Detection of extracellular neutrophil elastase in hamster lungs after intratracheal instillation of E. coli lipopolysaccharide using a fluorogenic, elastase-specific, synthetic substrate.

PubMed Central

Rudolphus, A.; Stolk, J.; van Twisk, C.; van Noorden, C. J.; Dijkman, J. H.; Kramps, J. A.

1992-01-01

Repeated intratracheal instillations of E. coli lipopolysaccharide (LPS) in hamster lungs cause an influx of polymorphonuclear leukocytes (PMNs) into the alveolar walls, with concomitant development of severe emphysema. It has been suggested that elastase, released by these PMNs, is involved in the development of emphysema. This study demonstrates the release of elastase from recruited PMNs in cryostat sections of hamster lungs, after being treated once, twice, or thrice with LPS, intratracheally. Elastase activity was visualized using two elastase-specific synthetic substrates, to which a methoxynaphthylamine (MNA) group had been bound covalently. Liberated MNA, when made insoluble by coupling with 5-nitrosalicylaldehyde, fluoresces strongly. The authors observed that the interval between start of incubation and appearance of fluorescence and the intensity of fluorescence correlated with the number of LPS administrations. Fluorescence was observed to be located in or in close vicinity to alveolar walls. No fluorescence was observed in sections of untreated hamsters. Liberation of MNA from synthetic substrates was delayed strongly by the addition of a recombinant secretory leukocyte proteinase inhibitor or a substituted cephalosporin neutrophil elastase inhibitor. The authors conclude that LPS-mediated PMN influx into the lung is accompanied by release of elastase from these cells and speculate that this PMN-elastase is involved in the development of LPS-mediated emphysema. Images Figure 1 Figure 2 Figure 3 PMID:1632460

Persistent elastase/proteinase inhibitor imbalance during prolonged ventilation of infants with bronchopulmonary dysplasia: evidence for the role of nosocomial infections.

PubMed

Walti, H; Tordet, C; Gerbaut, L; Saugier, P; Moriette, G; Relier, J P

1989-10-01

Acute imbalance between elastase and alpha-1-proteinase inhibitor (alpha 1Pi) may contribute to the development of bronchopulmonary dysplasia (BPD). The question of whether such an imbalance persists in BPD infants still requiring mechanical ventilation after 4 wk of life has not been previously addressed. We studied 14 infants still on mechanical ventilation at 4 wk of age: nine had BPD and five did not. Weekly (4 to 9 wk) serum and bronchoalveolar lavage (BAL) specimens were taken. alpha 1Pi and alpha-2-macroglobulin were measured in serum and BAL by immunoturbidimetric assay. BAL elastase activity was measured by cleavage of a synthetic substrate and expressed as ng of porcine pancreatic elastase equivalent. Infants with BPD had higher levels of serum alpha 1Pi and alpha-2-macroglobulin than those without BPD. In contrast, the corresponding BAL levels were either similar or even decreased (alpha 1Pi). Moreover, there was a 3-fold increase in elastase-1Pi imbalance expressed as the BAL ng of porcine pancreatic elastase equivalent/2 alpha 1Pi ratio. The role of nosocomial infections was evident in a subgroup of 11 infected BAL aspirates in BPD infants. In such cases we found a 3-fold increase in the BAL ng of porcine pancreatic elastase equivalent/alpha 1Pi ratio as compared to 35 noninfected BAL in BPD infants. These data suggest a persistent alveolitis with imbalance between elastase and proteinase inhibitors in prolonged severe BPD. Such an imbalance is, in part, explained by a local destruction and/or inactivation of alpha 1Pi. Our results also emphasize the increase in proteolysis with nosocomial pneumonia.

Distribution of pancreatic elastase and metalloproteinase in vertebrates.

