Human Neutrophil Elastase Levels Research Articles

Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios

In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 μg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.

Salivary and serum levels of neutrophil proteases in periodontitis and rheumatoid arthritis.

The aim was to profile serum and salivary levels of active-matrix metalloproteinase (aMMP)-8, tissue inhibitor MMP (TIMP)-1, aMMP-8/TIMP-1 ratio, total MMP (tMMP)-9, tMMP-9/TIMP-1 ratio, myeloperoxidase (MPO), and human neutrophil elastase (HNE) in periodontitis and rheumatoid arthritis (RA). Rheumatoid arthritis patients with periodontitis (RA + P, n = 26), periodontally healthy RA patients (RA, n = 23), systemically healthy periodontitis patients (P, n = 24), and controls (C, n = 24) were included. aMMP-8 levels were determined by a time-resolved immunofluorescence assay (IFMA), TIMP-1, tMMP-9, MPO, and HNE levels were measured by enzyme-linked immunosorbent (ELISA) assays. Higher salivary aMMP-8 (p < 0.001), aMMP-8/TIMP-1 ratio (p = 0.043), tMMP-9 (p = 0.011), tMMP-9/TIMP-1 ratio (p = 0.022), MPO (p = 0.026) and HNE (p < 0.001) levels were detected in P relative to the controls. Salivary TIMP-1 was increased in RA patients regardless of periodontal status (RA + P vs. P: p = 0.038; RA vs. C: p = 0.020). Serum neutrophil proteases were increased in RA groups (RA + P, RA) compared to systemically healthy groups (P, C) (p < 0.05). Serum levels of neutrophil proteases were increased in RA study groups; however rheumatologic status seemingly does not affect salivary levels of these proteins.

Effects of Pendrin Protein in Nasal Epithelial Cells on Mucin Production in the Context of Type 2 Inflammation.

Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. A total of 40 patients (10 CRS patients with nasal polyps,10 CRS patients without nasal polyps, and 20 nasal septum deviation patients) were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation.

Serological Biomarkers of Extracellular Matrix Turnover and Neutrophil Activity Are Associated with Long-Term Use of Vedolizumab in Patients with Crohn's Disease.

Crohn’s disease (CD) is a relapsing-remitting inflammatory disease of the gastrointestinal (GI) tract characterized by increased extracellular matrix (ECM) remodeling. The introduction of the α4β7-integrin inhibitor vedolizumab (VEDO) has improved disease management, although there is a high rate of primary non-response in patients with CD. We studied whether ECM biomarkers of neutrophil activity and mucosal damage could predict long-term response to VEDO in patients with CD. Serum levels of human neutrophil elastase (HNE)-derived fragments of calprotectin (CPa9-HNE), and matrix metalloproteinase (MMP)-derived fragments of type I (C1M), III (C3M), IV (C4M), and VI (C6Ma3) collagen, type III collagen formation (PRO-C3), basement membrane turnover (PRO-C4) and T-cell activity (C4G), were measured using protein fingerprint assays in patients with CD (n = 32) before VEDO therapy. Long-term response was defined as VEDO treatment of at least 12 months. CPa9-HNE was significantly increased at baseline in non-responders compared with responders (p < 0.05). C1M, C3M, C4M, C6Ma3, and PRO-C4 were also significantly increased at baseline in non-responders compared with responders (all p < 0.05). All biomarkers were associated with response to VEDO (all p < 0.05). To conclude, baseline levels of serum biomarkers for neutrophil activity and mucosal damage are linked to the pathology of CD, and are associated with long-term use of VEDO in patients with CD. Therefore, these biomarkers warrant further validation and could aid in therapeutic decision-making concerning vedolizumab therapy.

