, emphasizing the need to consider lung toxicity in studies involving STZ.

The Coexistence of Klebsiella pneumoniae and Candida albicans Enhanced Biofilm Thickness but Induced Less Severe Neutrophil Responses and Less Inflammation in Pneumonia Mice Than K. pneumoniae Alone

Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi.

Quercetin Protects Goat Sperm Motility by Inhibiting Neutrophil Extracellular Traps and Maintaining Plasma Membrane and Acrosome Integrity

Enhancing or protecting sperm motility has always been a pivotal approach to improving the ewe pregnancy rate. Sperm motility is highly susceptible to the immune status of the reproductive tract. Neutrophil extracellular traps (NETs) have been demonstrated to capture sperm and impair its motility in human, swine, and goat species. Quercetin is a flavonoid derived from Cuscuta Chinensis Lam., which can protect sperm from oxidative damage. In this study, we investigated whether inflammation decreases sperm motility and tried to clarify the potential protective mechanism of quercetin on goat sperm motility. Sperm-triggered NETs were analyzed by immunofluorescence analysis. Sperm acrosome integrity was detected by using giemsa staining. Quercetin exhibited no cytotoxicity towards sperm and PMNs within the concentration range of 20–80 μM. PMNs impaired both the survival rate and rapid linear motility of sperm, while quercetin significantly enhanced these parameters. PMNs captured sperm through NETs composed of DNA, citrullinated histone 3 (citH3), and neutrophil elastase (NE); however, quercetin effectively inhibited the release of sperm-stimulated NETs. The stimulation of PMNs with sperm resulted in a significant increase in levels of ROS and MDA, which decreased by quercetin. Moreover, PMNs caused integrity violation to both the plasma membrane and acrosome in sperm; this effect was significantly alleviated by quercetin. In conclusion, quercetin effectively ameliorated PMN-reduced sperm motility through the inhibition of NETs and oxidative stress, and preserving sperm plasma membrane and acrosome integrity, thereby providing preliminary insights into the underlying mechanisms and theoretical support for the development of potential sperm protectors.

Observation of neutrophil extracellular traps in the development of diabetic nephropathy using diabetic murine models.

Diabetic nephropathy (DN) is a progressive complication among patients with diabetes and the most common cause of end-stage kidney disease. Neutrophil extracellular traps (NETs) are known to play a role in kidney disease, thus this study aimed to determine their role in the development of diabetic kidney disease using diabetic murine models. Protein and histological analyses revealed that db/db mice and streptozotocin DN models expressed no significant NET-related proteins, myeloperoxidase, citrullinated histone H3 (citH3), neutrophil elastase, and lymphocyte antigen 6 complex locus G6D (Ly6G). However, the inflamed individuals in the DN model showed that citH3 and Ly6G were highly deposited in the renal system based on immunohistochemistry images. In vitro, NET treatment did not induce apoptosis in glomerular endothelial and renal tubular epithelial cells. NET inhibition by DNase administration demonstrated no significant changes in cell apoptosis. NET-related proteins were only expressed in the DN model with tubulointerstitial inflammation. Our study revealed that NETs are only induced in mice with hyperglycemia-induced inflammation.

Neutrophil Elastase Activates Macrophage Calpain as a Mechanism for Phagocytic Failure.

Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease Calpain-2 in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte derived macrophages (hMDM) from persons with CF (PwCF) and non-CF subjects, were treated with NE or control vehicle and cell lysates prepared to evaluate Calpain-2 protein abundance by Western, and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. Human MDM were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin, and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.

Neutrophil Elastase, Neuron-Specific Enolase, and S100B Protein as Potential Markers of Long-Term Complications Caused by COVID-19 in Patients with Type 2 Diabetes Mellitus (T2DM) and Advanced Stage of Diabetic Nephropathy (NfT2DM)-Observational Studies.

