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Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP-dependent manner. In vitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

BACKGROUND AND OBJECTIVES Assessment of plasma quality often focuses on the common safety tests for minimizing the risk of transmitting blood-borne pathogens. Little attention is paid to the possible quality attributes that ensure a consistent biochemical composition of plasma for fractionation. We therefore investigated the suitability of selected biochemical and haematological attributes that could be used as markers of plasma quality obtained by different separation and pre-treatment procedures. MATERIAL AND METHODS We characterized six plasma types, including source plasma, plasma recovered by classic means and in-line filtered plasma, by determining the analytical attributes protein content, coagulation factors and markers of coagulation, contact and complement activation. Residual cell content and cell-specific variables were also measured. RESULTS We found relevant differences between the plasma types in complement activation, as indicated by C3a measurements, while thrombin antithrombin complex values and, to a minor extent, activated factor XII concentrations indicated only moderate differences in activation levels of coagulation and contact systems. The most striking differences, however, were detected in residual cell content and concentrations of the platelet-associated proteins, platelet factor 4 and β-thromboglobulin. We showed that leucocyte reduction filters disrupt cells. This includes platelets, thereby releasing the platelet-associated proteins platelet factor 4 and β-thromboglobulin, and leucocytes as demonstrated by the release of elastase from polymorphonuclear leucocytes. Furthermore, the filtration processing of whole blood can lead to activation of the complement system. CONCLUSION Our results show that biochemical and cellular surrogate markers are valuable discriminators of plasma types.

Background Recent evidence suggests that generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, is associated with the pathogenesis of both venous thromboembolism and sickle cell disease (SCD). NETosis is a complex process regulated by several proteins such as peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), and myeloperoxidase (MPO). Among these regulators, PADI4 is responsible of histone citrullination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a promising therapeutic strategy for diseases such as SCD. Although attractive, this strategy requires supportive evidence of its role in the pathogenesis of SCD. Patients and Methods Patients from two independent cohorts were enrolled in this study. Samples were obtained at steady state (53 patients) or during acute episodes of vaso‐occlusive crisis (VOC; 28 patients) in patients from cohort 1. mRNA was extracted from granulocytes to analyze PADI4, ELANE, and MPO expression by qPCR. Furthermore, plasma activity of PADI4 was assessed from an independent cohort in 15 patients, within 24 hours from admission for VOC. Race‐matched healthy individuals from the same geographic regions were used as controls for each cohort. Results and Conclusions Higher levels of gene expression of PADI4 and ELANE were observed during VOC. Furthermore, plasma activity of PADI4 was higher in acute VOC when compared to healthy individuals. These results demonstrate that NETosis regulators are modulated during acute VOC, and pave the way for studies of PADI4 inhibition as a therapeutic strategy for acute VOC in SCD.

Objective To evaluate the role of immune cells and their effector cytokines in the pathogenesis and progression of knee osteoarthritis (OA) in matched OA synovial fluid (SF) and synovial tissue samples. Methods Cells from matched samples of synovial tissue and SF acquired from individuals undergoing total knee replacement for OA (n = 39) were characterized for immune cell-associated surface markers and intracellular cytokine expression using polychromatic flow cytometry. Additional individuals with radiographic knee OA (Kellgren/Lawrence severity grades ≥1) who had available etarfolatide (inflammatory cell) imaging (n = 26) or baseline and 3-year data on progression of radiographic knee OA (n = 85) were also assessed. SF cytokine concentrations in all cohorts were evaluated for associations with synovial tissue and SF cell phenotypes and severity of radiographic knee OA. Results Macrophages (predominant in the synovial tissue, 53% of total cells) and neutrophils (predominant in the SF, 26% of total cells) were the major immune cell populations identified in the OA knee joints, exhibiting expression of or association with transforming growth factor β1 (TGFβ1) and elastase, respectively, in the SF. Expression levels of TGFβ1 and elastase were significantly associated with severity of radiographic knee OA. Baseline SF concentrations of TGFβ1 and elastase along with radiographic knee OA severity scores were predictive of knee OA progression, with areas under the receiver operating characteristic curves of 0.810 (for TGFβ1), 0.806 (for elastase), and 0.846 (for both TGFβ1 and elastase combined), with greater stability of prediction when both markers were utilized. Conclusion Our findings demonstrate the hitherto underappreciated role of neutrophils in the sterile inflammatory process and progression of OA. Two soluble mediators, SF elastase and TGFβ1, are strong predictors of knee OA progression, reflecting a synergistic role of neutrophil and macrophage populations in the pathogenesis and worsening of OA that could potentially be utilized to identify patients who may have a greater risk of more rapid disease progression.

