Academic literature on the topic 'Human neutrophil elastase inhibitors'
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Journal articles on the topic "Human neutrophil elastase inhibitors"
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Mendez, Deena, Prasad SR, and Kutty AVM. "Differential Inhibition of Human Neutrophil Elastase and Bacterial Elastase by endogenous Protease Inhibitors." JOURNAL OF CLINICAL AND BIOMEDICAL SCIENCES 08, no. 1 (March 15, 2018): 10–13. http://dx.doi.org/10.58739/jcbs/v08i1.2.
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Human Neutrophil Elastase (HNE) is released from neutrophils when foreign substances are encoun-tered. Elastase is also released by invasive pathogenic microorganisms and is an important virulence factor. Serine protease inhibitors counter the elastase released in excess to prevent the damage of tissues. Aim: The aim of this study was to assess the specificity of the endogenous protease inhibitors α1 Anti trypsin( α1AT) and α2 Macroglobulin( α2MG) on Human Neutrophil Elastase and elastase released by Pseudomonas aeruginosa. Result: It was observed that Pseudomonal elastase exhibited resistance to inhibition in com-parison to Human Neutrophil Elastase particularly by α1 Anti trypsin. However, the inhibition patterns of these enzymes were more or less similar in both the cases by α2 Macroglobulin. Conclusion; α1AT is a less potent inhibitor of Pseudonomas aeruginosa elastase and might require exogenous inhibitory substances to control the damaging effects of this enzyme. Keywords: α1 Anti trypsin, α2 Macroglobulin, Human Neutrophil Elastase, Pseudomonas aeruginosa
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Dubin, A., J. Potempa, and J. Travis. "Structural and functional characterization of elastases from horse neutrophils." Biochemical Journal 300, no. 2 (June 1, 1994): 401–6. http://dx.doi.org/10.1042/bj3000401.
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In order better to understand the pathophysiology of the equine form of emphysema, two elastinolytic enzymes from horse neutrophils, referred to as proteinases 2A and 2B, have been extensively characterized and compared with the human neutrophil proteinases, proteinase-3 and elastase. Specificity studies using both the oxidized insulin B-chain and synthetic peptides revealed that cleavage of peptide bonds with P1 alanine or valine residues was preferred. Further characterization of the two horse elastases by N-terminal sequence and reactive-site analyses indicated that proteinases 2A and 2B have considerable sequence similarity to each other, to proteinase-3 from human neutrophils (proteinase 2A), to human neutrophil elastase (proteinase 2B) and to a lesser extent to pig pancreatic elastase. Horse and human elastases differed somewhat in their interaction with some natural protein proteinase inhibitors. For example, in contrast with its action on human neutrophil elastase, aprotinin did not inhibit either of the horse proteinases. However, the Val15, alpha-aminobutyric acid-15 (Abu15), alpha-aminovaleric acid-15 (Nva15) and Ala15 reactive-site variants of aprotinin were good inhibitors of proteinase 2B (Ki < 10(-9) M) but only weak inhibitors of proteinase 2A (Ki > 10(-7) M). In summary, despite these differences, the horse neutrophil elastases were found to resemble closely their human counterparts, thus implicating them in the pathological degradation of connective tissue in chronic lung diseases in the equine species.
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Janusz, M. J., and N. S. Doherty. "Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G." Journal of Immunology 146, no. 11 (June 1, 1991): 3922–28. http://dx.doi.org/10.4049/jimmunol.146.11.3922.
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Abstract The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.
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Potempa, J., J. J. Enghild, and J. Travis. "The primary elastase inhibitor (elastasin) and trypsin inhibitor (contrapsin) in the goat are serpins related to human α1-anti-chymotrypsin." Biochemical Journal 306, no. 1 (February 15, 1995): 191–97. http://dx.doi.org/10.1042/bj3060191.
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Two primary serine proteinase inhibitors in goat plasma have been isolated and characterized. The N-terminal sequence analysis of the purified proteins revealed that they are closely related to each other and are highly homologous to human alpha 1-anti-chymotrypsin rather than alpha 1-proteinase inhibitor. However, despite structural similarities the inhibitory specificity of the goat inhibitors differed from each other and from that of anti-chymotrypsin. In contrast with human anti-chymotrypsin, one of the goat inhibitors was shown to be a strong and specific inhibitor of trypsin (k(ass.) = 1.9 x 10(6) M-1.s-1), whereas the other was an efficient inhibitor of neutrophil elastase (k(ass.) = 1.5 x 10(6) M-1.S-1). Differences in the inhibitory specificity of each protein could readily be attributed to the amino acid sequence within the reactive site region. The trypsin inhibitor with an assumed arginine residue at the P1 position of the reactive-site peptide bond is referred to as ‘contrapsin’, and indicates that the occurrence of contrapsins is not restricted to rodents. In contrast, the inhibitory specificity, resistance to oxidative and proteolytic inactivation and the presence of a P1 leucine residue in the elastase inhibitor is unique among inhibitory serpins that have been characterized to date. Because this serpin is apparently the major elastase inhibitor in goat plasma, it is likely to be involved in the control of goat neutrophil elastase. Therefore, we suggest the name ‘elastasin’, and extend it to any other anti-chymotrypsin related serpins possessing neutrophil-elastase- inhibitory activity.
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Marinaccio, Lorenza, Azzurra Stefanucci, Giuseppe Scioli, Alice Della Valle, Gokhan Zengin, Angelo Cichelli, and Adriano Mollica. "Peptide Human Neutrophil Elastase Inhibitors from Natural Sources: An Overview." International Journal of Molecular Sciences 23, no. 6 (March 8, 2022): 2924. http://dx.doi.org/10.3390/ijms23062924.
