Elastase Activity Research Articles

Synovial fibroblasts from children with oligoarticular juvenile idiopathic arthritis induce migration and prolong viability of neutrophils.

Little is known of the processes that trigger neutrophil activation in the joint of patients with oligoarticular juvenile idiopathic arthritis (oJIA), and if synovial fibroblasts (S-Fib) play an important role in the activation. Therefore, we aimed to investigate whether S-Fib derived from oJIA patients drive neutrophil activation. Synovial fluid (SF) was collected from patients with oJIA. S-Fib were isolated from the SF of n = 7 patients through passaging. Subsequently, the S-Fib were primed or not with 20% of pooled SF. Supernatants were used to study migration of neutrophils in a transwell system. Additionally, the influence of S-Fib on neutrophils were studied in co-cultures. Phenotype and viability were assessed by flow cytometry. Neutrophil function was tested through the production of reactive oxygen species (ROS), and supernatants were tested for myeloperoxidase (MPO) release and elastase activity. Supernatants of S-Fib induced neutrophil migration (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (p = 0.0063). Additionally, co-culture between SF-primed S-Fib and neutrophils resulted in prolonged viability (n = 5, p = 0.0094), with little effect on activation markers, e.g., CD11b. Conversely, co-culture did not induce functional alterations (n = 4), such as production of ROS (p > 0.1570), release of MPO (p > 0.4934) or elastase activity (p > 0.0904). Finally, supernatant stimulation did not replicate the results of prolonged viability (p = 0.9102), suggesting a role of cell-contact. S-Fib from patients with oJIA induce migration of neutrophils via soluble mediators and, in addition, S-Fib prolong neutrophil viability in a cell-contact dependent manner. These mechanisms could be important for accumulation of neutrophils during arthritis.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Green synthesis of silver nanoparticles using food supplement from Avena sativa L., and their antioxidant, antiglycation, and anti-aging activities: In vitro and in silico studies

Food supplement of Avena sativa L. dry extract (AsDE) is produced with green leaves of wild oats and it is commonly used in patients with cognitive disorders and muscle deficits. These symptoms and pathologies can be promoted and/or aggravated by oxidative stress and protein glycation. In order to prevent or treat these disorders, new formulations that could enhance and benefit AsDE have been developed and the formation of metallic nanoparticles by green synthesis has shown to be an alternative. Thus, this study aimed to prepare silver nanoparticles (AgNPs) by green synthesis using AsDE and characterize these AgNPs. Besides, it aimed to evaluate in vitro antioxidant and antiglycation activities of AsDE and AgNPS, and analyze in vitro and in silico inhibitory action on aging enzymes (collagenase, elastase, and tyrosinase). UV–visible spectroscopy, Zeta potential and scanning transmission electron microscopy were used to characterize AgNPs. Antioxidant activity was determined by DPPH free radical scavenging, ferric ion reducing power (FRAP), lipid peroxidation inhibition, ABTS radical scavenging, and oxygen radical absorption capacity (ORAC). Molecular docking analyses were performed to evaluate interactions between the major compounds of AsDE and aging enzymes or DNA. Antioxidant tests demonstrated antioxidant activity of 19.67 % for AsDE and 42.98 % for AgNPs in DPPH test; and 115.67 µM Trolox Equivalent (TE)/g for AsDE and 554.33 µM TE/g for AgNPs in FRAP test. In addition, AsDE and AgNPs inhibited 25.40 % and 11.33 % of lipid peroxidation, respectively, and presented 255.22 µM TE/g for AsDE and 317.44 µM TE/g for AgNPs in ABTS test. AsDE and AgNPs exhibited antioxidant potential observed in ORAC assay. In addition, AsDE presented inhibitory action on collagenase, elastase and tyrosinase activities (78.23 %, 68.14 % and 62.27 % inhibition, respectively). Antiglycation tests demonstrated that bovine serum albumin exposed to ribose and treated with AsDE at 0.5 mg/mL exhibited the highest percentage of free amino groups (45.21 %), while samples treated with AgNPs showed 35.41 % free amino groups. On the other hand, AgNPs exhibited the highest percentage of inhibition of advanced glycation end-products formation (85.75 %), which did not differ from aminoguanidine, a known antiglycation agent. Besides, relative electrophoretic mobility assay demonstrated that samples treated with AsDE at 0.1 mg/mL or AgNPs showed antiglycation activity comparable to aminoguanidine. The in silico assays showed interactions between the major compounds of AsDE and aging enzymes or DNA. Therefore, results represent an important indicator for the development and discovery of new nanostructured pharmaceutical and cosmetic formulations using plants and their bioactive compounds.