PubMed

Yoshinaka, R; Sato, M; Tsuchiya, N; Ikeda, S

1986-01-01

Elastase-like enzymes were detected as zymogens in all of the pancreatic extracts from the gummy shark, bullhead shark, angel shark, smooth hammerhead, bestel, rainbow trout, carp, eel, Japanese mackerel, yellowtail, sea bass, parrotfish, bullfrog, chicken, bluewhite dolphin, hog, rat, cat, and dog. The distribution of pancreatic elastase and metalloproteinase was examined on the basis of the effect of specific inhibitors on elastase like-activity in each extract. The results indicate that pancreatic elastases are present in all the species examined and pancreatic metalloproteinases are present only in the teleost fishes.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice.

PubMed

Almeida-Reis, Rafael; Theodoro-Junior, Osmar A; Oliveira, Bruno T M; Oliva, Leandro V; Toledo-Arruda, Alessandra C; Bonturi, Camila R; Brito, Marlon V; Lopes, Fernanda D T Q S; Prado, Carla M; Florencio, Ariana C; Martins, Mílton A; Owen, Caroline A; Leick, Edna A; Oliva, Maria L V; Tibério, Iolanda F L C

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL / 6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNF α -, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2 α , collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNF α -positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

PubMed Central

Theodoro-Junior, Osmar A.; Oliveira, Bruno T. M.; Oliva, Leandro V.; Toledo-Arruda, Alessandra C.; Bonturi, Camila R.; Brito, Marlon V.; Prado, Carla M.; Florencio, Ariana C.; Martins, Mílton A.; Owen, Caroline A.; Oliva, Maria L. V.

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL/6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNFα-, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2α, collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNFα-positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease. PMID:28466019

A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice

PubMed Central

Theodoro-Júnior, Osmar Aparecido; Righetti, Renato Fraga; Almeida-Reis, Rafael; Martins-Oliveira, Bruno Tadeu; Oliva, Leandro Vilela; Prado, Carla Máximo; Saraiva-Romanholo, Beatriz Mangueira; Leick, Edna Aparecida; Pinheiro, Nathalia Montouro; Lobo, Yara Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Tibério, Iolanda de Fátima Lopes Calvo

2017-01-01

Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management. PMID:28216579

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

PubMed Central

2013-01-01

Background Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice. Methods BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge. Results Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice. Conclusion These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge. PMID:23347423

Cefoperazone Prevents the Inactivation of α1-Antitrypsin by Activated Neutrophils

PubMed Central

Dallegri, Franco; Dapino, Patrizia; Arduino, Nicoletta; Bertolotto, Maria; Ottonello, Luciano

1999-01-01

At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor α1-antitrypsin. In the present study, cefoperazone prevented α1-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with α1-antitrypsin without affecting the cells’ ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with α1-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation. PMID:10471586

Synthetic serine elastase inhibitor reduces cigarette smoke-induced emphysema in guinea pigs.

PubMed

Wright, Joanne L; Farmer, Stephen G; Churg, Andrew

2002-10-01

To test whether a serine elastase inhibitor could prevent or reduce emphysema, we exposed guinea pigs to cigarette smoke acutely, or daily for 6 months, and treated some animals with the neutrophil elastase inhibitor ZD0892. Acute smoke exposure increased lavage neutrophils and increased desmosine and hydroxyproline, measures of elastin and collagen breakdown; all these measures were reduced by ZD0892. Long-term smoke exposure produced emphysema and increases in lavage neutrophils, desmosine, hydroxyproline, and plasma tumor necrosis factor alpha (TNF-alpha). ZD0892 treatment returned lavage neutrophils, desmosine, and hydroxyproline levels to control values, and decreased airspace enlargement by 45% and TNF-alpha by 30%. Animals exposed to smoke for 4 months and then to smoke plus ZD0892 for 2 months were not protected against emphysema. Mice exposed to smoke showed increases in gene expression of neutrophil chemoattractant macrophage inflammatory protein-2, macrophage chemoattractant protein-1, and TNF-alpha at 2 hours along with increased plasma TNF-alpha; ZD0892 prevented the increases in macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 expression and reduced plasma TNF-alpha levels to baseline. These data demonstrate that a serine elastase inhibitor ameliorates the inflammatory and destructive effects of cigarette smoke, and that these effects are mediated in part by neutrophils and by smoke-driven TNF-alpha production.