Background: The overexposure to sun ultraviolet (UV) radiation produce photoaging by effect of free radicals on lipid, protein, and nucleic acid or by direct activation of responsible enzymes of cleavage of extracellular matrix components (EMC). Aims: To develop new anti-photoaging agents, the anti-aging activity of four “jarillas” standardized extracts from Argentina (Zuccagnia punctata Cav., Larrea divaricata Cav., Larrea cuneifolia Cav., and Larrea nitida Cav.) were examined. Methods: The effect on the activity and expression of enzymes related to photoaging (collagenase, hyaluronidase, elastase, tyrosinase) and the antioxidant capacity of four “jarillas” extracts were examined. Results: The results suggest that “jarillas” extracts could protect EMC by inhibition of skin aging-related enzymes and suppression of the expression of metalloproteinases (MMP-1) in human skin fibroblasts from photoinduced damage. Modulation of MMP-1 expression induced by UV radiation in human skin cells could be associated, at least in part, with the ROS scavenging capacity of “jarilla” extracts. The extract of Z punctata was more active than Larrea species extracts in all the trials. The activity of Z punctata and Larrea extracts could be ascribed to chalcones and lignans, major constituents of “jarillas,” respectively. Conclusions: The results obtained would indicate the potential of these adapted species to live in arid zones of Argentina, exposed to high ultraviolet radiation in production of cosmetic products and justify the promotion of their sustainable use and the development of crops. Fil: Orqueda, Maria Eugenia. Universidad Nacional de Tucumán. Instituto de Bioprospección y Fisiología Vegetal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto de Bioprospección y Fisiología Vegetal; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina Fil: Moreno, María Alejandra. Universidad Nacional de Tucumán. Instituto de Bioprospección y Fisiología Vegetal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto de Bioprospección y Fisiología Vegetal; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina Fil: Zampini, Iris Catiana. Universidad Nacional de Tucumán. Instituto de Bioprospección y Fisiología Vegetal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto de Bioprospección y Fisiología Vegetal; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina Fil: Bravo, Karent. Universidad de Antioquia; Colombia Fil: Osorio, Edison. Universidad de Antioquia; Colombia Fil: Isla, Maria Ines. Universidad Nacional de Tucumán. Instituto de Bioprospección y Fisiología Vegetal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto de Bioprospección y Fisiología Vegetal; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina

The loss of structural elastin due to intrinsic and extrinsic ageing results in the skin's inability to stretch and recoil (decrease in elasticity) and manifests as loss of skin firmness and sagging. While other extracellular matrix (ECM) components such as collagen and hyaluronic acid are continually synthesized and assembled through life, elastic fibres are not. Elastic fibre assembly and functionality require fibre cross-linking, induced by the lysyl oxidase-like (LOXL) enzymes, which sharply decrease during ageing.To evaluate the enhanced elastogenic effect of a blackberry-dill extract combination, which was hypothesized to induce elastin fibre component synthesis, fibre cross-linking and reduce elastin fibre degradation.The blackberry and the dill extracts were tested separately and in combination to confirm single ingredient bioactivity and synergistic benefits. Human skin explants, dermal fibroblasts, elastase assays, ELISAs, quantitative real-time PCRs and spectrofluorometer measurements were used. Moreover, a double-blinded, placebo-controlled clinical study was carried out to assess skin elasticity using Cutometer and histologically from biopsies.The blackberry extract induced elastin gene expression, elastin promoter activity and inhibited elastic fibre degradation by matrix metalloproteinases (MMPs) 9 and 12. The dill extract induced elastin, collagen and LOXL1 gene expression, resulting in enhanced fibre cross-linking in human skin explants. Clinically, the blackberry and dill combination treatment displayed synergistic pro-elasticity activity as compared to each ingredient alone and placebo.Taken together, these results demonstrated the two multimodal plant-based extracts complemented each other in terms of bioactivity and resulted in a synergistic elastogenesis induction.CONTEXTE: la perte de l’élastine structurelle causée par un vieillissement intrinsèque et extrinsèque provoque l’incapacité de la peau à s’étirer et à rebondir (diminution de l’élasticité) et se manifeste comme une perte de fermeté et un relâchement de la peau. Alors que d’autres composants de la matrice extracellulaire (MEC), tels que le collagène et l’acide hyaluronique sont continuellement synthétisés et assemblés tout au long de la vie, les fibres élastiques ne le sont pas. L’assemblage et la fonctionnalité des fibres élastiques nécessitent une réticulation des fibres, causée par les enzymes de type lysyle oxydase (LOXL), qui diminuent fortement au cours du vieillissement. OBJECTIF: évaluer l’effet élastogène amélioré d’une combinaison d’extrait de mûre et d’aneth, qui était supposée induire la synthèse des composants des fibres d’élastine, la réticulation des fibres et réduire la dégradation des fibres d’élastine. MÉTHODES: les extraits de mûre et d’aneth ont été testés séparément et ensemble pour confirmer la bioactivité d’un seul ingrédient et les avantages synergiques. Des explants de peau humaine, des fibroblastes cutanés, des dosages d’élastase, des ELISA, des analyses PCR quantitatives en temps réel et des mesures de spectrofluorimètre ont été utilisés. De plus, une étude clinique en double aveugle, contrôlée par placebo, a été réalisée pour évaluer l’élasticité de la peau à l’aide du cutomètre et histologiquement à partir de biopsies. RÉSULTATS: l’extrait de mûre a induit l’expression génique de l’élastine, l’activité de promoteur de l’élastine et a inhibé la dégradation des fibres élastiques par des métalloprotéinases matricielles (MPM) 9 et 12.L’extrait d’aneth a causé l’expression génique de l’élastine, du collagène et du gène LOXL1, entraînant une amélioration de la réticulation des fibres dans les explants de peau humaine. Cliniquement, le traitement par une combinaison de mûre et d’aneth a montré une activité de pro-élasticité synergique par rapport à chaque ingrédient seul et au placebo. CONCLUSION: ensemble, ces résultats ont démontré que les deux extraits de plantes multimodales se complètent en termes de bioactivité et ont entraîné une induction synergique de l’élastogenèse.