Plectranthus scutellarioides (L.) R.Br. Leaf Extract as Sunscreen, Skin Lightening, and Antiaging

Plectranthus scutellaroides is a plant with attractive colored leaves. It contains secondary metabolite compounds such as flavonoids and phenolics, which provide skin protection against ultraviolet rays from the sun. The research aims to investigate the activity of P. scutellarioides leaf extract from various variants and different solvents on sunscreen, skin lightening, and anti aging activities. The best extract is formulated in gel dosage form. Each P. scutellaroides leaf was extracted using ethanol, ethyl acetate, and hexane as solvents. The obtained extract was assessed for Sun Protecting Factor (SPF), UVA Protection grade (PA), antioxidant activity, and inhibition of tyrosinase, collagenase, elastase, and hyaluronidase. Determination was conducted in vitro using spectrophotometric methods. The highest sunscreen activity was observed in the ethanol extract Va (Va-ET), which exhibited SPF and PA values of 25.618±0.265, and 0.681±0.007 (star 3), respectively at a concentration of 100 ug/mL. The most significant skin-lightening activity was observed in the ethanol extract Vc (Vc-ET), with an IC50 value inhibiting tyrosinase of 39.059 ug/mL. Regarding the anti-aging activity of the extracts, as determined by their antioxidant activity and inhibition of collagenase, elastase, and hyaluronidase, the most promising extracts were obtained from ethanol extract Va (Va-ET) and ethyl acetate extract Va (Va-EA), with IC50 values of 79.734, 76.838, 143.384, and 122.467 ug/ mL, respectively. The Va-ET was formulated into a gel dosage form, and there was a significant effect of the gelling agent on the SPF value of the extract after formulation (p < 0.05). All extracts exhibit activity as sunscreen, skin lightening, and anti-aging, with the Va-ET showing the highest efficacy. Gelling agents significantly influence the SPF value of extracts after formulation into the gel dosage form. Among them, F3, formulated from the Va-ET of P. scutellaroides using Carbopol-940 as a gelling agent, exhibits the highest efficacy.

Effect of phenylalanine arginyl β-naphthylamide on the imipenem resistance, elastase production, and the expression of quorum sensing and virulence factor genes in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl β-naphthylamide (PAβN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAβN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAβN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAβN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAβN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAβN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.

Roles of transcriptional factor PsrA in the regulation of quorum sensing in Pseudomonas aeruginosa PAO1.

The transcription factor PsrA regulates fatty acid metabolism, the type III secretion system, and quinolone signaling quorum sensing system in Pseudomonas aeruginosa. To explore additional roles of PsrA in P. aeruginosa, this study engineered a P. aeruginosa PAO1 strain to carry a recombinant plasmid with the psrA gene (pMMBpsrA) and examined the impact of elevated psrA expression to the bacterium. Transcriptomic analysis revealed that PsrA significantly downregulated genes encoding the master quorum-sensing regulators, RhlR and LasR, and influenced many quorum-sensing-associated genes. The role of PsrA in quorum sensing was further corroborated by testing autoinducer synthesis in PAO1 [pMMBpsrA] using two reporter bacteria strains Chromobacterium violaceum CV026 and Escherichia coli [pSB1075], which respond to short- and long-chain acyl homoserine lactones, respectively. Phenotypic comparisons of isogenic ΔpsrA, ΔlasR, and ΔpsrAΔlasR mutants revealed that the reduced elastase, caseinase, and swarming activity in PAO1 [pMMBpsrA] were likely mediated through LasR. Additionally, electrophoretic mobility shift assays demonstrated that recombinant PsrA could bind to the lasR promoter at a 5'-AAACGTTTGCTT-3' sequence, which displays moderate similarity to the previously reported consensus PsrA binding motif. Furthermore, the PsrA effector molecule oleic acid inhibited PsrA binding to the lasR promoter and restored several quorum sensing-related phenotypes to wild-type levels. These findings suggest that PsrA regulates certain quorum-sensing phenotypes by negatively regulating lasR expression, with oleic acid acting as a crucial signaling molecule.