High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae.

PubMed

Yamashita, Nobuo; Komori, Yumiko; Okumura, Yoshiyuki; Uchiya, Kei-Ichi; Matsui, Takeshi; Nishimura, Akira; Ogawa, Kenji; Nikai, Toshiaki

2011-08-01

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain.

PubMed

Rose, C; Camus, A; Schwartz, J C

1988-11-01

A serine endopeptidase was characterized as a major inactivating enzyme for endogenous cholecystokinin (CCK) in brain. CCK-8 released by depolarization of slices of rat cerebral cortex, as measured by its immunoreactivity (CCK-ir), undergoes extensive degradation (approximately 85% of the amount released) before reaching the incubation medium. However, recovery of CCK-ir is enhanced up to 3-fold in the presence of serine-alkylating reagents (i.e., phenylmethylsulfonyl fluoride) as well as selected active site-directed inactivators (i.e., peptide chloromethyl ketones) or transition-state inhibitors (i.e., peptide boronic acids) of serine peptidases. Among these compounds, elastase inhibitors were the most potent protecting agents, whereas trypsin or chymotrypsin inhibitors were ineffective. HPLC analysis of endogenous CCK-ir recovered in media of depolarized slices indicated that endogenous CCK-5 [CCK-(29-33)-pentapeptide] was the most abundant fragment and that its formation was strongly decreased in the presence of an elastase inhibitor. HPLC analysis of fragments formed upon incubation of exogenous CCK-8 [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 whose formation was prevented or at least diminished in the presence of the elastase inhibitor. It is concluded that there is an elastase-like serine endopeptidase in brain that cleaves the two peptide bonds of CCK-8 where the carboxyl group is donated by a methionine residue and constitutes a major inactivation ectoenzyme for the neuropeptide.

A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain.

PubMed Central

Rose, C; Camus, A; Schwartz, J C

1988-01-01

A serine endopeptidase was characterized as a major inactivating enzyme for endogenous cholecystokinin (CCK) in brain. CCK-8 released by depolarization of slices of rat cerebral cortex, as measured by its immunoreactivity (CCK-ir), undergoes extensive degradation (approximately 85% of the amount released) before reaching the incubation medium. However, recovery of CCK-ir is enhanced up to 3-fold in the presence of serine-alkylating reagents (i.e., phenylmethylsulfonyl fluoride) as well as selected active site-directed inactivators (i.e., peptide chloromethyl ketones) or transition-state inhibitors (i.e., peptide boronic acids) of serine peptidases. Among these compounds, elastase inhibitors were the most potent protecting agents, whereas trypsin or chymotrypsin inhibitors were ineffective. HPLC analysis of endogenous CCK-ir recovered in media of depolarized slices indicated that endogenous CCK-5 [CCK-(29-33)-pentapeptide] was the most abundant fragment and that its formation was strongly decreased in the presence of an elastase inhibitor. HPLC analysis of fragments formed upon incubation of exogenous CCK-8 [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 whose formation was prevented or at least diminished in the presence of the elastase inhibitor. It is concluded that there is an elastase-like serine endopeptidase in brain that cleaves the two peptide bonds of CCK-8 where the carboxyl group is donated by a methionine residue and constitutes a major inactivation ectoenzyme for the neuropeptide. PMID:3186727

Elastase inhibitors as potential therapies for ELANE-associated neutropenia.