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Elastases are a broad group of enzymes involved in the lysis of elastin, the main component of elastic fibres. They are produced and released in the human body, mainly by neutrophils and the pancreas. The imbalance between elastase activity and its endogenous inhibitors can cause different illnesses due to their excessive activity. The main aim of this review is to provide an overview of the latest advancements on the identification, structures and mechanisms of action of peptide human neutrophil elastase inhibitors isolated from natural sources, such as plants, animals, fungi, bacteria and sponges. The discovery of new elastase inhibitors could have a great impact on the pharmaceutical development of novel drugs through the optimization of the natural lead compounds. Bacteria produce mainly cyclic peptides, while animals provide for long and linear amino acid sequences. Despite their diverse natural sources, these elastase inhibitors show remarkable IC50 values in a range from nM to μM values, thus representing an interesting starting point for the further development of potent bioactive compounds on human elastase enzymes.
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Inauen, W., D. N. Granger, C. J. Meininger, M. E. Schelling, H. J. Granger, and P. R. Kvietys. "Anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell injury: role of elastase." American Journal of Physiology-Heart and Circulatory Physiology 259, no. 3 (September 1, 1990): H925—H931. http://dx.doi.org/10.1152/ajpheart.1990.259.3.h925.
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The aim of this study was to assess the role of neutrophilic elastase in anoxia-reoxygenation-induced, neutrophil-mediated injury to microvascular endothelium. Cultured bovine microvascular endothelial cells were grown to confluence and labeled with 51Cr. The endothelial cells were exposed to a 30-min period of anoxia and subsequently reoxygenated. Endothelial cell injury, quantitated as 51Cr release and cell detachment, was determined 8 h after reoxygenation. Addition of neutrophils upon reoxygenation enhanced the anoxia-reoxygenation-induced increase in 51Cr release and cell detachment. The neutrophil-mediated injury was associated with elastase release from the neutrophils. Four agents were used to inhibit neutrophilic elastase activity: Eglin C, methoxysuccunyl-Ala2-Pro-Val-CH2Cl, L658,758, and a monoclonal antibody against neutrophilic elastase. All elastase inhibitors attenuated the neutrophil-mediated endothelial cell detachment but not 51Cr release. Addition of purified human neutrophilic elastase, at a level that mimicked the release from neutrophils, increased cell detachment in endothelial cells exposed to anoxia-reoxygenation but did not affect 51Cr release. Our results indicate that elastase plays an important role in anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell dysfunction.
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Rosengren, S., and K. E. Arfors. "Neutrophil-mediated vascular leakage is not suppressed by leukocyte elastase inhibitors." American Journal of Physiology-Heart and Circulatory Physiology 259, no. 4 (October 1, 1990): H1288—H1294. http://dx.doi.org/10.1152/ajpheart.1990.259.4.h1288.
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Application of leukotriene B4 (LTB4) to hamster cheek pouches induces neutrophil-dependent vascular leakage of macromolecules as well as leukocyte intravascular adherence and emigration. The effect of inhibitors of neutrophil elastase on these reactions was studied with intravital microscopy. Anesthetized hamsters were pretreated with the elastase inhibitors L 658,758, Eglin C, or dextran sulfate, and LTB4 (10 nM) was superfused over the cheek pouches. Neither Eglin C nor L 658,758 had any effect on the resulting vascular leakage of a macromolecular marker; in contrast, dextran sulfate suppressed this leakage by 85%. None of the compounds affected LTB4-induced leukocyte adherence or neutrophil diapedesis. The inhibitors were able to inhibit both hamster and human neutrophil elastase as estimated in crude neutrophil extracts. These results suggest that neutrophils extravasate and generate vascular leakage without the use of their elastase activity. The inhibitory effect of dextran sulfate on macromolecular leakage may be due to interaction with cationic proteins released from the neutrophils.
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Edwards, Philip D., and Chris A. Veale. "Inhibitors of human neutrophil elastase." Expert Opinion on Therapeutic Patents 7, no. 1 (January 1997): 17–28. http://dx.doi.org/10.1517/13543776.7.1.17.
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Filep, Janos G., Natalie Edner, Everton de Oliveira Lima Dos Santos, Meriem Sekheri, and Driss El Kebir. "TLR9 activation impairs phagocytosis-induced neutrophil apoptosis and prolongs E. coli-induced acute lung injury." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 129.1. http://dx.doi.org/10.4049/jimmunol.198.supp.129.1.
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Abstract Neutrophil dysfunction, resulting in delayed apoptosis and inefficient bacterial clearance, is a characteristic feature of severe pathologies, including sepsis and cystic fibrosis. Human neutrophils detect and respond to bacterial DNA (CpG DNA) through TLR9. We investigated the impact of CpG DNA on phagocytosis, phagocytosis-induced neutrophil apoptosis and clearance of E coli. Culture of human neutrophils with CpG DNA (0.1–3.2 μg/ml) resulted in decreased phagocytosis of opsonized E. coli or yeast. CpG DNA upregulated C3R (CD11b) expression, downregulated C5aR (CD88) expression and induced release of neutrophil elastase. C5aR cleavage was prevented by a specific neutrophil elastase inhibitor and the broad-spectrum serine protease inhibitor PMSF. Consistently, CpG DNA reduced phagocytosis-induced NADPH oxidase-mediated activation of caspase8 and caspase-3. These actions of CpG DNA were blocked by the telomere-derived TLR9 inhibitory oligonucleotide 5′-TTT AGG GTT AGG GTT AGG G-3′. In mice, CpG DNA impaired pulmonary clearance of E coli, suppressed neutrophil apoptosis and delayed resolution of lung injury evoked by intratracheal instillation of live E. coli. Genetic deletion of TLR9 rendered mice unresponsive to CpG DNA. These results identify a novel mechanism, neutrophil elastase-mediated inactivation of C5aR-mediated phagocytosis, by which CpG DNA could contribute to neutrophil dysfunction and prolongation of tissue injury. Our findings also suggest a therapeutic potential for TLR9 antagonists or neutrophil elastase inhibitors for enhancing clearance of bacterial infections in an environment where bacterial DNA is abundantly present.