Synthesis and characterization of TEMPO-oxidized peptide-cellulose conjugate biosensors for detecting human neutrophil elastase

Here we describe the synthesis and characterization of a peptide-cellulose conjugate biosensor based on TEMPO-oxidized nanofibrillated cellulose (tNFC) for detecting elevated levels of human neutrophil elastase (HNE) in chronic wounds. The fluorescent peptide HNE substrate constructed from n-succinyl-Ala-Pro-Ala-7-amino-4-methyl-coumarin was attached to the TEMPO-oxidized cellulose surface via polyethylene glycol linker. The characterization of the biosensor conjugate shows a high degree of peptide incorporation onto the surface with the degree of substitution of 0.057. The relatively small crystallite size of 26.0 Å compared to other cellulose- and nanocellulose-based materials leads to a large specific surface area which can promote access of HNE to the enzyme substrates due to decreased steric interactions. Likewise, the porosity for tNFC was found to be higher than all other samples, including the nanocellulosic aerogel, lending to its hydrogel-like nature. The properties of tNFC were compared to other cellulose-based materials. The volume of each crystallite and volume ratio to the largest sample was calculated. tNFC was found to occupy the smallest space resulting in high amounts of sensors per crystallite unit volume. With a small crystallite volume and large number of sensors, the tNFC peptide-cellulose conjugate biosensor could provide a more sensitive system and is a good candidate for point of care diagnostic devices for detecting elevated protease levels in humans.

Study of Human Neutrophil Elastase Levels and the Correlation with ARDS Severity in Thoracic Trauma Patients

Introduction: Thoracic trauma is trauma that hits the thoracic wall or intra-thoracic organs, either due to blunt trauma or due to sharp trauma. In thoracic trauma often causes impaired ventilation perfusion due to damage to the lung parenchyma. This results in impaired tissue oxygenation, which is one of the causes of Acute respiratory distress Syndrome (ARDS). These changes are caused by the release of pro-inflammatory mediators, plasmatic proteins and proteases into the alveolar space associated with ongoing edema, as well as oxidative products that ultimately result in severe inhibition of the surfactant system. Method: This study will examine the relationship between plasma elastase levels as a predictor of the incidence of ARDS in thoracic trauma patients in Malang. This study is an observational cohort study. The research subjects were thoracic trauma patients who fit the criteria. Result: I t can be concluded that there is a significant relationship between elastase levels and BGA-3. The elastase level is below the value of 11.65 ±1.85, the patient is likely not to experience ARDS in the future. If the value of elastase levels is limited to 23.79 ±3.95, the patient will experience mild type ARDS. Meanwhile, if the value of elastase levels is limited to 57.68 ±18.55, in the future the patient will experience moderate type of ARDS. Meanwhile, if the elastase level is between 107.85 ± 5.04, the patient will likely experience severe ARDS. Conclusion: Based on the characteristic test of the sample characteristics, diagnosis and action, there are significant differences in the causes of ARDS. Neutophil elastase levels correlate with the degree of ARDS incidence.

The role of autophagy in the overexpression of MUC5AC in patients with chronic rhinosinusitis

BackgroundAutophagy is a lysosomal degradation pathway that protects the body and is essential for cell survival and differentiation. Mucins (MUCs) are important components of secreted mucus, mucin (MUC)5 AC is the major MUC secreted in the normal airway. ObjectiveInvestigated the role of autophagy in pathogenic mucin (MUC)5 AC production during chronic rhinosinusitis (CRS). MethodsThe expression of human neutrophil elastase (HNE) and the autophagic proteins microtubule-associated protein 1 light chain (LC)3B-II, c-Jun N-terminal kinase (JNK), c-Jun, and MUC5AC were analyzed in the sinonasal mucosa and human nasal epithelial cells (HNECs) using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR). Autophagic vacuoles were studied using transmission electron microscopy (TEM). Primary HNECs were treated with HNE, bafilomycin A1, and SP600125. In some experiments, cultured primary HNECs were transfected with small interfering RNAs (siRNAs) to target Beclin-1 (BECN1; BECN1-siRNA), autophagy-related gene 5 (Atg5; Atg5-siRNA), and c-Jun (c-Jun-siRNA). Cultured cells were analyzed using western blotting, qRT-PCR, and ELISA. ResultsIn CRS patients, both with and without nasal polyps, the expression levels of HNE, LC3B, JNK, c-Jun, and MUC5AC were upregulated. Bafilomycin A1 upregulated LC3B-II expression and inhibited MUC secretion in HNE-treated normal primary HNECs. Autophagosomes were observed in HNE-treated primary HNECs using TEM. HNE-induced secretion of MUC5AC was suppressed in normal primary HNECs by BECN1-siRNA, Atg5-siRNA, c-Jun-siRNA, and SP600125. ConclusionsIn HNE-induced CRS, autophagy increases the secretion of MUC5AC by promoting the phosphorylation of JNK and c-Jun.