Efficacy of the Coordinative compound of Aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid on the Model of Chronic Alcohol-Induced Pancreatitis

The aim of the study is to determine the efficacy of the innovative coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (CCAA, possessing hepatoprotective and antioxidative activity) on the model of chronic alcohol-induced pancreatitis in rats (Lieber–DeCarli isocaloric alcohol liquid diet for 10 weeks + LPS administration once a week for the three last weeks, 3 mg/kg intravenously). CCA was administered for 21 days at doses of 30, 60, and 120 mg/kg intragastrically. Leucocyte content in the peripheral blood and erythrocyte sedimentation rate, basal glycemia, total lipids level, α-amylase and pancreatic elastase activity in blood serum and the duodenal contents as well as histological structure of the pancreas were evaluated. In the blood serum and the homogenate of the pancreas, glutathione content, superoxide dismutase (SOD), catalase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase activities were measured. The dose-dependent normalizing effect of CCAA was registered by the influence on the non-specific inflammatory markers, pancreatic exocrine function, and cytoarchitectonics of the pancreas (the highest efficacy – at a dose of 120 mg/kg). The normalizing influence on the prooxidant-antioxidant balance markers was not dose-dependent with an especially significant normalizing influence on SOD activity. The results substantiate expanding indications for the of the studied drug.

Neutrophils insert elastase into hepatocytes to regulate calcium signaling in alcohol-associated hepatitis.

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

Impaired neutrophil migration underpins host susceptibility to infectious colitis

Citrobacter rodentium models infection with enteropathogenic E. coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here we found that bone marrow, blood and colonic C57 neutrophils expressed CD11bHi and reached the infected colonic lumen where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonisation site resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.

DNA of neutrophil extracellular traps promote NF-κB-dependent autoimmunity via cGAS/TLR9 in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However, whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD, employing human neutrophils, airway epithelial cells (AECs), dendritic cells (DCs), and a long-term CS-induced COPD mouse model, alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice (cGAS-−/−, TLR9−/−); Additionally, bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs, containing oxidatively-damaged DNA (NETs-DNA), promoted AECs proliferation, nuclear factor kappa B (NF-κB) activation, NF-κB-dependent cytokines and type-I interferons production, and DC maturation, which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model, blocking NETs-DNA-sensing via cGAS−/− and TLR9−/− mice, inhibiting NETosis using mitoTEMPO, and degrading NETs-DNA with DNase-I, respectively, reduced NETs infiltrations, airway inflammation, NF-κB activation and NF-κB-dependent cytokines, but not type-I interferons due to IFN-α/β receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels, but not type-I interferon levels. In conclusion, NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore, targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Effect of Trehalose Disaccharide on Activation of Microglia and Indices of Systemic Inflammation in an Experimental Model of Alzheimer's Disease.

In an experimental model of Alzheimer's disease in mice, oral administration of trehalose disaccharide reduces neuroinflammation assessed by the expression level of microglia activation marker Iba1 and affects the neutrophil degranulation activity. A potential anti-inflammatory effect of 4% trehalose solution associated with a decrease in the activity of leukocyte elastase in plasma was revealed.