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Trainor, Diane Amy. "Synthetic inhibitors of human neutrophil elastase." Trends in Pharmacological Sciences 8, no. 8 (August 1987): 303–7. http://dx.doi.org/10.1016/0165-6147(87)90123-4.
Full textDissertations / Theses on the topic "Human neutrophil elastase inhibitors"
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Roghanian, Ali. "Modulation of dendritic cells by human neutrophil elastase and its inhibitors in pulmonary inflammation." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/4398.
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Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs.
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Leahy, Darren. "Probing the role of methionine oxidation in substrate and inhibitor interactions with native and recombinant Human Neutrophil Elastase." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/204141/1/Darren_Leahy_Thesis.pdf.
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This thesis was an exploration of how Human Neutrophil Elastase (HNE) activity can be modulated by oxidation of methionine residues located on substrates and inhibitors. Research focused on producing a molecular toolbox of innovative HNE substrates and inhibitors specifically engineered to include methionine, then assessing the mechanism by which oxidation leads to targeted interaction with HNE. This may be an important biochemical process in chronic obstructive pulmonary disease, which is linked to HNE destruction of elastic lung tissue together with oxidative damage by cigarette smoke and neutrophil-mediated inflammation.
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Morla, Shravan. "Glycosaminoglycan Mimetics for the Treatment of Cancer and Lung Inflammation." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5948.
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Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar and either uronic acid or galactose. They are expressed on virtually all mammalian cells, usually covalently attached to proteins, forming proteoglycans. GAGs are highly negatively charged due to an abundance of sulfate and carboxylic acid groups, and are structurally very diverse, with differences arising from chain length, the type of monomeric units, the linkages between each monomeric unit, the position of sulfate groups, and the degree of sulfation. GAGs are known to interact with a multitude of proteins, impacting diverse physiological and pathological processes. In addition, most of the biological interactions mediated by proteoglycans are believed to be primarily because of the GAG chains present on their surface. Considering the involvement of GAGs in multiple diseases, their use in the development of drugs has been of significant interest in the pharmaceutical field. Heparin, the first GAG-based drug developed in 1935, is still the most widely used anticoagulant in the world. The therapeutic potential of GAGs for the treatment of many other disease states, including cancer, inflammation, infection, wound healing, lung diseases, and Alzheimer’s disease, is being actively studied with many GAGs currently in clinical trials. However, challenges associated with the heterogeneous and complex structure of GAGs, limit their successful development. To combat such issues, our lab has focused on developing Non- Saccharide GAG Mimetics (NSGMs) as structural mimics of GAGs. NSGMs, being synthetic molecules, offer multiple advantages over GAGs. The studies mentioned here describe our efforts in the development of NSGMs as potential therapeutics for cancer, and cystic fibrosis.
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Lin, Hong. "Regulation of the human neutrophil elastase gene." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398446.
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Klimecki, Haley M. "Expression of Human Neutrophil Elastase in K. Lactis." Digital Commons @ East Tennessee State University, 2010. https://dc.etsu.edu/honors/2.
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Human neutrophils are the most abundant type of white blood cell and provide the body with a line of defense against foreign, infectious microorganisms. Contained within the azurophilic granules in the cytoplasm of neutrophils are three serine proteases, Human Neutrophil Elastase, Cathepsin G, and Protease 3. Once a foreign bacterium is engulfed by white blood cells, these enzymes attack and degrade the invading body, thus killing it (Reeves et al., 2002). The focus of this research is centered on the production of one of the serine proteases, human neutrophil elastase (HNE), and while the importance of HNE can be seen, genetic mutations or improper regulation can compromise a person’s immunity. Neutropenia (a low neutrophil count) is one such disease caused by a genetic mutation of HNE that results in susceptibility to infection (Li and Horwitz, 2001). Additionally, HNE is a powerful enzyme that can attack the elastin of the lung if not properly controlled. Consequently, genetic deficiencies of alpha-1 proteinase inhibitor protein in the blood can result in emphysema because active HNE released from neutrophils is free to degrade lung tissue (Laurell and Eriksson, 1965).
Recombinant HNE is not currently available, and the enzyme must be isolated from human blood cells, which has inherent hazards. Additionally, the lack of recombinant HNE has prevented studies involving site–directed mutagenesis to study the intracellular processing of HNE near its C-terminal end where mutations have been found to result in neutropenia. Kinetic studies of the full-length HNE might shed some light on why its C-terminal region is removed before storage in cytoplasmic granules.
The HNE DNA sequence was first codon optimized for yeast and commercially synthesized. It was then fused with DNA for eGFP (enhanced green fluorescent protein) via an enterokinase cleavage site (D4K). This DNA construct (eGFP-D4K-HNE) was then inserted into the Kluyveromyces lactis (K. lactis) pKLAC1 vector, downstream of the alpha mating factor which directs proteins for secretion. Then, chemically competent GG799 cells (a strain of K. lactis) were transformed with the linearized pKLAC1-eGFP-D4K-HNE insert through a protocol from New England Biolabs. Theoretically, the gene integrates into the yeast genome upon transformation via sequences within the pKLAC1 vector that are homologous with the LAC4 gene promoter that allows for galactose utilization (Colussi 2005). Acetamide was used as a selectable marker because wild type K. lactis cells are not able to use acetamide as a nitrogen source. The pKLAC1 vector, however, contains the Aspergillus nidulans gene acetamidase (amdS) that allows only transformants to grow on plates with acetamide as the sole nitrogen source (Read 2007).
Selected colonies were transferred to both liquid and agar-based synthetic media with galactose to induce transcription and translation of the HNE gene to produce the eGFP-D4K-HNE fusion, and screened via fluorescence microscopy for production of eGFP. None of the screened colonies tested positive for the presence of the fusion protein.