Electrochemical sensing of human neutrophil elastase and polymorphonuclear neutrophil activity

Human neutrophil elastase (HNE) is a serine protease, produced by polymorphonuclear neutrophils (PMNs), whose uncontrolled production has been associated with various inflammatory disease states as well as tumour proliferation and metastasis. Here we report the development and characterisation of an electrochemical peptide-based biosensor, which enables the detection of clinically relevant levels of HNE. The sensing platform was characterised in terms of its analytical performance, enzymatic cleavage kinetics and cross-reactivity and applied to the quantitative detection of protease activity from PMNs from human blood.

Faster Diagnosis of Alpha-1-Antitrypsin Deficiency through Electrochemical and Quartz Crystal Microbalance Testing

Alpha-1-Antitrypsin (A1AT) Deficiency (A1AD) is a potentially debilitating inherited condition that can lead to reduced levels of A1AT in the lungs and increased levels in the liver. In the lungs, the condition is routinely associated with an imbalance in levels of Human Neutrophil Elastase (HNE) and its native inhibitor A1AT (predisposing those affected to early-onset emphysema). In the liver, certain genetic mutations lead to an accumulation of A1AT. No cure exists, and current approaches for diagnosis are insufficient as illustrated by the fact that A1AD is underdiagnosed and diagnosis is often delayed.1New methods for diagnosis are needed, and one possible route is the use of electrochemical techniques combined with quartz crystal microbalance (QCM) approaches. Often, QCM and electrochemistry are combined using a gold working electrode that has been functionalized with a particular molecule. In our case, Porcine Pancreatic elastase (PPE), a surrogate of HNE, has been used as an enzymatic marker. When a potentiostat was combined in parallel to the apparatus, measurements of both the frequency and the current responses were simultaneously performed. PPE was immobilized to the surface of the gold electrode through the use of a crosslinker. The immobilized PPE was then incubated with various concentrations of N-Succinyl-Ala-Ala-Ala-p-Nitroanalide (SApNA), a chromogenic substrate for elastase, allowed to react, and the absorbance measured to determine the activity of the enzyme. This same test was also completed with the A1AT inhibitor in the solution with the SApNA and the absorbance measured again. From these results, the frequency was measured and converted to mass to determine the extent of inhibition. This data was then examined via kinetics-based models to estimate the kinetic parameters of inhibition. Electrochemical measurements have been performed concurrently with the frequency measurements. Using phosphate buffered saline (PBS) in the presence of ferrocyanide/ferricyanide as the redox species, cyclic voltammetry was performed to determine the extent that the electrode surface is covered. As the SApNA and A1AT was added to the solution, the change in the ferrocyanide/ferricyanide peaks were observed. It is the ultimate goal of this work to develop a test that can be used to determine the inhibition kinetics of HNE via A1AT, and thus shed light on new approaches to diagnose A1AD. Such an approach could also serve as a platform that could be useful in the early detection and diagnosis of many different diseases that could allow for preemptive treatment and could potentially improve life expectancy outcomes for those afflicted. American Thoracic Society/European Respiratory Society Statement: Standards for the Diagnosis and Management of Individuals with Alpha-1-Antitrypsin Deficiency. (2003). Am. J. Respir. Crit. Care Med. 168(7): 818-900.