PubMed

Makaryan, Vahagn; Kelley, Merideth L; Fletcher, Breanna; Bolyard, Audrey Anna; Aprikyan, A Andrew; Dale, David C

2017-10-01

Mutations in ELANE , the gene for neutrophil elastase (NE), a protease expressed early in neutrophil development, are the most frequent cause of cyclic (CyN) and severe congenital neutropenia (SCN). We hypothesized that inhibitors of NE, acting either by directly inhibiting enzymatic activity or as chaperones for the mutant protein, might be effective as therapy for CyN and SCN. We investigated β-lactam-based inhibitors of human NE (Merck Research Laboratories, Kenilworth, NJ, USA), focusing on 1 inhibitor called MK0339, a potent, orally absorbed agent that had been tested in clinical trials and shown to have a favorable safety profile. Because fresh, primary bone marrow cells are rarely available in sufficient quantities for research studies, we used 3 cellular models: patient-derived, induced pluripotent stem cells (iPSCs); HL60 cells transiently expressing mutant NE; and HL60 cells with regulated expression of the mutant enzyme. In all 3 models, the cells expressing the mutant enzyme had reduced survival as measured with annexin V and FACS. Coincubation with the inhibitors, particularly MK0339, promoted cell survival and increased formation of mature neutrophils. These studies suggest that cell-permeable inhibitors of neutrophil elastase show promise as novel therapies for ELANE -associated neutropenia. © Society for Leukocyte Biology.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

LTB(4)-induced nasal gland serous cell secretion mediated by neutrophil elastase.

PubMed

Cardell, L O; Agustí, C; Takeyama, K; Stjärne, P; Nadel, J A

1999-08-01

Local allergen challenge causes nasal hypersecretion and also causes local leukotriene (LT) release, including LTB(4). Because LTB(4) causes leukocyte recruitment, and because neutrophil elastase is a potent secretagogue, we examined the hypothesis that LTB(4) causes nasal hypersecretion via neutrophil elastase. We developed a method for isolating and superfusing a nasal segment in dogs. Instillation of LTB(4) into the nasal segment caused a time-dependent increase in the volume of airway fluid, in lysozyme secretion, and in the recruitment of neutrophils. ICI 200,355, a selective inhibitor of neutrophil elastase, prevented LTB(4)-induced nasal secretion and lysozyme secretion, but it had no effect on neutrophil recruitment. We conclude that LTB(4) causes potent nasal secretion via release of elastase, and therefore LTB(4) may play a major role in allergic nasal hypersecretion.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Neutrophil elastase inhibitor, ONO-5046, modulates acid-induced lung and systemic injury in rabbits.

PubMed

Kaneko, K; Kudoh, I; Hattori, S; Yamada, H; Ohara, M; Wiener-Kronish, J; Okumura, F

1997-09-01

Acid instillation leads to direct lung and to secondary systemic organ injury, probably via activated macrophages and neutrophils. This study investigated the effects of neutrophil elastase on organ injury after unilateral lung acid instillation by administrating a specific neutrophil elastase inhibitor, ONO-5046, before acid instillation. Three groups of anesthetized rabbits (n = 12 in each group) underwent tracheostomies, and instillations were made into their right lower lobe airspaces with either phosphate buffered saline (pH, 7.4; volume, 1.2 ml/kg; n = 12) or HCl (pH, 1.25; volume, 1.2 ml/kg; n = 24). In half of the acid-instilled rabbits, ONO-5046, 10 mg/kg, was given intravenously 15 min before the HCl instillation, and then 10 mg x kg(-1) x h(-1) of the drug was continuously infused throughout the experiment. The other groups of animals received the vehicle intravenously. Anesthesia and mechanical ventilation was continued for 8 h, whereas arterial blood gases were sampled intermittently. Eight hours after saline or acid instillation, the animals were killed, and their lungs, heart, kidneys, liver, and small intestines were harvested. Wet-to-dry weight ratios (W/ D) and myeloperoxidase (MPO) assays of these organs were done, and elastase assays on the bronchoalveolar lavage fluids (BALF) obtained from each lung also were performed. Pretreatment with ONO-5046 attenuated the physiologic changes seen in the vehicle-treated animals. Significant decreases in W/D of the noninstilled lungs and of the small intestine and normalization of the oxygenation of the experimental animals occurred. The ONO-5046 pretreatment did not affect the neutrophil sequestration in the lungs or in the other organs as determined by neutrophil counts in BALF and by the MPO assays. A neutrophil elastase inhibitor, ONO-5046, administered immediately before acid instillation attenuated the physiologic changes seen in the vehicle-treated animals. The drug blocked neutrophil elastase but did not block neutrophil sequestration in the lungs, although the drug improved measurements of lung injury.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