Background and purpose Chronic obstructive pulmonary disease, encompassing chronic airway obstruction and lung emphysema, is a major worldwide health problem and a severe socio-economic burden. Evidence previously provided by our group has shown that inhibition of inducible NOS (iNOS) prevents development of mild emphysema in a mouse model of chronic tobacco smoke exposure and can even trigger lung regeneration. Moreover, we could demonstrate that pulmonary hypertension is not only abolished in cigarette smoke-exposed iNOS-/- mice but also precedes emphysema development. Possible regenerative effects of pharmacological iNOS inhibition in more severe models of emphysema not dependent on tobacco smoke, however, are hitherto unknown. Experimental approach We have established a mouse model using a single dose of porcine pancreatic elastase or saline, intratracheally instilled in C57BL/6J mice. Emphysema, as well as pulmonary hypertension development was determined by both structural and functional measurements. Key results Our data revealed that (i) emphysema is fully established after 21 days, with the same degree of emphysema after 21 and 28 days post instillation, (ii) emphysema is stable for at least 12 weeks and (iii) pulmonary hypertension is evident, in contrast to smoke models, only after emphysema development. Oral treatment with the iNOS inhibitor N(6)-(1-iminoethyl)-l-lysine (L-NIL) was started after emphysema establishment and continued for 12 weeks. This resulted in significant lung regeneration, evident in the improvement of emphysema and reversal of pulmonary hypertension. Conclusion and implications Our data indicate that iNOS is a potential new therapeutic target to treat severe emphysema and associated pulmonary hypertension. Linked articles This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

β-amyloid (Aβ) aggregates involved in Alzheimer's disease (AD) are resistant to proteases but could be destabilized by small peptides designed to target specific hydrophobic regions of Aβ that take part in aggregate assembly. Since thrombin and AD are intricately connected, and elastase modulates thrombin activity, elastase-digested thrombin peptides were verified for intervention in the Aβ-aggregation pathway. Intact or elastase-digested thrombin destabilized Aβ fibril, as demonstrated by thioflavin T assay. Peptides were synthesized employing thrombin as a template, of which, a hexapeptide (T3) showed maximum destabilization at 1 µm. ExPASy peptide cutter software coupled with mass spectrometric analysis confirmed the generation of T3 peptide from elastase-digested thrombin. TEM micrographs revealed that 30-day incubation of preformed Aβ fibrils or monomers with T3 resulted in destabilization or inhibition, respectively, leading mostly to particles of 1.74 ± 0.17 nm, which roughly corresponded to Aβ monomer. Surface plasmon resonance employing CM5 chip coupled with Aβ40 mouse monoclonal antibody showed a drop in response when T3 was incubated with Aβ fibrils between 2 and 8 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and confocal microscopy demonstrated the ability of T3 to rescue neuroblastoma cells from Aβ oligomer-induced cytotoxic damage. Although no [Aβ-T3] adduct could be detected by mass spectrometry, an initial interaction appeared to facilitate the process of destabilization/inhibition of aggregation. T3 was comparable to standard β-sheet breaker peptides, LPFFD and KLVFF in terms of Aβ aggregate destabilization. High hydrophobicity values coupled with recognition and breaking elements make T3 a potential candidate for future therapeutic applications.

Diseases bring about the need for interventions that pinpoint each specific aspect of the illness. Commonly, remission of a complex disease is accomplished by mixing treatments, medications, and therapeutics together in a fashion where they may negatively interact with each other or never arrive at the diseased site as a systemic heterogeneous mixture. Chronic wounds display intricacy as they are very localized and have their own environment where tissue deconstruction due to high levels of numerous proteases outweighs normal tissue reconstruction. This idea leads to the necessity of a protein that contains low diffusivity rates for localized treatment, strength against high concentrations of proteolytic species that lead to degradation of short chain peptides, while encompassing broad inhibitory effects against multiple proteases. Elastin-Like Peptides are an attractive, thermoresponsive, protein-based drug delivery partner as they contain low diffusivity and serve as a stable architecture for short chain peptide fusion. In this project, a novel elastin-like peptide-based protein has been created to target the inhibition of both human neutrophil elastase and matrix metalloprotease-2. As a biologic, this is unique as it is a protein with specific biological activities against multiple proteases, ultimately displaying the potential to mix and match differing biologically active peptides within one amino acid sequence.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism

Surviving in the gastrointestinal tract is crucial for biopeptides to exert physiological effects in vivo. To enrich elastase inhibitory peptides with high digestive stability, elastin peptides was separated by using ultrafiltration and macroporous resin column, then changes in elastase inhibitory activity, amino acid composition and peptide profile of elastin peptides during in vitro digestion were measured. The results revealed that the fraction F3 eluted by 40% ethanol displayed high digestive stability possibly due to the high content of Pro‐containing peptides (20.25%). Particularly, thirty peptides in F3 exhibited high digestive stability and most of them contained GV and/or PG fragment. Since GV and PG could inhibit 35.70% and 51.77% elastase activity at the concentration of 20 mm, respectively, peptides in F3 survived in in vitro digestion which contained GV and PG sequence might be potential elastase inhibitors. This provides an approach for preparation of peptides with high bioactivity and digestive stability.

Elastin is an amorphous protein highly resistant to elastase degradation and is believed to be the most stable component among the extracellular matrix (ECM) members. Thus the excessive deposition of elastin in advanced liver fibrosis may contribute to the declining reversibility of the disease. Our previous study has found that elastin crosslinking inhibition can effectively arrest liver fibrosis progression. To further understand the roles of elastin involved in liver fibrosis, we systematically investigated the expression, accumulation, and degradation based on dynamic and bidirectional CCl4 -induced liver fibrosis mouse models and visualized the ultrastructure of elastin globules in cultured LX-2 cells. We found that the expression pattern of tropoelastin (soluble elastin) and collagen I was not completely comparable at both the transcriptional and posttranscriptional levels during liver fibrosis progression and regression. Elastin mainly accumulated onto the internodular fibrous septa and enlarged portal areas and intertwined with collagen I at the late stage of liver fibrosis. Three-dimensional analysis of elastin and collagen I by confocal immunofluorescence coupled with biochemical analyses revealed that with respect to collagen, elastin deposition was characterized by late aggregation in progression and slow turnover in regression. In addition, we visualized the dynamic ultrastructure of ECM fibers during liver fibrogenesis and fibrolysis and the ultrastructure of elastin globules self-aggregated by tropoelastin crosslinking. Our current study established new general hallmarks of elastin levels and forms in progressive and regressive liver fibrosis and provided a foundation for further experimental investigation of the growing role of elastin in liver fibrosis regression.

BACKGROUND Trappin-2/pre-elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological process of systemic sclerosis (SSc). OBJECTIVES To clarify the potential role of trappin-2 in SSc. METHODS Serum trappin-2 levels were determined by enzyme-linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin-2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription-real-time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation. RESULTS Since serum trappin-2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin-2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin-2 levels than in those with normal levels. Furthermore, serum trappin-2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin-2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin-2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin-2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter. CONCLUSIONS Endothelial trappin-2 up-regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.

BACKGROUND AND PURPOSE: Abdominal aortic aneurysm (AAA) is a degenerative disease affecting human health, but there are no safe and effective medications for AAA therapy. Cycloastragenol (CAG), derived from Astragali Radix, has various pharmacological effects. However, whether CAG can protect against AAA remains elusive. In this study, we investigated whether CAG has an inhibitory effect on AAA and its related mechanism. EXPERIMENTAL APPROACH: The AAA mouse model was induced by incubating the abdominal aorta with elastase. CAG was administered by gavage at different doses beginning on the same day or 14 days after inducing AAA to explore its preventive or therapeutic effects respectively. The preventive effects of CAG on AAA were verified in another AAA mouse model induced by angiotensin II in ApoE(−/−) mouse. In vitro experiments were implemented on rat vascular smooth muscle cells (VSMCs) stimulated by TNF‐α. KEY RESULTS: Compared to the control AAA model group, CAG (125 mg·kg(−1) body weight day(−1)) reduced the incidence of AAA, the dilatation of aorta and elastin degradation in media in both mouse models of AAA. CAG suppressed the inflammation, oxidation, phenotype switch and apoptosis in TNF‐α‐stimulated VSMCs, ameliorated the expression and activity of MMPs and decreased the activation of the ERK/JNK signalling pathway. CAG also inhibited the degradation of elastin in TNF‐α‐stimulated VSMCs. CONCLUSION AND IMPLICATIONS: CAG presents protective effects against AAA through down‐regulation of the MAPK signalling pathways and thus attenuates inflammation, oxidation, VSMC phenotype switch and apoptosis and the expression of MMPs as well as increasing elastin biosynthesis.