Despite numerous studies conducted by various research teams, predicting long-term outcomes (known as Post-COVID-19 Syndrome, PCS) that may result from Coronavirus Disease 2019 (COVID-19) remains challenging. PCS affects over a million people, primarily those with comorbid conditions. Therefore, it is crucial to undertake research aimed at developing a predictive model for early diagnosis of PCS, which in turn would enable faster preventive actions. The aim of this study was to assess the value of measuring and attempt a quantitative evaluation using Enzyme-Linked Immunosorbent Assay (ELISA) tests of three non-serum proteins, whose presence in the blood during COVID-19 was associated with severe disease progression: neutrophil elastase (NE), calcium-binding protein S100B, and neuron-specific enolase (NSE). The concentrations of these proteins were measured in blood serum samples collected before the COVID-19 pandemic from (1) patients with type 2 diabetes (T2DM); (2) advanced stage diabetic nephropathy (NfT2DM); (3) a healthy group; and in blood serum samples collected two years after recovering from COVID-19 from patients with (4) T2DM and (5) NfT2DM. It was found that elevated levels of NE and NSE were significantly more common (p < 0.05) in patients with NfT2DM after recovering from COVID-19 compared to the other groups, while elevated levels of S100B were significantly more frequently observed in patients with T2DM after recovering from COVID-19 (p < 0.05). Demonstrating differences in the prevalence of NE, NSE, and S100B in individuals who recovered from COVID-19 with T2DM and NfT2DM makes these proteins important components of the developing predictive model for early detection of PCS. To our knowledge, this is the first study showing the significance of NE, NSE, and S100B in PCS in the context of T2DM and NfT2DM.

Dexamethasone and IFN-γ primed mesenchymal stem cells conditioned media immunomodulates aberrant NETosis in SLE via PGE2 and IDO.

Systemic Lupus Erythematosus (SLE) is characterized by dysregulated immune responses, with neutrophil extracellular traps (NETs) playing a significant role. NETs are recognized by autoantibodies in SLE patients, exacerbating pathology. Both excessive NET formation and impaired degradation contribute to SLE pathophysiology. To investigate the immunomodulatory effects of Dexamethasone-primed Wharton's jelly (WJ) derived MSCs CM (DW) and IFN-γ-primed WJ-MSCs-CM (IW) on NETosis and associated protein markers in SLE patients' LPS or ribonucleoprotein immune complexes (RNP ICs) induced neutrophils and in pristane induced lupus (PIL) model. And to elucidate the mechanism involved therein. We investigated the immunomodulatory effects of DW and IW on NETosis in SLE. Utilizing ex vivo and in vivo models, we assessed the impact of preconditioned media on NET formation and associated protein markers neutrophil elastase (NE), citrullinated histone (citH3), myeloperoxidase (MPO), cytoplasmic and mitochondrial ROS production. We also examined the involvement of key immunomodulatory factors present in DW and IW, including prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor-beta (TGF-β). Preconditioned media effectively suppressed NETosis and reduced ROS generation in SLE neutrophils, indicating their immunomodulatory potential. Inhibition studies implicated IDO and PGE2 in mediating this effect. Combined treatment with DW or IW together with hydroxychloroquine (HCQ) demonstrated superior efficacy over HCQ alone, a standard SLE medication. In PIL mouse model, DW and IW treatments reduced NETosis, ROS generation, as evidenced by decreased NET-associated protein expression in vital organs. Our study highlights the multifaceted impact of IW and DW on NETosis, ROS dynamics, and lupus severity in SLE. These findings underscore the potential of preconditioned media for the development of targeted, personalized approaches for SLE treatment.

High-dose atorvastatin and rosuvastatin reduce the levels of neutrophil extracellular trap-related proteins in coronary artery disease: association with prothrombotic state.