NASA Astrophysics Data System (ADS)

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-07-01

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT.

PubMed

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-07-16

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

PubMed Central

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-01-01

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway. PMID:26177797

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed Central

Llewellyn-Jones, C. G.; Lomas, D. A.; Stockley, R. A.

1994-01-01

BACKGROUND--Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS--The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS--When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS--It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage. Images PMID:7912452

Effect of urinary trypsin inhibitor on preterm labor with high granulocyte elastase concentration in cervical secretions.

PubMed

Hayashi, Masako; Oya, Atsuko; Miyake, Hidehiko; Nakai, Akihito; Takeshita, Toshiyuki

2010-04-01

To explore whether intravaginal treatment with urinary trypsin inhibitor (UTI) prevents preterm delivery in patients in preterm labor with increased levels of granulocyte elastase in cervical secretions. The subjects were patients in preterm labor with increased levels of granulocyte elastase in cervical secretions from 16 to 33 weeks gestation. Maternal and neonatal outcomes were compared between patients receiving UTI treatment (UTI group; n=33) and those not receiving UTI treatment (control group; n=40). In patients receiving UTI, the mean gestational age at delivery was greater than that in the control group (37.8 vs. 35.6 weeks, p=0.003), and the rates of premature delivery before 34 and 37 weeks gestation were lower (3% vs. 20%, p=0.028; and 18% vs. 47%, p=0.008, respectively). The percentage of neonates weighing more than 2,500 g was significantly higher in the UTI group, with no neonates weighing less than 1,500 g. The neonatal hospitalization rate was lower in the UTI group (9% vs. 42%, p=0.001). In patients in preterm labor with a high elastase concentration in cervical secretions, treatment with UTI reduced the risk of preterm delivery and improved neonatal outcomes.

Pulmonary deposition and disappearance of aerosolised secretory leucocyte protease inhibitor.

PubMed Central

Stolk, J.; Camps, J.; Feitsma, H. I.; Hermans, J.; Dijkman, J. H.; Pauwels, E. K.

1995-01-01

BACKGROUND--The neutrophil elastase inhibitor, secretory leucocyte protease inhibitor (SLPI), is a potential therapeutic tool in inflammatory lung diseases such as cystic fibrosis and pulmonary emphysema. The distribution and disappearance in the lung of aerosolised recombinant SLPI (rSLPI) was investigated in healthy humans and in patients with cystic fibrosis or alpha 1-antitrypsin-associated emphysema. METHODS--To distinguish aerosolised rSLPI from endogenous SLPI the recombinant inhibitor was radiolabelled with 99m-technetium (99mTc) pertechnetate. Distribution and disappearance of aerosolised 99mTc-rSLPI in the lungs were studied by gamma radiation imaging. RESULTS--The deposition of 99mTc-rSLPI in normal volunteers was homogeneous in all lung lobes, while in patients with cystic fibrosis or emphysema only well ventilated areas showed deposition of the aerosol. The disappearance rate of 99mTc-rSLPI was biexponential. The half life of the rapid phase was 0.2-2.8 hours, while that of the slow phase was more than 24 hours. CONCLUSIONS--Future aerosol therapy with rSLPI will be most beneficial for well ventilated lung tissue that needs protection against neutrophil derived elastase. It may be more difficult to neutralise the burden of elastase in poorly ventilated, highly inflamed areas as are seen in cystic fibrosis. Images PMID:7638807

Chemically modified tetracycline-3 (CMT-3): a novel inhibitor of the serine proteinase, elastase.