Background Many arguments suggest that neutrophils could play a prominent role in COVID‐19. However, the role of key components of neutrophil innate immunity in severe forms of COVID‐19 has deserved insufficient attention. We aimed to evaluate the involvement of neutrophil elastase, histone‐DNA, and DNases in systemic and multi‐organ manifestations of COVID‐19. Methods We performed a multicenter study of markers of neutrophil innate immunity in 155 cases consecutively recruited in a screening center, local hospitals, and two regional university hospitals. The cases were evaluated according to clinical and biological markers of severity and multi‐organ manifestations and compared to 35 healthy controls. Results Blood neutrophil elastase, histone‐DNA, myeloperoxidase‐DNA, and free dsDNA were dramatically increased, and DNase activity was decreased by 10‐fold, compared with controls. Neutrophil elastase and histone‐DNA were associated with intensive care admission, body temperature, lung damage, and markers of cardiovascular outcomes, renal failure, and increased interleukin‐6 (IL‐6), IL‐8, and CXCR2. Neutrophil elastase was an independent predictor of the computed tomography score of COVID‐19 lung damage and the number of affected organs, in multivariate analyses. The increased blood concentrations of NE and neutrophil extracellular traps were related to exacerbation of neutrophil stimulation through IL‐8 and CXCR2 increased concentrations and increased serum DAMPs, and to impaired degradation of NETs as a consequence of the dramatic decrease in blood DNase activity. Conclusion Our results point out the key role of neutrophil innate immunity exacerbation in COVID‐19. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID‐19., Blood levels of neutrophil elastase and histone‐DNA are associated with severe and systemic and multi‐organ manifestations of COVID‐19. Increased blood concentrations of neutrophil elastase and neutrophil extracellular traps are related to exacerbation of neutrophil stimulation through activated IL‐8/CXCR2 pathway. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID‐19.

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H2 O2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H2 O2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019.

We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n = 8) and mid luteal-MLP (n = 7) phases explants were cultured for 24-48 hr with medium alone (Control), ELA (0.5 μg/ml,1 μg/ml), sivelestat - ELA inhibitor (INH,10 μg/ml), or ELA (0.5 μg/ml,1 μg/ml) + INH (10 μg/ml). COL1 gene transcription was done by qRT-PCR and PGE2 and PGF2 α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2−/− mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.

IL-1 family cytokines act as apical initiators of inflammation in many settings and can promote the production of a battery of inflammatory cytokines, chemokines and other inflammatory mediators in diverse cell types. IL-36 alpha, IL-36 beta and IL-36 gamma, which belong to the extended IL-1 family, have been implicated as key initiators of skin inflammation in psoriasis. IL-36 gamma is highly upregulated in lesional skin from psoriatic individuals, and heritable mutations in the natural IL-36 receptor antagonist result in a severe form of psoriasis. IL-36 family cytokines are initially expressed as inactive precursors that require proteolytic processing for activation. The neutrophil granule-derived protease elastase proteolytically processes and activates IL-36 alpha and IL-36 gamma, increasing their biological activity similar to 500-fold, and also robustly activates IL-1 alpha and IL-33 through limited proteolytic processing. Consequently, inhibitors of elastase activity may have potential as anti-inflammatory agents through antagonizing the activation of multiple IL-1 family cytokines. Using in silico screening approaches, we have identified small-molecule inhibitors of elastase that can antagonize activation of IL-36 gamma by the latter protease. The compounds reported herein may have utility as lead compounds for the development of inhibitors of elastase-mediated activation of IL-36 and other IL-1 family cytokines in inflammatory conditions, such as psoriasis.

BACKGROUND: The aim of this study is to separate polyphenols from grape pomace using a surfactant-based separation, Colloidal Gas Aphrons (CGA) and to investigate their inhibitory activity against skin relevant enzymes, collagenase and elastase. Ethanolic (EE) and hot water crude extracts (HWE) were produced first and then the CGA generated using TWEEN20 were applied resulting in polyphenols enriched fractions (CGA-EE and CGA-HWE, ethanol and hot water extracts derived fractions respectively). RESULTS: Both crude extracts inhibited the enzymes in a dose-dependent manner however, further extraction by CGA led to fractions with higher inhibitory efficiency against collagenase. Although gallic acid was the main component of the CGA-HWE, others such as kaempferol must have contributed to its potency which was over six times more than gallic acid's. The CGA-EE was found to be about four times more efficient than its crude extract and over six times more efficient than gallic acid in collagenase's inhibition; quercetin was the major polyphenol in this fraction. CONCLUSION: It is evident that ethanol and hot water extraction processes led to different polyphenols composition and thus different inhibitory activity against collagenase and elastase. Further separation with CGA increased the inhibitory potency of both extracts against collagenase. Overall the results here showed the potential application of the CGA fractions from grape extracts in cosmetics.

An ability to predict the response to conventional non-surgical treatment of a periodontal site would be advantageous. However, biomarkers or tests devised to achieve this have lacked sensitivity. The aim of this study is to assess the ability of a novel combination of biomarkers to predict treatment outcome of patients with chronic periodontitis.Gingival crevicular fluid (GCF) and subgingival plaque were collected from 77 patients at three representative sites, one healthy (probing depth [PD] ≤3 mm) and two diseased (PD ≥6 mm), at baseline and at 3 and 6 months after treatment. Patients received standard non-surgical periodontal treatment at each time point as appropriate. The outcome measure was improvement in probing depth of ≥2 mm. Concentrations of active enzymes (matrix metalloproteinase [MMP]-8, elastase, and sialidase) in GCF and subgingival plaque levels of Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum were analyzed for prediction of the outcome measure.Using threshold values of MMP-8 (94 ng/μL), elastase (33 ng/μL), sialidase (23 ng/μL), and levels of P. gingivalis (0.23%) and T. forsythia (0.35%), receiver operating characteristic curves analysis demonstrated that these biomarkers at baseline could differentiate healthy from diseased sites (sensitivity and specificity ≥77%). Furthermore, logistic regression showed that this combination of these biomarkers at baseline provided accurate predictions of treatment outcome (≥92%).The "fingerprint" of GCF enzymes and bacteria described here offers a way to predict the outcome of non-surgical periodontal treatment on a site-specific basis.