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Huynh, Thi Ngoc Tram. "Nouvelle approche du traitement de l’emphysème ;Synthèse et activité biologique d’inhibiteurs de l’élastase neutrophile humaine." Thesis, Reims, 2014. http://www.theses.fr/2014REIMP201/document.
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Dans le but d'obtenir des composés originaux ayant une activité inhibitrice envers la NE, des triazoles et des 2-aminofuranes diversement substitués ont été synthétisés. Une réaction de "click chemistry" catalysée par le cuivre entre un azide et un alcyne a été appliquée pour la synthèse des triazoles. La synthèse des composés de type 2-aminofuranes est basée sur l'attaque nucléophile d'un isonitrile sur un γ-oxo butynoate d'alkyle en présence d'un aldéhyde aromatique.Les tests biologiques ont été réalisés tout au long de la thèse. Les composés présentant les meilleures IC50 envers la NE ont été sélectionnées pour une évaluation en terme de sélectivité vs les autres sérines protéases du PN (la cathepsine G et la protéinase 3). Enfin, les meilleurs inhibiteurs ont subi une évaluation de leur activité anti-oxydante selon deux méthodes: méthode au DPPH (1,1-diphényl-2-picryl-hydrazyl) et méthode au thiocyanate ferriqueTo prepare new compounds with an inhibitory activity towards NE, diversely substituted triazoles and 2-aminofuranes were synthesized. Triazoles were obtained using the copper catalyzed "click chemistry" between an azide and an alkyne. The synthesis of 2-aminofuranes is based on the nucleophilic attack of an isonitrile on an alkyl γ-oxo-butynoate in the presence of an aromatic aldehyde.Biological assays were carried out throughout the thesis. Compounds owning the best IC50 towards NE were selected for evaluation in terms of selectivity vs the two other serine-proteases of the PN (cathepsin G and proteinase 3). Finally, evaluation of antioxidant activity of the best compounds was achieved using by two approachs: DPPH (1,1-diphenyl -2-picrylhydrazyl) method and ferric thiocyanate method
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Novak, Tanya. "Oral ulceration in Behçet's disease : an investigation of neutrophil elastase and its inhibitors." Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8810.
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Behçet’s disease (BD) is a vasculitis of unknown aetiology typified by recurrent oral and genital ulcers, skin and ocular lesions. Debilitating manifestations can also affect vascular, gastrointestinal and neurological systems. Previous BD investigations showed increased circulating neutrophils and neutrophil elastase (NE), a serine protease. NE can digest connective tissues compromising their integrity if not regulated. In this study, NE and its two main inhibitors, secretory leukocyte protease inhibitor (SLPI) and alpha1- antitrypsin (α1AT), were investigated to determine if NE dysregulation is triggering oral mucosal damage. Findings were compared to healthy controls (HC) and recurrent aphthous stomatitis (RAS) patients, a disorder of episodic oral ulceration. FlowCytoMixTMmultiplex-assays compared saliva and serum inflammatory cytokines measurements where salivary levels reflected disease activity and correlated with published serum levels. Salivary NE, SLPI, and α1AT were measured by ELISA. Patients with oral ulcers had increased NE. Unexpectedly, BDq (quiescent, without ulceration) had increased NE, but SLPI was significantly lower than RASq and HC. RASq NE levels were similar to HC. Overall, NE correlated with α1AT levels, but showed an inverse relationship with SLPI. Quantitative PCR revealed significantly increased SLPI mRNA expression in both BDq and RASq buccal epithelium. High mRNA/low SLPI protein expression during ulceration could be explained by deficient translation, blocked ELISA antibody binding, or SLPI depletion. Despite high α1AT, all study groups had enzymatically active salivary NE which was successfully inhibited by recombinant SLPI. Confocal microscopy revealed BD patients’ blood neutrophils readily release neutrophil extracellular traps (NETs) in vitro compared to HCs. Antimicrobial NETs have mixed granule contents coating decondensed chromatin fibres and are associated with autoimmunity. During NET production, our novel observation that intracellular SLPI but not α1AT co-localised with NE suggests a regulatory role. This study supports the theory that a protease-antiprotease imbalance may play a role in BD oral and systemic pathology.
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Averhoff, Petra. "Characterization of the specificity of human neutrophil elastase for Shigella flexneri virulence factors." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2006. http://dx.doi.org/10.18452/15650.