PubMed

Gu, Ying; Lee, Hsi-Ming; Simon, Sanford R; Golub, Lorne M

2011-12-01

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α(1)-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.

PubMed

Garratt, Luke W; Sutanto, Erika N; Ling, Kak-Ming; Looi, Kevin; Iosifidis, Thomas; Martinovich, Kelly M; Shaw, Nicole C; Kicic-Starcevich, Elizabeth; Knight, Darryl A; Ranganathan, Sarath; Stick, Stephen M; Kicic, Anthony

2015-08-01

Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease. Copyright ©ERS 2015.

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis.

PubMed

Viskupicova, Jana; Danihelova, Martina; Majekova, Magdalena; Liptaj, Tibor; Sturdik, Ernest

2012-12-01

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e-m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a-d, 3a-d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

PubMed

Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

2012-03-01

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Role of neutrophilic elastase in ethanol induced injury to the gastric mucosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kvietys, P.R.; Carter, P.R.

1990-02-26

Intragastric administration of ethanol (at concentrations likely to be encountered by the mucosa during acute intoxication) produces gastritis. Recent studies have implicated neutrophils in the gastric mucosal injury induced by luminal ethanol. The objective of the present study was to assess whether neutrophilic elastase contributes to the ethanol-induced gastric mucosal injury. Sprague-Dawley rats were instrumented for perfusion of the gastric lumen with saline or ethanol. Mucosal injury was quantitated by continuously measuring the blood-to-lumen clearance of {sup 51}Cr-EDTA. The experimental protocol consisted of a 40 minute control period (saline perfusion) followed by three successive 40 minute experimental periods (ethanol perfusion).more » During the three experimental periods the concentration of ethanol was progressively increased to 10, 20, and 30%. The experiments were performed in untreated animals and in animals pretreated with either Eglin c (an inhibitor of elastase and cathepsin G activity) or L 658 (a specific inhibitor of elastase activity). The effects of ethanol on EDTA clearance (x control) in untreated (n = 9) and L658 treated (n = 5) animals are shown in the Table below. Pretreatment with L 658 significantly attenuated the ethanol-induced increases in EDTA clearance. Pretreatment with Eglin c (n = 6) also provided some protection against ethanol-induced injury, but not to the extent as that provided by L658. The results of the authors studies suggest that neutrophilic elastase contributes to a gastric mucosal injury induced by luminal perfusion of the stomach with physiologically relevant concentrations of ethanol.« less

Application of chemical arrays in screening elastase inhibitors.

PubMed

Gao, Feng; Du, Guan-Hua

2006-06-01

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).

Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L.

PubMed

Hernández González, Jorge Enrique; García-Fernández, Rossana; Valiente, Pedro Alberto

2015-01-01

The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, Ki = 2.35·10-8 M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (Ki = 1.3·10-9 M, and Ki = 1.2·10-8 M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor's P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.

Coagulofibrinolytic changes in patients with disseminated intravascular coagulation associated with post-cardiac arrest syndrome--fibrinolytic shutdown and insufficient activation of fibrinolysis lead to organ dysfunction.