Background and Aim A previous UK study showed that 6.1% of patients with diarrhea-predominant irritable bowel syndrome (IBS-D) had evidence of severe pancreatic exocrine insufficiency (PEI), but these findings need replication. We aimed to identify the prevalence of PEI based on fecal elastase stool testing in consecutive outpatients presenting with chronic unexplained abdominal pain and/or diarrhea and/or IBS-D. Methods Patients aged over 40 years presenting to hospital outpatient clinics from six sites within Australia with unexplained abdominal pain and/or diarrhea for at least 3 months and/or IBS-D were studied. Patients completed validated questionnaires and donated a stool sample in which elastase concentration was measured by ELISA. A concentration of

Human neutrophil elastase impacts on atherosclerotic plaque stability by inducing apoptosis in endothelial cells. Our aim was to investigate the proapoptotic mechanism of elastase on endothelial cells and to evaluate the presence of elastase in human plaque material. Human endothelial cells were treated with purified human neutrophil elastase. Apoptosis was assayed by capsase-3/7 activation, TUNEL, and sub-G1 assay. Activation of unfolded protein response (UPR) effector molecules binding Ig protein, soluble X-binding protein-1, protein kinase RNA-like ER kinase (PERK), and C/EBP-homologous protein (CHOP) was analyzed by RT-PCR, immunocytochemistry, and Western blot. Genetic silencing of CHOP was achieved by small interfering RNA. Elastase induces autophagic-apoptotic forms of endothelial cell death in a time- and dose-dependent manner, in conjunction with a significant increase in phosphorylation/expression of the canonical UPR-activation markers PERK and CHOP. By using CHOP knockdown, we identified CHOP as a key mediator of elastase-induced endothelial cell death. Immunohistochemical analysis of human rupture-prone plaque specimens confirmed the presence of elastase and colocalization with apoptosis. We have demonstrated for the first time that the PERK-CHOP branch of the UPR is causally involved in elastase-induced apoptosis of endothelial cells. Ex vivo analysis of human rupture-prone plaques confirmed the presence of elastase and its colocalization with markers of apoptosis. This novel role of elastase underlines the potential of combined targeting of elastase and endoplasmic reticulum stress in the prevention of plaque progression and cardiovascular events.-Grechowa, I., Horke, S., Wallrath, A., Vahl, C.-F., Dorweiler, B. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

Sustained high levels of activated polymorphonuclear leukocytes (PMNs) and PMN-derived proteases in the microenvironment of chronic venous leg ulcers (CVLUs) are linked to chronic inflammation and delayed healing. Uncontrolled PMN activity eventually destroys newly developed tissue and degrades critical growth factors. The bioactive components of fish oil (n-3 eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) have strong inflammation-resolving actions and have been shown to assuage PMN activity, but have not been tested in CVLU patients. This randomized controlled study compared the effectiveness of oral EPA+DHA therapy to a placebo for reducing PMN activation in CVLU microenvironments. At Days 0, 28 and 56, markers of PMNs (CD15) and activated PMNs (CD66b), and levels of PMN-derived proteases human neutrophil elastase (HNE) and matrix metalloproteinase-8 (MMP-8) were measured in CVLU fluid from patients receiving standard compression therapy and 1) EPA+DHA therapy (n=16) or 2) placebo (n=19). By Day 56, the EPA+DHA Group had a significantly lower percentage of CD66b+ cells in CVLU fluid compared to Day 0 (p = 0.02) and to Day 28 (p= 0.05). Importantly, there were downward trends in levels of both MMP-8 and HNE over time in the EPA+DHA Group, which also demonstrated greater reductions in wound area by Day 28 (57% reduction) and Day 56 (76% reduction) than the Control Group (35% and 59%, respectively). Moreover, reductions in wound area had significant negative relationships with CD15+ cells in wound fluid at Days 28 (ρ = 0.008) and 56 (p

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 109 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed.

Background and Purpose Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Experimental Approach Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI). Key Results MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries. Conclusions and Implications These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS.

Tendinopathy of the long head of the biceps (TLHB) involves various types of extracellular matrix degeneration, but previous studies have not evaluated elastic fibers. The purpose of this study was to investigate elastic fiber distribution in long head of the biceps (LHB). The TLHB tendons of 16 consecutive patients (eight men and eight women; average age of 55.75 years; age range of 40-71 years) were transected and harvested. Three cadaveric LHB tendons were used as the control group. The expression of collagen type I was decreased, but type III was increased in TLHB. Disruption of elastic fibers was particularly observed in grade II specimens where the level of elastase-positive staining was significantly higher than in grade I specimens. Elastic fibers were not observed in the grade III area, implying a higher expression of elastase than in the grade I area. Results of Western blotting showed that the expression of elastin was higher in the control group and the levels of elastin significantly decreased in grades II and III of TLHB. Levels of osteopontin and elastase were increased in primary culture of human tenocytes after experiencing elastic derived peptide treatment. These results suggested that elastase may be caused by the disruption of elastic fibers in the development of chronic tendinopathy and that elastic derived peptide may enhance elastase and osteopontin expression. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1919-1926, 2017.