Validation of a method for measuring the antielastolytic activity of human circulating alpha1-antitrypsin

The existence of alpha-1 antitrypsin variants with apparently unremarkable phenotypes and serum concentrations, contrasting with a clinical picture suggestive of a severe deficiency, led us to investigate whether in these cases there was a reduction or even suppression of the capacity of alpha-1 antitrypsin to inhibit elastase. To this end, in two different laboratories, we adapted and validated a method for measuring the functional activity of alpha-1 antitrypsin, based on spectrophotometric kinetic analysis of the inhibition by serum alpha-1 antitrypsin of the hydrolytic activity of porcine pancreatic elastase on a chromogenic substrate. This method has proved to be robust, reproducible and transferable and made possible to define, on the basis of an analysis of a hospital population, a functionality index with a confidence interval comprised between 0.87 and 1.2, allowing to identify subjects likely to have a functional deficiency of alpha-1 antitrypsin, whether this deficiency being of a genetic origin without any quantitative or phenotypic translation, or whether being acquired under the effect of external agents (cigarette smoke or viruses).

New Polymeric Hydrogels with Cannabidiol and α-Terpineol as Potential Materials for Skin Regeneration-Synthesis and Physicochemical and Biological Characterization.

Dermatology and cosmetology currently prioritize healthy, youthful-looking skin. As a result, research is being conducted worldwide to uncover natural substances and carriers that allow for controlled release, which could aid in the battle against a variety of skin illnesses and slow the aging process. This study examined the biological and physicochemical features of novel hydrogels containing cannabidiol (CBD) and α-terpineol (TER). The hydrogels were obtained from ε-caprolactone (CL) and poly(ethylene glycol) (PEG) copolymers, diethylene glycol (DEG), poly(tetrahydrofuran) (PTHF), 1,6-diisocyanatohexane (HDI), and chitosan (CHT) components, whereas the biodegradable oligomers were synthesized using the enzyme ring-opening polymerization (e-ROP) method. The in vitro release rate of the active compounds from the hydrogels was characterized by mainly first-order kinetics, without a "burst release". The antimicrobial, anti-inflammatory, cytotoxic, antioxidant, and anti-aging qualities of the designed drug delivery systems (DDSs) were evaluated. The findings indicate that the hydrogel carriers that were developed have the ability to scavenge free radicals and impact the activity of antioxidant enzymes while avoiding any negative effects on keratinocytes and fibroblasts. Furthermore, they have anti-inflammatory qualities by impeding protein denaturation as well as the activity of proteinase and lipoxygenase. Additionally, their ability to reduce the multiplication of pathogenic bacteria and inhibit the activity of collagenase and elastase has been demonstrated. Thus, the developed hydrogel carriers may be effective systems for the controlled delivery of CBD, which may become a valuable tool for cosmetologists and dermatologists.

Evaluation of potential anti-aging effects of Achillea phrygia Boiss. & Balansa (Asteraceae)

This study aims to determine the anti-aging effects of Achillea phrygia, an endemic plant, by evaluating its sun protection factor (SPF) level, antioxidant activity, total phenolic content, extracellular matrix-degrading enzymes (ECM) inhibition, genotoxic/anti-genotoxic, and cytotoxic activities. The SPF level was assessed using an in vitro quantitative method, while antioxidant capacity was determined through DPPH, β-carotene, and hydroxyl-radical (H2O2) scavenging assays. The total phenolic content was quantitatively conducted using the Folin Ciocalteu reagent. The inhibition of ECM-degrading enzymes was determined using matrix metalloproteinase-1 (MMP-1), hyaluronidase, and elastase enzymes. Genotoxic/anti-genotoxic properties were assessed using the AMES Salmonella/microsome assay, and cytotoxicity effects were assessed through the MTT assay. The results indicated that A. phrygia showed moderate SPF activity (SPF = 4.013) and exhibited IC50 values of 0.183 ± 0.03, 0.079 ± 0.51, and 1.18 ± 0.35 mg/mL for DPPH, β-carotene, and hydroxyl-radicals, respectively. The total phenolic content was measured to be 23.56 ± 1.42 mg GAE/g dry extract. Furthermore, the extract demonstrated inhibition of MMP-1 (47.98%) and elastase (39.2%) activities. Importantly, it did not induce DNA damage and showed antigenotoxic activity ranging from 10% to 65.6%. The cytotoxicity assay revealed an IC50 value of 42.41±4.05 µg/mL. These findings suggest that A. phrygia could be utilized as a cosmetic ingredient in skincare products due to its ability to protect against UV radiation, exhibit antioxidant properties, prevent extracellular matrix degradation, and inhibit DNA damage.

Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1β, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.

Neutrophil-mediated Inflammatory Plasminogen Degradation, Rather Than High Plasminogen-Activator Inhibitor-1, May Underly Failures and Inefficiencies of Intrapleural Fibrinolysis

Complex pleural space infections often require treatment with multiple doses of intrapleural tissue plasminogen activator (tPA) and deoxyribonuclease, with treatment failure frequently necessitating surgery. Pleural infections are rich in neutrophils, and neutrophil elastase degrades plasminogen, the target substrate of tPA, that is required to generate fibrinolysis. We hypothesized that pleural fluid from patients with pleural space infection would show high elastase activity, evidence of inflammatory plasminogen degradation, and low fibrinolytic potential in response to tPA that could be rescued with plasminogen supplementation. Does neutrophil elastase degradation of plasminogen contribute to intrapleural fibrinolytic failure? We obtained infected pleural fluid and circulating plasma from hospitalized adults (n= 10) with institutional review board approval from a randomized trial evaluating intrapleural fibrinolytics vssurgery for initial management of pleural space infection. Samples were collected before the intervention and on days 1, 2, and 3 after the intervention. Activity assays, enzyme-linked immunosorbent assays, and Western blot analysis were performed, and turbidimetric measurements of fibrinolysis were obtained from pleural fluid with and without exogenous plasminogen supplementation. Results are reported as median (interquartile range) or number (percentage) as appropriate, with an α value of .05. Pleural fluid elastase activity was more than fourfold higher (P= .02) and plasminogen antigen levels were more than threefold lower (P= .04) than their corresponding plasma values. Pleural fluid Western blot analysis demonstrated abundant plasminogen degradation fragments consistent with elastase degradation patterns. We found that plasminogen activator inhibitor 1 (PAI-1), the native tPA inhibitor, showed high antigen levels before the intervention, but the overwhelming majority of this PAI-1 (82%) was not active (P= .003), and all PAI-1 activity was lost by day 2 after the intervention in patients receiving intrapleural tPA and deoxyribonuclease. Finally, using turbidity clot lysis assays, we found that the pleural fluid of 9 of 10 patients was unable to generate a significant fibrinolytic response when challenged with tPA and that plasminogen supplementation rescued fibrinolysis in all patients. Our findings suggest that inflammatory plasminogen deficiency, not high PAI-1 activity, is a significant contributor to intrapleural fibrinolytic failure. ClinicalTrials.gov; No.: NCT03583931; URL: www. gov.

Abstract 1015: Knockout Of P27 Kip1 Accelerates Topical Elastase/3-aminopropionitrile Fumarate Salt-induced Abdominal Aortic Aneurysm Rupture In Mice

Background: Abdominal aortic aneurysms (AAAs) are multifactorial diseases, and unknown genetic factors could contribute to aneurysms. The cell cycle inhibitor CDKN1B gene, also known as p27 Kip1 (p27), affects the arterial response to injury. Previous studies have shown that the loss of p27 exacerbates atherosclerosis progression by increasing vascular smooth muscle cells and macrophages. To assess the role of p27 in AAA development, another form of arterial response to inflammatory injury, we conducted experiments on the AAA model using p27 knockout (p27 -/- ) and wild-type (WT) mice. Methods: AAAs were induced in male and female p27 -/- and WT (C57BL/6J) mice by topically applying porcine pancreatic elastase and administrating 0.2% 3-aminopropionitrile fumarate salt (BAPN) in the drinking water. Topical elastase application was divided into active and heat-inactivated elastase groups. The aortic diameter was evaluated by ultrasound examination preoperatively and every 7 days postoperatively ( Fig. A ). Surviving mice were sacrificed after the final ultrasound evaluation, and aortic samples were collected for histological analysis. Results: Fig. B shows the aortic diameter change evaluated by ultrasound examination. AAA rupture occurred in one male p27-/- mice (33%, 1 of 3) and two female p27-/- mice (40%, 2 of 5), but in no WT mice of either gender. Macrophages were assessed using anti-F4/80 antibody. In the elastase-active group, both male and female p27 -/- mice had a significantly higher accumulation of macrophage than WT mice (P<0.01 and P<0.01, respectively), but there was no significant staining of F4/80 in the elastase-inactive group ( Fig. C and D ). Conclusions: Knockout of p27 exacerbated aneurysm rupture in a topical elastase/BAPN-induced AAA model and was associated with increased macrophage infiltration.