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Neutrophile Granulocyten wirken als einer der ersten Abwehrmechanismen gegen invasive Mikroorganismen im angeborenen Immunsystem von Mammalia. Aktiviert durch inflammatorische Signale verlassen diese Granulocyten das vaskuläre System und migrieren durch das Gewebe zum Infektionsherd. Dort binden sie die Mikroorganismen, phagozytieren und eliminieren diese schließlich mit hoher Effizienz. Humane Neutrophile Elastase (NE) ist Bestandteil der neutrophilen Granula und spielt eine entscheidende Rolle im Abbau von Virulenzfaktoren enteroinvasiver Bakterien, einschließlich der Shigella Virulenzfaktoren IpaB (invasion antigen plasmid B) und IcsA (intracellular spread A). NE gehört zu der Familie der Chymotrypsin-ähnlichen Serinproteasen, die sich durch Sequenz- und Strukurähnlichkeit auszeichnen, jedoch sehr unterschiedliche biologische Funktionen aufweisen. Cathepsin G (CG) ist wie NE eine Chymotrypsin-ähnliche Serinprotease und ebenfalls in neutrophilen Granula lokalisiert. Allerdings zeigt CG keine Aktivität gegenüber Virulenzfaktoren von Shigella. Obwohl die Kristallstrukturen von CG und NE fast identisch sind, konnten einzelne oder mehrere Aminosäuren in der Substratbindungsspalte identifiziert werden, die zwischen den beiden Enzymen differieren. Dies legte die Vermutung nahe, dass die Spezifität von NE gegenüber Virulenzfaktoren in diesen Unterschieden codiert sein könnte. Daher wurden diese Aminosäuren durch die analogen CG Aminosäuren oder durch Alanin ersetzt. Der Vergleich der funktionellen Eigenschaften der NE Mutanten mit wildtyp NE zeigte, dass die Aminosäuren an den Positionen 98 und 216-224 entscheidend für die Substratspezifität von NE sind. Die NE Mutanten N98A, 216-218 und 216-224 waren nicht mehr in der Lage, die Virulenzfaktoren IcsA und IpaB sowie das NE Peptidsubstrat abzubauen. Stattdessen haben diese Mutanten die Fähigkeit erlangt, das CG Peptidsubstrat abzubauen. Zusammenfassend konnten wir Aminosäuren in NE identifizieren, die sowohl die Spezifität von NE für das Peptidsubstrat als auch für die Virulenzfaktoren von Shigella flexneri determinieren.Neutrophil granulocytes are one of the first lines of defense of the mammalian innate immune system against invading microorganisms. In response to inflammatory stimuli, neutrophils migrate from the blood stream to infected tissues where they bind, engulf and inactivate microorganisms efficiently. Human neutrophil elastase (NE), a neutrophil granule component, is a key host defense protein that rapidly destroys virulence factors of enteroinvasive pathogens including IpaB (invasion plasmid antigen B) and IcsA (intracellular spread A) from Shigella. NE belongs to the family of chymotrypsin-like serine proteases with sequence and structural similarity but with very different biological functions. Cathepsin G (CG) is another abundant chymotrypsin-like serine protease in neutrophil granules. However, in contrast to NE, CG does not cleave virulence factors of Shigella. The crystallographic structures of NE and CG are very similar but we identified single or multiple residues in the substrate-binding cleft to differ in these two enzymes. We hypothesized that NE specificity for bacterial virulence factors resides within these structural differences. Therefore these specific residues in NE were replaced with the analogous amino acids of CG or with alanine. By comparing the functional properties of these NE mutants to wildtype NE we were able to show that the amino acids at position 98 and 216-224 are crucial for the substrate specificity of NE. The NE mutants N98A, 216-218 and 216-224 did not cleave the virulence factors IcsA and IpaB as well as the NE peptide substrate but cleaved the CG peptide substrate. In summary, we identified residues in NE that determine the specificity of NE for the peptide substrate and for the Shigella flexneri virulence factors.
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Hannah, Sharon. "Investigation of the peptides produced from human elastin by digestion with neutrophil elastase and with cathepsin G." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/19822.
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Emphysema is a degenerative lung disease which, it is suggested, results probably from repeated periods of proteinase:antiproteinase imbalance during which excess of enzyme attacks the extracellular matrix. Of the many enzymes produced by inflammatory cells human neutrophil elastase (HNE) is thought to be the major offending enzyme. It attacks elastin, which is responsible for the elastic recoil of the lung. If emphysema was simply a result of the destruction of elastin by HNE then degradation products of elastin would inevitably be present, at least transiently, in the serum of patients. The aim of this project was to separate and characterise the soluble peptides resulting from the digestion of elastin with HNE and/or human neutrophil cathepsin G (HNCG), another neutrophilic enzyme, which is primarily bactericidal and, to determine if any of the peptides were characteristic of digestion by one enzyme or combination of enzymes. HNE and HNCG were isolated from purulent sputum, and elastin was isolated by two methods from post-mortem lungs. The digestion of the elastin by the enzymes was followed by measuring the amino groups liberated during the course of the digestion. A method was developed for the measurement of the insoluble as well as the soluble products of digestion. Initially, the amounts of soluble and insoluble products were similar, but the amount of soluble products soon exceeded the amount of insoluble products. The soluble products of digestion were separated by reverse-phase chromatography. The peptides separated into two groups (A and B), regardless of which enzyme was involved in the initial digestion. Both groups were heterogeneous mixtures of peptides. Filtration experiments and amino acid analysis showed that the groups of peptides differed in size and composition.
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Smith, Eliot T. "Bioengineering the Expression of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, and C-Reactive Protein in Yeast." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etd/1198.
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The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human cathepsin G (hCatG) and human neutrophil elastase (HNE) were expressed in P. pastoris and HNE was also expressed in K. lactis. The recombinant variants rhCatG and rHNE, with intact C-terminal extensions, were expressed as fusion proteins with the soluble heme-binding domain of cytochrome B5 (CytB5) and an N-terminal hexahistidine (6xHis) tag for purification. The CytB5 domain was linked to the native N-termini of active rhCatG and rHNE by the EPLcleavable substrate sequence DDDDK~I, where ~ is the sessile bond. These fusion proteins were directed for secretion. The yeast P. pastoris expressed up to 3.5 mg/L of EPL-activable rHNE in fermentation broth; however, only 200 μg/L of rhCatG could be produced by this method. Recombinant expression in K. lactis never surpassed 100 μg/L of activable rHNE. The CytB5 fusion domain was present in the heme-bound form, conferring a red color and 410 nm absorbance peak to solutions containing the fusion proteins. This absorbance pattern was most readily visible during the purification of CytB5-rHNE from P. pastoris. Human C-reactive protein (hCRP) and the substitution variant CRP E42Q were expressed in recombinant form and secreted by P. pastoris. Both products were found to bind phosphocholine (PCh) in the same manner as native hCRP. Difficulties encountered during purification revealed that wild type recombinant CRP (rCRP) was produced at 2 different molecular masses. The P. pastoris recombinant expression system yielded better results than K. lactis. Bioreactor-scale fermentation in a 5 L vessel facilitated expression and characterization of these recombinant proteins.
Book chapters on the topic "Human neutrophil elastase inhibitors"
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Powers, J. C., C. M. Kam, H. Hori, J. Oleksyszyn, and E. F. Meyer. "Synthetic Mechanism-Based and Transition-State Inhibitors for Human Neutrophil Elastase." In Biochemistry of Pulmonary Emphysema, 123–41. London: Springer London, 1992. http://dx.doi.org/10.1007/978-1-4471-3771-9_10.