The receptor for advanced glycan end products (RAGE) has been identified as a susceptibility gene for chronic obstructive pulmonary disease (COPD) in genome-wide association studies (GWASs). However, less is known about how RAGE is involved in the pathogenesis of COPD. To determine the molecular mechanism by which RAGE influences COPD in experimental COPD models, we investigated the efficacy of the RAGE-specific antagonist FPS-ZM1 administration in in vivo and in vitro COPD models. We injected elastase intratracheally and the RAGE antagonist FPS-ZM1 in mice, and the infiltrated inflammatory cells and cytokines were assessed by ELISA. Cellular expression of RAGE was determined in protein, serum, and bronchoalveolar lavage fluid of mice and lungs and serum of human donors and patients with COPD. Downstream damage-associated molecular pattern (DAMP) pathway activation in vivo and in vitro and in patients with COPD was assessed by immunofluorescence staining, Western blot analysis, and ELISA. The expression of membrane RAGE in initiating the inflammatory response and of soluble RAGE acting as a decoy were associated with up-regulation of the DAMP-related signaling pathway via Nrf2. FPS-ZM1 administration significantly reversed emphysema in the lung of mice. Moreover, FPS-ZM1 treatment significantly reduced lung inflammation in Nrf2+/+ , but not in Nrf2-/- mice. Thus, our data indicate for the first time that RAGE inhibition has an essential protective role in COPD. Our observation of RAGE inhibition provided novel insight into its potential as a therapeutic target in emphysema/COPD.-Lee, H., Park, J.-R., Kim, W. J., Sundar, I. K., Rahman, I., Park, S.-M., Yang. S.-R. Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling.

Abnormalities in lungs caused by emphysema might alter their response to sepsis and the occurrence of acute lung injury (ALI). This study compared the extension of ALI in response to intraperitoneal lipopolysaccharide (LPS) injection in Wistar rats with and without emphysema induced by elastase. Adult male Wistar rats were randomized into four groups: control, emphysema without sepsis, normal lung with sepsis and emphysema with sepsis. Sepsis was induced, and 24 h later the rats were euthanised. The following analysis was performed: blood gas measurements, bronchoalveolar lavage (BAL), lung permeability and histology. Animals that received LPS showed significant increase in a lung injury scoring system, inflammatory cells in bronchoalveolar lavage (BAL) and IL‐6, TNF‐α and CXCL2 mRNA expression in lung tissue. Animals with emphysema and sepsis showed increased alveolocapillary membrane permeability, demonstrated by higher BAL/serum albumin ratio. In conclusion, the presence of emphysema induced by elastase increases the inflammatory response in the lungs to a systemic stimulus, represented in this model by the intraperitoneal injection of LPS.

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes and is considered to be a multifunctional enzyme. HNE is also involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrrolo[2,3-b]pyridine derivatives of our previously reported potent HNE inhibitors. Our results show that position 2 of the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted, and modifications of this position resulted in loss of HNE inhibitory activity. Conversely, the introduction of certain substituents at position 5 was tolerated, with retention of HNE inhibitory activity (IC(50) = 15–51 nM) after most substitutions, indicating that bulky and/or lipophilic substituents at position 5 probably interact with the large pocket of the enzyme site and allow Michaelis complex formation. The possibility of Michaelis complex formation between Ser195 and the ligand carbonyl group was assessed by molecular docking, and it was found that highly active HNE inhibitors are characterized by geometries favorable for Michaelis complex formation and by relatively short lengths of the proton transfer channel via the catalytic triad.

There is a significant unmet need for safe and effective anti-inflammatory treatment for cystic fibrosis. The aim of this study was to evaluate the safety of acebilustat, a leukotriene A4 hydrolase inhibitor, and its effect on inflammation biomarkers in patients with cystic fibrosis. Seventeen patients with mild to moderate cystic fibrosis were enrolled and randomized into groups receiving placebo or doses of 50 mg or 100 mg acebilustat administered orally, once daily for 15 days. Sputum neutrophil counts were reduced by 65% over baseline values in patients treated with 100 mg acebilustat. A modestly significant 58% reduction vs. placebo in sputum elastase was observed with acebilustat treatment. Favorable trends were observed for reduction of serum C-reactive protein and sputum neutrophil DNA in acebilustat-treated patients. No changes in pulmonary function were observed. Acebilustat was safe and well tolerated. The results of this study support further clinical development of acebilustat for treatment of cystic fibrosis.