Neutrophil extracellular trap (NET) formation is involved in atherothrombosis. We sought to investigate whether statins affect neutrophil extracellular traps activation and release (NETosis) in coronary artery disease (CAD), and whether such changes show associations with statin‑induced additional effects. We studied 130 patients with advanced CAD before and at least 6 months after initiation of high‑dose statin therapy with rosuvastatin 40 mg/d or atorvastatin 80 mg/d. The levels of circulating citrullinated histone H3 (H3cit), myeloperoxidase (MPO), and neutrophil elastase (NE) were assessed as proteins associated with NETosis along with thrombin generation, plasma clot permeability (Ks), clot lysis time (CLT), and fibrinolysis inhibitors. Following statin therapy intensification, we observed reduced accumulation of H3cit (-30.4%), MPO (-28.1%), and NE (-25.5%; all P <0.001), all not associated with low‑density lipoprotein cholesterol (LDL‑C) lowering (-25%). However, H3cit level was lower in 50 patients (38.5%) who achieved the target LDL‑C below 1.8 mmol/l (-16.5%; P = 0.004), and in 19 patients (14.6%) with LDL‑C below 1.4 mmol/l (-25.5%; P = 0.001), as compared with the remaining individuals. The reductions in H3cit and MPO levels were associated with a 42.9% decrease in C‑reactive protein (CRP) level on high‑dose statins (R = 0.855; P <0.001 and R = 0.25; P = 0.004, respectively), along with increases in Ks and reduction in thrombin activatable fibrinolysis inhibitor (TAFI) activity, but not with CLT or thrombin generation (all |R|, 0.24-0.4; P <0.01). In multivariable analysis, changes in CRP (β = 0.771; P <0.001), TAFI activity (β = 0.125; P = 0.013), and fibrinogen level (β = 0.106; P = 0.034) were independently associated with a decrease in H3cit concentration. We showed for the first time that high‑dose statins can reduce the level of NET‑related proteins in association with anti‑inflammatory and antithrombotic actions in CAD patients.

Targeting neutrophil serine proteases in bronchiectasis.

Persistent neutrophilic inflammation is a central feature in both the pathogenesis and progression of bronchiectasis (BE). Neutrophils release neutrophil serine proteases (NSPs), such as neutrophil elastase, cathepsin G and proteinase 3. When chronically high levels of free NSP activity exceed those of protective antiproteases, structural lung destruction, mucosal-related defects, further susceptibility to infection and worsening of clinical outcomes can occur. Despite the defined role of prolonged, high levels of NSPs in BE, no drug that controls neutrophilic inflammation is licensed for the treatment of BE. Previous methods of suppressing neutrophilic inflammation (such as direct inhibition of neutrophil elastase) have not been successful; however, an emerging therapy designed to address neutrophil-mediated pathology, inhibition of the cysteine protease cathepsin C (CatC, also known as dipeptidyl peptidase 1), is a promising approach to ameliorate neutrophilic inflammation, since this may reduce the activity of all NSPs implicated in BE pathogenesis, and not just neutrophil elastase. Current data suggest that CatC inhibition may effectively restore the protease-antiprotease balance in BE and improve disease outcomes as a result. Clinical trials for CatC inhibitors in BE have reported positive Phase III results. In this narrative review, we discuss the role of high NSP activity in BE, and how this feature drives the associated morbidity and mortality seen in BE. This review discusses therapeutic approaches aimed at treating neutrophilic inflammation in the BE lung, summarising clinical trial outcomes, and highlighting the need for more treatment strategies that effectively address chronic neutrophilic inflammation in BE.

Neutrophil extracellular traps are an important component of chronic myocardial inflammation in patients with heart failure

Abstract Background and aim We have previously shown that heart failure (HF) is characterized by chronic inflammation. However, the role of neutrophils (N) and extracellular neutrophil traps (NETs) in the myocardium of patients with HF is largely unknown. The present study investigated the number of neutrophils (N) and their proportion that develop NETs in HF. Methods We used quantitative confocal microscopy and NETs markers in the left ventricular biopsies obtained from 5 controls and from patients with HF due to dilated (DCM, n=7), ischemic (ICM, n=7) and inflammatory (infCMP, n=7) cardiomyopathy. We used immunolabeling for CD45 (N) and for NETs (citrullinated histone 3 (CitH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO). Results Compared to the control, the number of N was 4.3-5.7 times higher in HF. Using single labeling for NETs marker revealed that 41.1±4.6% of N in DCM, 42.9±4.1% in ICM and 47.6±2.9% in infCMP developed NETs. The use of dual labeling (MPO with CitH3, MPO with PAD-4 and CitH3 with NE) resulted in 54.5±4.7% of N-developing NETs in DCM, 59.5±4.1% in ICM and 77.6%±4.2 in infCMP. The difference between N-undergoing NETs was statistically different in infCMP than in DCM.and ICM. The proportion of N-forming NETs in control tissue was less than 5% and was significantly different from that in HF patients regardless of etiology. Conclusions This is the first study to show the presence of NETs in human hearts in situ and demonstrates that NETs are an important component of chronic inflammation in HF due to cardiomyopathies.