Assessing salivary innate immune responses in adolescents with dentofacial abnormalities and central precocious puberty

Relevance. Central precocious puberty (CPP), characterized by a high incidence of 60% among endocrinopathies during puberty, significantly influences dentofacial development and the local immune defense mechanisms in adolescents. However, the impact on saliva's innate immune function remains underexplored.This study aims to evaluate the innate immune function of saliva in individuals with CPP coexisting with dentofacial abnormalities.Materials and methods. An analysis of saliva biochemical markers was conducted on 59 adolescents diagnosed with CPP and dentofacial abnormalities, alongside a control group of 21 age-matched individuals with dentofacial abnormalities but without systemic conditions, aged 13-18 years. The evaluation focused on lysozyme and urease levels, dysbiosis markers indicating oral microbiota status, malondialdehyde (MDA) levels, and catalase activity to gauge the balance between pro-oxidant and antioxidant systems, alongside proteolytic enzyme elastase activity.Results. Adolescents with combined CPP and dentofacial abnormalities exhibited weakened antioxidant and antibacterial defenses, evidenced by decreased catalase activity (0.105 ± 0.020 mkat/l, p < 0.001) and lysozyme levels (54.7 ± 3.2 U/l, p < 0.02), against a backdrop of increased lipoperoxidation and microbial contamination (MDA levels at 0.48 ± 0.11 μmol/l, p < 0.05, urease levels at 0.713 ± 0.015 μkat/l, p < 0.01), and heightened inflammation (elastase activity up to 2.71 ± 0.12 μkat/l, p < 0.01).Conclusion. The diminished innate immune function in saliva necessitates the development of strategies to enhance it, serving as a preventive measure against gingivitis in patients with CPP.

Synergistic Quorum quenching activity of Cinnamaldehyde against biofilm forming Pseudomonas aeruginosa

Quorum sensing is an essential mechanism for the pathogenesis of Pseudomonas aeruginosa, which causes opportunistic infections in humans. Quorum sensing inhibition has been considered a unique strategy to treat opportunistic infections and prevent an early event of infection without killing bacterial cells. The present study has demonstrated that cinnamaldehyde is able to inhibit biofilm formation by interfering with the quorum sensing activity of P. aeruginosa and is also able to disperse biofilms if they have already formed. It was also proven that synergistic action between 2.5 μM of cinnamaldehyde and 1 μM of cephalexin was effective in controlling biofilm formation. Cephalexin combined with cinnamaldehyde repressed the virulence factors associated with biofilms in Pseudomonas aeruginosa, such as Quorum sensing mediated motility, rhamnolipid, pyocyanin, elastase and rhamnolipid activities. A combination of cinnamaldehyde and cephalexin showed better biofilm inhibition (52.80%) than individual treatments and there was a reduction in the biofilm thickness and surface coverage among P. aeruginosa strains treated with cinnamaldehyde plus cephalexin. Further, it was also reported that the N-Hexanoylhomoserine lactone concentration was lower in P. aeruginosa strains treated with cinnamaldehyde plus cephalexin, while the control set-up showed a high amount of N-Hexanoylhomoserine lactone concentration. However, cinnamaldehyde with amoxycillin, oxacillin, or streptomycin showed a comparatively weaker quorum quenching effect when tested using checkerboard assays. Combination of CAD with CEPH can be a better alternative to treat P. aeruginosa infection.