Rapeseed proteins have been considered as being poorly digestible in the gut of non-ruminants. The aim of the study was to assess the digestibility of napin and cruciferin in ileal digesta of broiler chickens, testing sixteen samples of rapeseed co-products with protein levels ranging from 293 g/kg to 560 g/kg dry matter. Each sample was included into a semi-synthetic diet at a rate of 500 g/kg and evaluated with broiler chickens in a randomised design. Dietary and ileal digesta proteins were extracted and identified by gel-based liquid chromatography–tandem mass spectrometry (LC-MS/MS). Three isomers of napin (a 2S albumin) and nine cruciferins (an 11S globulin) were identified in the rapeseed co-products, whereas six endogenous enzymes such as trypsin (I-P1, II-P29), chymotrypsin (elastase and precursor), carboxypeptidase B and α-amylase were found in the ileal digesta. It is concluded that as none of the rapeseed proteins were detected in the ileal digesta, rapeseed proteins can be readily digested by broiler chickens, irrespective of the protein content in the diet.

Background and Purpose Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. Experimental Approach We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. Key Results Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca2+]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. Conclusions and Implications GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

Preclinical Research A number of N-benzoylindoles were designed and synthesized as deaza analogs of previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC50 = 3.8 μM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc.

PURPOSE To demonstrate the feasibility to quantify the lung respiratory airway in vivo with hyperpolarized xenon diffusion magnetic resonance imaging (MRI), which is able to detect mild emphysema in the rat model. MATERIALS AND METHODS The lung respiratory airways were quantified in vivo using hyperpolarized xenon diffusion MRI (7T) with eight b values (5, 10, 15, 20, 25, 30, 35, 40 s/cm2 ) in five control rats and five mild emphysematous rats, which were induced by elastase. The morphological results from histology were acquired and used for comparison. RESULTS The parameters DL (longitudinal diffusion coefficient), r (internal radius), h (alveolar sleeve depth), Lm (mean linear intercept), and S/V (surface area to lung volume ratio) derived from the hyperpolarized xenon diffusion MRI in the emphysematous group showed significant differences from those in the control group (P 0.8). CONCLUSION The lung respiratory airways can be quantified by hyperpolarized xenon diffusion MRI, showing the potential for mild emphysema diagnosis. Also, the study suggested that the hyperpolarized xenon DL is more sensitive than DT (transverse diffusion coefficient) to detect mild emphysema. LEVEL OF EVIDENCE 1 J. Magn. Reson. Imaging 2017;45:879-888.

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa–secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non–cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.—Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Objective To determine changes in blood granulocyte counts and in plasma myeloperoxidase (MPO) and elastase (ELT) concentrations in surgical colic cases, and to determine the relationship between these changes and the surgical procedure performed, occurrence of postoperative ileus, and final outcome. Design Prospective clinical study conducted over a 12-month period. Setting University teaching hospital. Animals Fifty-three horses undergoing emergency laparotomy and surviving at least 12 hours postoperatively. Interventions Blood samples were taken before surgery, during surgery, at the recovery from anesthesia, and then serially until the 150th hour after the first blood sampling. Granulocyte counts were performed by an automated cell hematology analyzer. Specific ELISAs were performed for the MPO and ELT measurements. Mixed models were used to compare the time-trends of the 3 parameters. Measurements and Main Results Taking all horses together, the time-trends of MPO and ELT were not significantly different from each other, but they were significantly different from the granulocyte time-trend. The type of surgical procedure did not influence the time-trends of the 3 parameters. Significant changes in the granulocyte time-trends were associated with postoperative ileus and outcome. Significant changes in the MPO time-trends were associated with outcome. The ELT time-trends were not influenced by ileus or outcome. Conclusions Granulocyte counts and MPO change over time and are related to the severity of the inflammatory reaction in surgical colic cases. These time-trends may allow evaluation of treatment efficacy in an effort to modulate excessive granulocyte activation and degranulation.

It is widely accepted that elevated protease activity (EPA) in chronic wounds impedes healing. However, little progress has occurred in quantifying the level of protease activity that is detrimental for healing. The aim of this study was to determine the relationship between inflammatory protease activity and wound healing status, and to establish the level of EPA above which human neutrophil-derived elastase (HNE) and matrix metalloproteases (MMP) activities correlate with nonhealing wounds. Chronic wound swab samples (n = 290) were collected from four wound centers across the USA to measure HNE and MMP activity. Healing status was determined according to percentage reduction in wound area over the previous 2–4 weeks; this was available for 211 wounds. Association between protease activity and nonhealing wounds was determined by receiver operating characteristic analysis (ROC), a statistical technique used for visualizing and analyzing the performance of diagnostic tests. ROC analysis showed that area under the curve (AUC) for HNE were 0.69 for all wounds and 0.78 for wounds with the most reliable wound trajectory information, respectively. For MMP, the corresponding AUC values were 0.70 and 0.82. Analysis suggested that chronic wounds having values of HNE >5 and/or MMP ≥13, should be considered wound healing impaired. EPA is indicative of nonhealing wounds. Use of a diagnostic test to detect EPA in clinical practice could enable clinicians to identify wounds that are nonhealing, thus enabling targeted treatment with protease modulating therapies.