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Patton, Lavonne M. "In Vivo Evaluation of MDL 201,404YA, A Novel Inhibitor of Human Neutrophil Elastase." In Proteases, Protease Inhibitors and Protease-Derived Peptides, 83–96. Basel: Birkhäuser Basel, 1993. http://dx.doi.org/10.1007/978-3-0348-7397-0_7.
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Kirschenheuter, Gary P., Josef Oleksyszyn, Lyle W. Spruce, Maciej Wieczorek, Thomas M. Kloppel, Sanford R. Simon, and John C. Cheronis. "Synthesis and Characterization of Human Neutrophil Elastase Inhibitors Derived from Aromatic Esters of Phenylalkanoic Acids." In Proteases, Protease Inhibitors and Protease-Derived Peptides, 71–82. Basel: Birkhäuser Basel, 1993. http://dx.doi.org/10.1007/978-3-0348-7397-0_6.
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Gast, Alain, and Joseph G. Bieth. "Inhibition of Human Neutrophil Elastase by Acid-Soluble Inter-α-Trypsin Inhibitor." In Advances in Experimental Medicine and Biology, 75–82. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-1057-0_8.
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Smith, Robin A., Robert A. Stockley, and Simon T. Hodgson. "Neutrophil Elastase Inhibitors." In New Drugs for Asthma, Allergy and COPD, 173–76. Basel: KARGER, 2001. http://dx.doi.org/10.1159/000062153.
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Fitzgerald, Mary F. "Small-molecule neutrophil elastase inhibitors as therapies for respiratory disease." In New Drugs and Targets for Asthma and COPD, 225–30. Basel: KARGER, 2010. http://dx.doi.org/10.1159/000320823.
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Hornebeck, W., V. Bizot-Foulon, A. Meddahi, and B. Pellat. "Inhibition of Human Neutrophil Elastase by Lipophilic Heparin and Dextran Derivatives." In Proteolysis in Wound Repair, 37–49. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-61130-8_4.
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Simon, Sanford, Micha Vered, Alex Rinehart, John Cheronis, and Aaron Janoff. "Inhibition of Human Neutrophil Elastase by Polyguanylic Acid and other Synthetic Polynucleotides." In Advances in Experimental Medicine and Biology, 65–74. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-1057-0_7.
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Bode, W. "The Specific Interaction of Human Leukocyte Elastase with Various Protein Inhibitors." In Protein Structure and Protein Engineering, 75–85. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-74173-9_9.
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Skinner, Martha, Tsuranobu Shirahama, Phillip Stone, Lawreen Heller Connors, James Calore, and Alan S. Cohen. "Amyloid-Agarose Plate Test: Ultrastructural Changes in the Fibril and its Association with Human Neutrophil Elastase." In Amyloidosis, 261–65. Boston, MA: Springer US, 1986. http://dx.doi.org/10.1007/978-1-4613-2199-6_32.
Full textConference papers on the topic "Human neutrophil elastase inhibitors"
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Bell, D., M. Jackson, C. MacRae, A. L. Muir, and J. Dawes. "NEUTROPHIL ELASTASE IS A MARKER OF NEUTROPHIL ACTIVATION IN ACUTE MYOCARDIAL INFARCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643020.
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Elastase is released from human neutrophils as a result of active secretion, phagocytosis or cell lysis. It is a broad-spectrum protease which not only hydrolyses the major components of tissue matrix, but can also affect platelet aggregation, coagulation and fibrinolysis. Neutrophils localise at the site of myocardial infarction and have been implicated in subsequent cell damage. A radioimmunoassay was developed which detects elastase complexed to its inhibitors α2- antitrypsin and α2-macroglobulin as wen as the free enzyme. Plasma concentrations of elastase in 31 healthy controls were 21.3 ± 13.8 ng/ml, and did not differ significantly from those in 22 patients with stable angina (23.6 ± 8.6 ng/nl). In 19 patients with myocardial infarction, however, plasma elastase levels rose to a peak within 48 hours of infarct which was significantly higher than normal levels (95.1 < 83.7; P < 0.001), and then declined. Correction of the data for neutrophil count did not affect the significance of the observed differences. Measurement of whole blood elastase reflected the neutrophil count and was not otherwise informative. Thus, neutrophils are activated after myocardial infarction and release sufficient elastase for it to be detected systemically. This may extend tissue damage and affect coagulation and fibrinolysis at the site of infarction.
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Weitz, J., S. Landman, and S. Birken. "IDENTIFICATION OF A NEUTROPHIL ELASTASE CLEAVAGE SITE ON THE Act -CHAIN OF PRIMATE FIBRINOGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643896.
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Human neutrophil elastase (HNE) cleaves the Aα21-22 bond of fibrinogen thus releasing the fibrinopeptide A (FPA)-containing fragment Aαl-21. Plasma Aal-21 levels reflect in vivo HNE activity and peptide levels are increased in cigarette smokers and patients with chronic lung disease. To further explore the HNE-fibrinogen interaction, we set out to develop an animal model. The digestion of purified baboon and marmoset fibrinogen by human thrombin, HNE and extracts of baboon and marmoset neutrophils was monitored with a specific radioimmunoassay for human FPA. Thrcmbin produced quantitative release (2 mol/mol fibrinogen) of FPA. In contrast, HNE and the neutrophil extracts did not release FPA, but rather, produced quantitative release of a larger, FPA-containing fragment. Immunochemically, this fragment was clearly distinguishable from FPA in that in vitro thrombin treatment increased its immunoreactivity 1,000-fold (thrombin increasable FPA or TIFPA). TIFPA release by the neutrophil extracts was blocked by α1-proteinase inhibitor, a specific HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl) and an anti-HNE IgG, indicating that elastase was the responsible proteinase and that there was homology between the human and primate enzymes. The products of HNE and neutrophil extract proteolysis of the primate fibrinogens were then separated by high performance liquid chromatography and the TIFPA-containing fractions were subjected to amino acid sequence analysis. The FPA-containing fragments each consisted of 21 amino acids, had minor substitutions when compared with human A α] -21 [Baboon: Aα(3) Ser - Thr; Marmoset Aα(l) Ala - Thr, Aα(3) Ser - Thr, Aα(ll) Glu - Ala], and exhibited complete crossreactivity with the human peptide. Using the TIFPA assay, there was good recovery of primate or human Aαl-21 added to primate blood and the mean peptide level in 8 healthy marmosets was similar to that in man (0.5 nM and 0.4 nM, respectively). In conclusion, (1) the Aα;21 -22 bond of baboon and marmoset fibrinogen is a cleavage site for human and primate elastase, (2) baboon and marmoset Aal-21 can be measured with the assay for the human peptide, and (3) the primate serves as a useful model for the study of elastase-fibrinogen interactions.