Human neutrophil elastase detection with fluorescent peptide sensors conjugated to cellulosic and nanocellulosic materials: part II, structure/function analysis

Human neutrophil elastase (HNE) is one of a number of proteases that is receiving increased attention as a marker for inflammatory diseases and sensor-based point of care diagnostics. Integral to sensor-based detection is the transducer surface, which is the platform of the sensor’s signal transmittance. Here we describe the bioactivity and related transducer surface properties of cellulose and nanocellulose matrices as peptide–cellulose fluorescent sensors. Detection sensitivity of the sensor signals for HNE levels typically found in chronic wounds is characterized. The fluorescent elastase peptide substrate, Succinamidyl-Ala-Ala-Pro-Val-amidylcoumadin (Pep) was employed in both cellulose and nanocellulose transducer surfaces evaluated for biosensor sensitivity to HNE. The cellulose transducers selected are filter paper (FP) and print cloth (PC) fabric and are comprised of processed cotton fibers. The nanocellulose transducers are the wood cellulose nanocrystals (wCNC) and the wood nanocellulose composites (wNCC). The wNCCs consist of blended quantities of nanocrystalline and microfibrillated cellulose at 66/33 and 50/50, and are characterized as thin films. The biosensor activity was in the order of wCNC-Pep > FP-Pep = NCC-Pep (50/50) > NCC-Pep (66/33) > PC-Pep. Sensor sensitivity correlated with specific surface area. A depiction of peptide substitution on nanocellulosic and cellulosic surfaces is rendered through peptide–cellulose crystallite models derived from X-ray diffraction analysis of the material, and the models discussed in light of biosensor structure activity relationships. In addition, the overall morphology, pore size and porosity of the materials are discussed for their suitability as protease sensors.

Activated Neutrophils Are Associated with Pediatric Cerebral Malaria Vasculopathy in Malawian Children.

ABSTRACTMost patients with cerebral malaria (CM) sustain cerebral microvascular sequestration of Plasmodium falciparum-infected red blood cells (iRBCs). Although many young children are infected with P. falciparum, CM remains a rare outcome; thus, we hypothesized that specific host conditions facilitate iRBC cerebral sequestration. To identify these host factors, we compared the peripheral whole-blood transcriptomes of Malawian children with iRBC cerebral sequestration, identified as malarial-retinopathy-positive CM (Ret+CM), to the transcriptomes of children with CM and no cerebral iRBC sequestration, defined as malarial-retinopathy-negative CM (Ret-CM). Ret+CM was associated with upregulation of 103 gene set pathways, including cytokine, blood coagulation, and extracellular matrix (ECM) pathways (P < 0.01; false-discovery rate [FDR] of <0.05). Neutrophil transcripts were the most highly upregulated individual transcripts in Ret+CM patients. Activated neutrophils can modulate diverse host processes, including the ECM, inflammation, and platelet biology to potentially facilitate parasite sequestration. Therefore, we compared plasma neutrophil proteins and neutrophil chemotaxis between Ret+CM and Ret-CM patients. Plasma levels of human neutrophil elastase, myeloperoxidase, and proteinase 3, but not lactoferrin or lipocalin, were elevated in Ret+CM patients, and neutrophil chemotaxis was impaired, possibly related to increased plasma heme. Neutrophils were rarely seen in CM brain microvasculature autopsy samples, and no neutrophil extracellular traps were found, suggesting that a putative neutrophil effect on endothelial cell biology results from neutrophil soluble factors rather than direct neutrophil cellular tissue effects. Meanwhile, children with Ret-CM had lower levels of inflammation, higher levels of alpha interferon, and upregulation of Toll-like receptor pathways and other host transcriptional pathways, which may represent responses that do not favor cerebral iRBC sequestration.