The high-dose statin treatment reduces neutrophil extracellular traps formation in patients with coronary artery disease

Abstract Background Neutrophil extracellular traps formation (NETosis) is currently considered as an important mediator in atherosclerosis and atherothrombosis. However, the impact of high-dose statin treatment, as potent cholesterol-lowering agents with pleiotropic effects, on NETosis in coronary artery disease (CAD) has been poorly described. Purpose We sought to assess whether in CAD patients the recommended statin treatment intensification is associated with changes in NETs-related markers and if such changes can alter fibrin clot properties. Methods 130 patients with advanced CAD, who did not achieve the target low-density lipoprotein cholesterol (LDL-C) were included. Before and at least 6 months after initiation of high-dose statin therapy (rosuvastatin 40 mg/d or atorvastatin 80 mg/d) citrullinated histone H3 (H3cit), myeloperoxidase (MPO) and neutrophil elastase (NE) as markers associated with NETosis were assessed in relation to C-reactive protein (CRP), thrombin generation, plasma clot permeability (Ks), clot lysis time (CLT), and fibrinolysis proteins. Results At baseline, NETosis did not differ depending on gender and cardiovascular risk factors but H3cit correlated with MPO (R=0.543, P&amp;lt;0.001) and CRP (R=0.540, P&amp;lt;0.001). All NETosis markers were inversely correlated with Ks. Median LDL-C reduction by 25% (P&amp;lt;0.001) on high-dose statin treatment was independent from H3cit reduction by 30.4%, MPO by 28.1%, and NE by 25.5% (in all P&amp;lt;0.001, Figure 1). The ΔH3cit and ΔMPO, but not ΔNE were associated with ΔCRP (R=0.855, P&amp;lt;0.001; R=0.250, P=0.004, respectively), Δthrombin activatable fibrinolysis inhibitor (TAFI) (R=0.385, P&amp;lt;0.001; R=0.245, P=0.005), and inversely with ΔKs (R=-0.315, P&amp;lt;0.001; R=-0.395, P=0.001). In multivariable analysis the H3cit decrease was independently associated with ΔCRP (β=0.771, P&amp;lt;0.001), ΔTAFI (β=0.125, P=0.013) and Δfibrinogen (β=0.106, P=0.034) but not with ΔLDL-C. Conclusions As has been shown for the first time high-dose statin treatment reduces levels of NETs-related markers, regardless of lipid-lowering effect in CAD patients.

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Increased Lipocalin 2 detected by RNA sequencing regulates apoptosis and ferroptosis in COPD

BackgroundChronic obstructive pulmonary disease (COPD) is a complex respiratory condition influenced by environmental and genetic factors. Using next-generation sequencing, we aimed to identify dysregulated genes and potential therapeutic targets for COPD.MethodsPeripheral blood leukocyte RNA profiles from COPD patients and healthy controls were analyzed using next-generation sequencing. Key genes involved in COPD pathogenesis were identified through protein–protein interaction network analysis. In vitro, bronchial epithelial cells treated with cigarette smoke extract (CSE) were used to study the effects on gene expression, cell viability, apoptosis, and ferroptosis. Additionally, Lipocalin 2 (LCN2) inhibition experiments were conducted to elucidate its role in COPD-related cellular processes.ResultsAnalysis of RNA profiles revealed consistent downregulation of 17 genes and upregulation of 21 genes across all COPD groups. Among these, Cathelicidin Antimicrobial Peptide(CAMP), Defensin Alpha 4(DEFA4), Neutrophil Elastase(ELANE), LCN2 and Lactotransferrin(LTF) were identified as potentially important players in COPD pathogenesis. Particularly, LCN2 exhibited a close association with COPD and was found to be involved in cellular processes. In vitro experiments demonstrated that CSE treatment significantly increased LCN2 expression in bronchial epithelial cells in a concentration-dependent manner. Moreover, CSE-induced apoptosis and ferroptosis were observed, along with alterations in cell viability, Glutathione content, Fe2 + accumulation, ROS: Reactive Oxygen Species and Malondialdehyde levels, Lactate Dehydrogenase(LDH) release and Glutathione Peroxidase 4(GPX4) expression. Inhibition of LCN2 expression partially reversed these effects, indicating the pivotal role of LCN2 in COPD-related cellular processes.ConclusionOur study identified six candidate genes: CAMP, DEFA4, ELANE, LCN2, and LTF were upregulated, HSPA1B was downregulated. Notably, LCN2 emerges as a significant biomarker in COPD pathogenesis, exerting its effects by promoting apoptosis and ferroptosis in bronchial epithelial cells.