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Farache Trajano, Luiza, Rebecca Moore, and Quentin Sattentau. "The Presence of Chemical Cross-Linking Stabilises HIV-1 Envelope Glycoprotein Trimer Antigens in a Model of Intramuscular Immunisation." In Building Bridges in Medical Science 2021. Cambridge Medicine Journal, 2021. http://dx.doi.org/10.7244/cmj.2021.03.001.4.
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Background: The HIV-1 envelope glycoprotein (Env) is the target of antigen design for antibody- based vaccination. In 2019, four trimeric Env vaccines entered an experimental trial: ConM, ConS, and their cross-linked counterparts. The trimers were formulated with MPLA adjuvant. Studies have demonstrated that adjuvants trigger neutrophil infiltration. Neutrophils activate and degranulate releasing proteases, namely elastase and cathepsinG. Aims: To assess the stability and immunogenicity of these vaccines in the presence of adjuvant- recruited neutrophils and their proteolytic enzymes. Methods: Trimers were incubated with commercially-sourced proteases. To analyse stability, samples were reduced, denatured and separated using gel electrophoresis. To assess antibody binding, a trimer-protease incubation was followed by an ELISA. To establish more physiologically relevant conditions, harvested neutrophils were exposed to various adjuvants. The supernatant, shown to contain elastase, was incubated alongside the vaccines. The reducing and denaturing gels, as well as the ELISA, was repeated. Results: Gel analysis revealed that un-crosslinked trimers underwent significant digestion whereas cross-linking conferred enhanced stability. In the presence of neutrophil-sourced protease-containing-supernatant, trimers displayed resistance to digestion. The differential stability profile of Env trimers when exposed to commercially sourced compared to supernatant- derived proteases may be due to the inhibitory effect of human serum on elastase. Antibody epitopes were maintained in vitro. Conclusion: The vaccine antigens are sensitive to enzymatic degradation. This is reduced by cross-linking and human serum.
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Seitz, R., M. Wolf, R. Egbring, and K. Havemann. "Neutrophil Elastase, Thrombin and Plasmin in Septic Shock: Influence on Prognosis." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643894.
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The prognosis of septicaemia depends on the occurrence of disseminated disturbances of the microcirculation impairing organ function and haemorrhagic complications due to consumption of coagulation factors. The intravasal appearance of three potentially involved proteinases in active form can be detected by immunologic determination of their complexes with inhibitors: thrombin-antithrombin III (TAT), according to PELZER et al. (Thrombos. Haemostas. 54:24,1985); plasmin- antiplasmin (PAP), ldlE, antiserum donated by KARGES; human neutrophil elastase (HNE)- antitrypsin, ELISA, Merck, DarmstadtIn 47 patients with septic shock (19 survived, group A; 28 lethal, group B) the PAP levels were moderately elevated throughout the course without vaviations related to the outcome. TAT was initially strongly increased in both groups (18.8±6.3 ng/ml/16.5±7.2), and decreased towards the end of the course in both groups (2.7±0.5/3.7±0.8). Though the intial HNE levels were higher in group A (2458±348ng/ml) than in group B (1291±295, p=0.017), they decreased, in group A more rapidly and were at the end almost significantly lower than in group B (315±54/652&3x00B1;161, p=0,059). The decrease of TAT as well as HNE was associated with substitution of antithrombin III concentate (ATIII) and fresh frozen plasma (ffp) given with the aim of normalization of haemostasis and replacement of inhibitors. Factor XIII, a substrate of both thrombin and HNE, was initially equally low about 50% of normal in both groups, but increased only in group A (69.1±7.1/49.6±5.5, p=0.045) towards the end.Conclusions: Both TAT and HNE decreased after initial elevation under substitution of ATIII and ffp. A rapid decrease seems to be a favourable sign which is accompanied by rising levels of F XIII, while sustained elevation of HNE points to a poor prognosis
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Selak, M. A., M. Chignard, and J. B. Smith. "CHARACTERIZATION OF A NEUTROPHIL CPYMOTRYPSIN-LIKE ENZYME THAT ACTIVATES PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643157.