Disequilibrium of Blood Coagulation and Fibrinolytic System in Patients With Coronary Artery Ectasia

Thrombus formation and myocardial infarction are not uncommon in patients with coronary artery ectasia (CAE). In light of this, the present study aims to systemically evaluate the blood coagulation and fibrinolytic systems in CAE patients. In this study, we enrolled 30 patients with CAE, 30 patients with coronary atherosclerosis disease (CAD), and 29 subjects with normal coronary arteries (control). The coagulation system was evaluated using a routine coagulation function test performed in the hospital laboratory before coronary angiography, and measurements included prothrombin time, international normalized ratio, activated partial thromboplastin time, fibrinogen time, and thrombin time. The evaluation of the fibrinolytic system included measurements of D-dimer, euglobulin lysis time, plasminogen activator inhibitor 1, plasminogen, plasminogen activity assay, α1-antitrypsin (α1-AT), α2 plasmin inhibitor (α2-PI), and α2-macroglobulin (α2-MG). Alpha1-AT, α2-PI, and α2-MG also inhibit activities of 3 neutrophil serine proteases, namely human neutrophil elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3); therefore, the plasma levels of these 3 proteinases were also evaluated.In CAE patients, the circulating coagulation system was normal. For the fibrinolytic system, a decrease of plasminogen activity was observed (P = 0.029) when compared with CAD patients, and the concentrations of α1-AT (both P < 0.001), α2-PI (P = 0.002 and P = 0.025), and α2-MG (P = 0.034 and P < 0.001) were significantly elevated when compared with CAD patients and normal controls. Moreover, the plasma levels of HNE (both P < 0.001) and CG (P = 0.027 and 0.016) in CAE patients were also significantly higher than those of the CAD and control groups. There was no difference in plasma PR3 concentration among these 3 groups.Disequilibrium of the coagulation/fibrinolytic system may contribute to thrombus formation and clinical coronary events in patients with CAE. The increased plasma concentrations of α1-AT, α2-PI, and α2-MG might provide beneficial effects by inhibiting the proteinases and restraining the ectatic process; on other hand, they led to unfavorable results by inhibiting plasmin and decreasing thrombus degradation in CAE patients.

Neutrophil serine proteases and their endogenous inhibitors in coronary artery ectasia patients.

Objective:Proteolytic enzymes possibly contribute to coronary artery ectasia (CAE). This study aimed to determine whether neutrophils, neutrophil serine proteases (NSPs), and their endogenous inhibitors participated in the pathological process of CAE.Methods:The study consisted of 30 patients with CAE, 30 patients with coronary artery disease (CAD), and 29 subjects with normal coronary arteries (Control). The following circulating items were measured: the main NSPs, including human neutrophil elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3); soluble elastin (sElastin), which was a degradation product of elastin fibres; NSP inhibitors such as α1-protease inhibitor (α1-PI), α2-macroglobulin (α2-MG), secretory leucoprotease inhibitor (SLPI), and elafin; as well as two neutrophil activation markers (myeloperoxidase and lactoferrin) and three classic neutrophil activators [tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and bacterial endotoxin].Results:The levels of HNE, CG, and sElastin were elevated in the CAE group. The levels of α1-PI and α2-MG were also significantly increased in the CAE group. The levels of myeloperoxidase and lactoferrin were higher in the CAE group. The levels of TNF-α, IL-8, and endotoxin were unchanged in the CAE group compared with those in the CAD group.Conclusion:Neutrophils may participate in the process of vessel extracellular matrix destruction and coronary ectasia by releasing NSPs in a non-classical manner.