Development and validation of a nomogram for predicting bacterial infections in patients with acute exacerbation of chronic obstructive pulmonary disease.

Bacterial infection is a significant contributory factor in the pathogenesis of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and it has a pivotal role in exacerbating symptoms and precipitating mortality among patients with chronic obstructive pulmonary disease (COPD). The early identification of bacterial infection in individuals with COPD remains a challenge. Therefore, the present study aimed to create and validate a risk assessment tool using easily accessible serum biomarkers to predict bacterial infection in individuals with AECOPD. A retrospective cohort study was carried out at Pingxiang People's Hospital (Pingxiang, China) from January 2023 to December 2023, involving individuals diagnosed with AECOPD. A total of 544 patients with AECOPD were randomly allocated to the two following groups: The training set, which included 70% (n=384) of the patients, and the validation set, which included 30% (n=160) of the patients. Subsequently, a nomogram model was constructed using multivariate logistic regression analysis in the training set. Its discriminatory ability and calibration were internally validated, while decision curve analyses were employed to assess the clinical utility of the nomogram. The incidence of bacterial infection in hospitalized patients with AECOPD was 50% in the training set and 48.1% in the validation set. The nomogram model incorporated independent factors associated with bacterial infection, including C-reactive protein, neutrophil elastase, procalcitonin and eosinophils, identified by univariate and multivariate logistic regression analyses. The area under the curve of the nomogram model was 0.835 [95% confidence interval (CI): 0.795-0.875] in the training set and 0.785 (95% CI: 0.715-0.856) in the validation set. The model demonstrated excellent discrimination and calibration in the validation set [c-statistic: 0.79 (95% CI: 0.68-0.90)]. Furthermore, the discrimination and overfitting bias of the model were assessed through internal validation, revealing a C-index of 0.836 for the initial group and 0.788 for the subsequent validation set. The straightforward risk prediction model for early identification of bacterial infections is valuable for hospitalized patients with AECOPD.

Can switching from cigarettes to heated tobacco products reduce consequences of pulmonary infection?

While tobacco industry data suggests that switching from combustible cigarettes to heated tobacco products (HTPs), like IQOS, may reduce the users' exposure to respiratory toxicants, it is not known if using HTPs impacts the outcomes of acute respiratory infections. Does switching from cigarettes to HTPs improve responses to pulmonary infection. We conducted experiments in which 3 groups of mice were pre-exposed to cigarette smoke for 8 weeks, followed by 8-week exposure to (1) HTPs (tobacco product switching), (2) air (smoking cessation), or (3) continued exposure to cigarette smoke. Pulmonary bacterial clearance and surrogate markers of lung damage were assessed as study outcomes. Significantly compromised clearance of bacteria from the lungs post-acute challenge occurred in both the switching group and in mice continuously exposed to cigarette smoke. Bacterial clearance, inflammatory T-cell infiltration into the lungs, and albumin leak improved at 12h post-acute challenge in the switching group compared to mice continuously exposed to cigarette smoke. Bacterial clearance, total lung immune-cell infiltration, inflammatory T-cell infiltration into the lungs, the content of total proteins in the BAL, and albumin leak measured post-acute challenge were compromised in the switching group compared to mice in the cessation group. Switching from cigarettes to HTPs did not improve lung myeloperoxidase and neutrophil elastase levels (markers for lung inflammation and damage), which, however, were significantly reduced in the cessation group. This study reveals only a modest improvement in respiratory infection outcomes after switching exposure from cigarettes to HTPs and significantly compromised outcomes compared to a complete cessation of exposure to all tobacco products.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes.

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Soluble Urokinase Plasminogen Activator Receptor as a Predictor of All-Cause Death in Patients Undergoing Coronary Angiography at 10-Year Follow-Up.