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Communication between neutrophils and platelets was previously investigated by measuring platelet aggregation, serotonin release and changes in cytosolic free calcium subsequent to specific stimulation of neutrophils by fMet-Leu-Phe (FMLP) in a suspension of both cell types. The addition of the chemotactic peptide was shown to elicit secondary platelet activation as a consequence of primary stimulation of neutrophils. Cell-free supernatants from FMLP-stimulated neutrophils were capable of inducing platelet activation thus demonstrating that a factor released bv neutrophils was responsible for the observed platelet responses. After eliminating classical platelet agonists as the acitive agent, it was shown that an enzyme termed neutroohilin induced platelet calcium mobilization, secretion and aggregation. The current studies were conducted to characterize the mediator released bv neutrophils. Neutrophilin bound bo cation exchange resins but failed to bind to anion exchangers. The biological activity associated with neutroohilin was unaffected by leupeptin, only very weakly diminished by N-bosyl-Lvs-chloromethvl ketone and was strongly inhibited by N-tosvl-Phe-chloromethvl ketone, aloha-l-antitrvpsin, soybean trypsin inhibitor and Z-Glv-Leu-Phe-chloromethvl ketone. Neutroohilin was released from stimulated neutrophils only after cytochalasin B treatment, as was beta-glucuronidase, suggesting that both enzymes are located in azurophilic granules. Neutroohilin-induced platelet activation was inhibited bv antiserum to human catheosin G in a dose-deoendent manner but was unaffected by antiserum to human elastase or alpha-fetoprotein. The inhibitor sensitivity, immunological cross-reactivity, ionic properties and probable subcellular localization indicate that neutrophilin is a cationic chymotrvosin-like enzyme related, if not identical to, catheosin G. Neutroohilin-induced platelet activation could explain different pathological events in which platelets and neutroohils are known to be involved.
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Lagente, V., I. Guenon, I. Morel, O. Sellier-Kessler, and E. Chevalier. "A Novel Protein Epitope Mimetic (PEM) Neutrophil Elastase (NE) Inhibitor, POL6014, Inhibits Human NE-Induced Acute Lung Injury in Mice." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a5668.
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P. Felgueiras, Helena, Sílvia M. M. A. Pereira Lima, A. Francisca G. Silva, and Susana P. G. Costa. "Exploring the antibacterial potential of human neutrophil elastase inhibitor Ala-Ala-Pro-Val synthesized using microwave-assisted solid phase." In 6th International Electronic Conference on Medicinal Chemistry. Basel, Switzerland: MDPI, 2020. http://dx.doi.org/10.3390/ecmc2020-07414.
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Emerson, T. E., and T. B. Redens. "SYNERGISTIC EFFICACY OF COMBINING ANTITHROMBIN-III AND ALPHA 1-PROTEINASE INHIBITOR PROPHYLAXIS IN THE ENDOTOXEMIC SHEEP PULMONARY DYSFUNCTION MODEL." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644891.
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The adult respiratory distress syndrome (ARDS) isa serious, often fatal condition associated with septicemia/endotoxemia and Other inflammatory disease states. It is characterized by increased microvascular permeability to protein and pulmonary edema, amongother pathologies. Neutrophil elastase, thrombin andfibrin induced pulmonary microemboli are possible mediators of this pathology. The present study was to determine the effect of pretreatment with large doses of anti thrombin-111 (AT-III), a major inhibitor of coagulation and alpha 1-proteinase inhibitor (al-PI), a major inhibitor of neutrophil elastase, alone and in combination, on selected indices of ARDS in the sheep lung lymph fistula model. Range sheep weighing 35-45 kg were maintained under Halothane anesthesia and surgically prepared the day of the experiment. The sheep were randomized and given iv infusions of 250 U/kg purified human AT-III alone (n=7), 100 mg/kg purified human al-PI alone (n=6), AT-III/al-PI combined (n=5), or an equal volume of normal saline (n=6). Measurements were taken before and hourly after iv challenge with 2 ug/kg E. coli endotoxin. Lunglymph flow increased between 220% - 2802Tof control in the AT-III only, al-PI only, and saline control groups (P<0.05), but did not increase in the AT-III/al-PI combined group (P>0.05). The lymph to plasma protein concentration ratio increased significantly in all groups (P<0.05). Transvascular protein flow and clearance increased, but the increases were significantly greater in the AT-III alone, al-PI alone andsaline control groups compared to the AT-III/al-PI combined group. Pulmonary artery pressure increased initially in all groups (PC0.05) but returned to a level near baseline by 2 hours. Pulmonary artery wedgepressure did not change significantly in any group (P>0.05). This study shows that combined AT-III and al-PI prophylaxis prevented or significantly attenuated indices of ARDS, while neither AT-III alone nor al-PI alone did during 5 hours of endotoxemia. The data suggest that AT-III/al-PI combined protects against ARDS in this model and that the protective effectis synergistic.
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Sié, P., D. Dupouy, F. Dol, and B. Boneu. "INACTIVATION OF HEPARIN COFACTOR II (HC II) BY POLYMORPHONUCLEAR LEUKOCYTES (PMNL)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643868.
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Several authors have shown that antithrombin III (AT III) was catalytically inactivated by neutrophil elastase, an observation relevant to pathophysiological processes in the vicinity of inflammatory sites. The aim of this study was to investigate whether HC II, another natural thrombin inhibitor, is also inactivated by PMNL.A rapid loss of HC II activity occured upon incubation with fresh human PMNL stimulated by phorbol myristate acetate (Tl/2 1 uM HC II, 0.35 108 cells/mm : -2 min) or with PMNL extracts prepared by nitrogen cavitation. Antithrombin (dermatan sulfate cofactor) and antichymotrypsin activities of HC II were lost at the same rate. Resting PMNL were ineffective. Inactivation was prevented by several serine-protease inhibitors but was Ca++ /Mg++ independent. Inactivation coincided with the formation of a 54 KD peptide after a first non-inactivating degradation into a 62 KD peptide (native HC II : 76 KD). These reaction products are reminiscent of those described upon incubation with proteinase I from Echis carinatus venom. HC II was inactivated more rapidly than AT III (T 1/2 of AT III in the same conditions -15 min). However, heparin (1-10 ug/ml) strongly accelerated the rate of AT III inactivation and slightly protected HC II, thus reversing the order of inactivation. Dermatan sulfate had no effect on this process.In conclusion, this study shows ; l)both AT III and HC II are rapidly inactivated by PMNL enzymes, thus favoring locally thrombin-mediated processes ; 2) heparin increases AT III degradation by PMNL, a possible route of catabolism operating in patients with low AT III levels during heparin treatment.
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Holmes, W. E., H. R. Lijnen, and D. Collen. "CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644766.
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α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.