Pulmonary matrix metalloproteinase-9 activity in mechanically ventilated children with respiratory syncytial virus

Respiratory syncytial virus (RSV) infection is a potent stimulus for airway epithelial expression of matrix metalloproteinase (MMP)-9. MMP-9 activity in vivo is a predictor of disease severity in children with RSV-induced respiratory failure. Human airway epithelial cells were infected with RSV A2 strain and analysed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 (a natural inhibitor of MMP-9) release. In addition, endotracheal samples from children with RSV-RF and controls (non-RSV pneumonia and nonlung disease controls) were analysed for MMP-9, TIMP-1, human neutrophil elastase and myeloperoxidase activity. RSV infection of airway epithelia was sufficient to rapidly induce MMP-9 transcription and protein release. Pulmonary MMP-9 activity peaked at 48 h in infants with RSV-induced respiratory failure. In the RSV group, MMP-9 activity and MMP-9/TIMP-1 ratio imbalance predicted higher oxygen requirement and worse paediatric risk of mortality scores. The highest levels of human neutrophil elastase and myeloperoxidase activity were measured in the RSV cohort; however, unlike MMP-9, these neutrophil markers failed to predict disease severity. These results support the hypothesis that RSV is a potent stimulus for MMP-9 expression and release from human airway epithelium, and that MMP-9 is an important biomarker of disease severity in mechanically ventilated children with RSV lung infection.

Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity

In chronic wounds, elevated human neutrophil elastase (HNE) is a destructive protease that has been proposed as a biomarker. Numerous wound dressing designs have been introduced in an effort to lower HNE levels. The clinical detection of HNE as a point of care biomarker or an in situ colorimetric adjuvant to chronic wound dressings presents potential advantages in the management of chronic wounds. A colorimetric approach to the detection of HNE using peptide conjugated cotton cellulose nanocrystals (CCN) is reported here. For this purpose a HNE tripeptide substrate, n-Succinyl-Alanine–Alanine-Valine-para-nitroanilide (Suc-Ala–Ala-Val-pNA), was covalently attached to glycine esterified CCN and compared with a similar tetrapeptide analog for colorimetric HNE sensor activity. Visible HNE activity was significantly higher on CCN tripeptide conjugates when compared with similar analogs synthesized on paper. Upon enzymatic release of para-nitroaniline (pNA) from the Glycine-CCN conjugate of succinyl-Ala–Ala-Val-pNA, amplification of the colorimetric response from pNA with reactive dyes enhanced visible absorption of the chromogen. Two color amplifying dyes that react with pNA were compared for their ability to enhance the visual sensor response to HNE activity. The colorimetric detection of HNE with CCN tripeptide conjugates was sensitive at HNE levels previously reported in chronic wound fluid (0.05 U/mL HNE). The HNE sensor and the chromogen amplifying dyes were interfaced with 50 and 10 kD dialysis cellulose membranes (DCM) to model filtration of HNE and chromogen (pNA) from a model wound dressing surface before and after sensor reactivity. The detection sensitivity to HNE activity was assessed with the CCN-tripeptide conjugate interfaced at the DCM surface distal and proximal to a dressing surface. The HNE sensor interfaced proximal to the dressing surface was most efficient with 10 kD membrane filtration of pNA and subsequent reaction with amplifying dyes. When interfaced with the 10 kD cellulose membrane, elastase sensor activity remained sensitive to 0.05 U/mL HNE. The nanocellulose surface properties, performance and design issues of the biosensor approach are discussed.

Serum MMPs 7–9 and their inhibitors during glucocorticoid and anti-TNF-α therapy in pediatric inflammatory bowel disease