Background: We aimed to explore the predictive role of soluble urokinase plasminogen activator receptor (suPAR) in patients undergoing coronary angiography by systematically evaluating its association with adverse cardiovascular events at 10 years follow-up. Methods: The KORONEF study was a single-center, observational, prospective study with 492 subjects included. In the multivariable Cox regression model, we checked the impact of suPAR, neutrophil elastase, myeloperoxidase, and DNase 1 on long-term outcomes. Results: The mean study population age was 64.4 ± 9.9 years, and there were 37.2% women. We divided the population into tertiles of suPAR levels (T1 0.793-2.135 ng/mL; T2 2.136-2.868 ng/mL; and T3 2.872-8.677 ng/mL). Patients with higher suPAR concentrations were more often females (tertile 1 vs. tertile 3: 27.4% vs. 50.6%, p < 0.001) and older age (60.8 ± 8.7 years vs. 68.8 ± 9.5 years, p < 0.001). They also characterized higher incidence of diabetes (17.7% vs. 38.0%, p < 0.001), previous myocardial infarction (22% vs. 44.8%, p < 0.001), and chronic kidney disease (3% vs. 18.4%, p < 0.001), but lower incidence of dyslipidemia (54.3% vs. 35.6%). The 10-year all-cause death rates were 14.6% vs. 34.1%, HR 2.68, 95% CI 1.66-4.33, p < 0.001 for tertile 2, and 14.6% vs. 39.9%, HR 3.24, 95% CI 2.03-5.17, p < 0.001 for tertile 3. The optimal cut-off suPAR value of 2.39 ng/mL provided a sensitivity of 66.9% and a specificity of 54.6% in predicting all-cause death. Conclusions: The association of elevated suPAR with increased mortality risk suggests its potential relevance in predicting long-term outcomes and may help inform more individualized management strategies for high-risk patients.

Exo-Cleavable Linkers: Enhanced Stability and Therapeutic Efficacy in Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) combine cytotoxic payloads with monoclonal antibodies through chemical linkers. Finding linkers that both enhance circulatory stability and enable effective tumor payload release remains a challenge. The conventional valine-citrulline (Val-Cit) linker is associated with several inherent drawbacks, including hydrophobicity-induced aggregation, a limited drug-antibody ratio (DAR), and premature payload release. This study introduces an exolinker approach, repositioning the cleavable peptide linker at the exo position of the p-aminobenzylcarbamate moiety, as an advancement over conventional linear linkers. This design, which incorporates hydrophilic glutamic acid, addresses the limitations of the Val-Cit platform and improves the ADC in vivo profiles. In vitro and in vivo evaluations showed that exolinker ADCs reduced premature payload release, increased drug-to-antibody ratios, and avoided significant aggregation, even with hydrophobic payloads. Furthermore, the payload remained stably attached to the ADC even in the presence of enzymes like carboxylesterases and human neutrophil elastase, indicating the potential for a favorable safety profile.

Cytokines Measured in Nasal Lavage Compared to Induced Sputum in Patients with Mild Cystic Fibrosis

The measurement of cytokines in induced sputum and nasal lavage (NL) samples has been performed for years in people with cystic fibrosis (CF). The aim of this study was to directly compare sputum and NL samples and interpret results based on disease severity in patients who were categorized as having mild or severe lung disease. The categorization was based primarily on structural abnormalities detected on lung computed tomography and secondarily on lung function. The serum inflammatory markers neutrophil elastase (NE), IL-1β, 2, 6, 8, 10 and 17a were measured in each sputum and NL sample. Thirty-two sample pairs from 29 patients were included in this study (13 mild, 19 severe). In the patients classified as severe, many systemic inflammatory markers as well as sputum cytokines were significantly higher compared to those in the mild patients. However, all the markers measured in the NL were higher in the mild patients (p =&lt; 0.05 for NE, IL-6 and IL-8). In addition, many cytokines in the NL correlated negatively with those in the sputum samples. Major differences in the cytokine levels were shown although the samples were obtained at the same time in the same patient. Advanced structural lung disease was closely related to systemic and lower airway inflammation, whereas preserved lung function was associated with higher levels in the NL. We hypothesize that the main part of the immune response takes place in the nasal mucosa in patients with minor pulmonary changes. Our results suggest that inflammation must be interpreted individually depending on the compartment in which it is measured. Further research is needed to accurately understand inflammatory markers measured in NL.