Objectives.In inflammatory bowel diseases (IBD), matrix metalloproteinases (MMPs) participate in intestinal tissue damage and regenerative processes. MMP activity is inhibited by tissue inhibitors of MMPs (TIMPs) and plasma inhibitor, α2-macroglobulin (α2M). We evaluated serum MMPs, their inhibitors and markers of neutrophil activity, myeloperoxidase (MPO) and human neutrophil elastase (HNE), during glucocorticoid (GC) and anti-TNF-α therapies in pediatric IBD, in aim to find new tools for assessment of therapeutic response. Methods.Serum samples were collected before and within a month after the start of therapy with oral GC (n = 19) or anti-TNF-α agent (n = 16), and from 32 pediatric control patients. Serum levels of MMP-7, MMP-9, TIMP-1, TIMP-2, α2M, MPO, and HNE were analyzed with enzyme-linked immunoabsorbent assays (ELISA) and MMP-8 by immunofluorometric assay (IFMA). Disease activity was monitored with erythrocyte sedimentation rate (ESR), CRP, fecal calprotectin (FC), and physician's global assessment of clinical disease activity (PGA). Results. In IBD, pretreatment serum MMP-7, MMP-8, MMP-9, α2M, MPO, and HNE were elevated compared with controls. During GC therapy, MMP-7, TIMP-1, and MMP-7/TIMP-2 decreased (all p < 0.05). During anti-TNF-α therapy, MMP-7 decreased (p = 0.063), but remained higher than that after GC therapy (p < 0.05). α2M (p < 0.05) and HNE (p < 0.05) increased, the former higher than that in GC-treated patients. The levels of MMPs and their inhibitors did not markedly associate with inflammatory markers in blood or feces. Conclusions.In pediatric IBD, serum MMP-7 mirrors disease activity, and together with TIMP-1, reflects GC therapy response. α2-Macroglobulin expression parallels the anti-TNF-α response.

Age-related differences in the pathogenesis of chronic rhinosinusitis

Age-related differences in the pathogenesis of chronic rhinosinusitis

Characterization of potential elastase inhibitor-peptides regulated by a molecular switch for wound dressings applications

Elastase plays an important role in wound healing process, degrading damaged tissue and allowing complete tissue recovery. The levels of human neutrophil elastase (HNE) are usually controlled by endogenous inhibitors. However, in the presence of high levels of elastase, like the ones present in chronic wounds, the inhibitors cannot overcome this overproduction and the enzyme starts to degrade the surrounding healthy tissue. In this work we report the development of a molecular switch to control the elastase activity in the exudate of non-healing chronic wounds. A peptide library was generated and screened in a microarray format for protein kinase-mediated phosphorylation. Two peptides were identified as casein kinase Iδ (CKI) substrates: KRCCPDTCGIKCL and its analogous peptide KRMMPDTMGIKML, with cysteine residues replaced by methionine residues. These peptides were studied in solution, both in the phosphorylated and non-phosphorylated forms as potential inhibitors for elastase. The obtained results show that the reversible process of phosphorylation/dephosphorylation results in differential inhibitory activity of the peptides. Thus the reversible process of phosphorylation/dephosphorylation can be used as a kind of molecular switch to control elastase activity. Degradation studies reveal that both the inhibitor-peptides and CKI are degraded by elastase. These results envisage the safe utilisation of these inhibitor-peptides together with CKI in the formulation of wound dressings.

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.

Bornyl (3,4,5-trihydroxy)-cinnamate - An optimized human neutrophil elastase inhibitor designed by free energy calculations

Human neutrophil elastase (HNE), a serine protease, is involved in the regulation of inflammatory processes and controlled by endogenous proteinase inhibitors. Abnormally high levels of HNE can cause degradation of healthy tissues contributing to inflammatory diseases such as rheumatoid arthritis, and also psoriasis and delayed wound healing. In continuation of our research on HNE inhibitors we have used the recently developed binding mode model for a group of cinnamic acid derivative elastase inhibitors and created bornyl (3,4,5-trihydroxy)-cinnamate. This ligand exhibited improved binding affinity predicted by means of free energy calculations. An organic synthesis scheme for the ligand was developed and its inhibitory activity was tested toward the isolated enzyme. Its IC 50 value was found to be three times lower than that of similar compounds, which is in line with the computational result showing the high potential of free energy calculations as a tool in drug development.