PubMed

Wada, Takeshi; Gando, Satoshi; Mizugaki, Asumi; Yanagida, Yuichiro; Jesmin, Subrina; Yokota, Hiroyuki; Ieko, Masahiro

2013-07-01

Post-cardiac arrest syndrome (PCAS) is often associated with disseminated intravascular coagulation (DIC), thus leading to the development of multiple organ dysfunction syndrome (MODS). The aim of this study was to examine the pathophysiological relationships between coagulation, fibrinolysis and fibrinolytic shutdown by evaluating the levels of coagulofibrinolytic markers, including soluble fibrin, thrombin-activatable fibrinolysis inhibitor (TAFI), tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPAIC), plasmin-alpha2 plasmin inhibitor complex (PPIC), neutrophil elastase and fibrin degradation product by neutrophil elastase (EXDP). Fifty-two resuscitated patients were divided into two groups: 22 DIC and 30 non-DIC patients. The levels of soluble fibrin, PPIC, tPAIC, EXDP and neutrophil elastase in the DIC patients with PCAS were significantly higher than those observed in the non-DIC patients. The values of the tPAIC and JAAM DIC scores were found to be independent predictors of increased SOFA scores in the DIC patients. The MODS patients demonstrated significantly higher levels of soluble fibrin and tPAIC; however, the levels of TAFI and EXDP were identical between the patients with and without MODS. In addition, positive correlations were observed between the levels of tPAIC and EXDP in the patients with non-MODS; however, no correlations were observed between these markers in the MODS patients. Thrombin activation and fibrinolytic shutdown play important roles in the development of organ dysfunction in PCAS patients. Neutrophil elastase-mediated fibrinolysis cannot overcome the fibrinolytic shutdown that occurs in DIC patients with PCAS, thus resulting in the development of MODS. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack

PubMed Central

2018-01-01

ABSTRACT Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. PMID:29636440

Inactivation of Human Neutrophil Elastase by 1, 2, 5 – Thiadiazolidin-3-one 1, 1 Dioxide-based Sulfonamides

PubMed Central

Li, Yi; Yang, Qingliang; Dou, Dengfeng; Alliston, Kevin R.; Groutas, William C.

2008-01-01

The interaction of a series of 1, 2, 5 –thiadiazolidin-3-one 1, 1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S′ subsites, had a profound effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elastase (HNE) and was devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results of these studies demonstrate that exploitation of differences in the S′ subsites of HNE and PR 3 can lead to highly selective inhibitors of HNE. PMID:17976994

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

PubMed

Spinosa, Michael; Lu, Guanyi; Su, Gang; Bontha, Sai Vineela; Gehrau, Ricardo; Salmon, Morgan D; Smith, Joseph R; Weiss, Mark L; Mas, Valeria R; Upchurch, Gilbert R; Sharma, Ashish K

2018-05-29

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b + macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema.

PubMed

Doucet, Alain; Bouchard, Dominique; Janelle, Marie France; Bellemare, Audrey; Gagné, Stéphane; Tremblay, Guy M; Bourbonnais, Yves

2007-08-01

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1' methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1' subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

Peptide inhibitor of CXCL4-CCL5 heterodimer formation, MKEY, inhibits experimental aortic aneurysm initiation and progression.

PubMed

Iida, Yasunori; Xu, Baohui; Xuan, Haojun; Glover, Keith J; Tanaka, Hiroki; Hu, Xiaolei; Fujimura, Naoki; Wang, Wei; Schultz, Joshua R; Turner, Court R; Dalman, Ronald L

2013-04-01

Macrophages are critical contributors to abdominal aortic aneurysm (AAA) disease. We examined the ability of MKEY, a peptide inhibitor of CXCL4-CCL5 interaction, to influence AAA progression in murine models. AAAs were created in 10-week-old male C57BL/6J mice by transient infrarenal aortic porcine pancreatic elastase infusion. Mice were treated with MKEY via intravenous injection either (1) before porcine pancreatic elastase infusion or (2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited migration of adaptively transferred leukocytes into aneurysmal aortae in recipient mice. Although all vehicle-pretreated mice developed AAAs, aneurysms formed in only 60% (3/5) and 14% (1/7) of mice pretreated with MKEY at 10 and 20 mg/kg, respectively. MKEY pretreatment reduced aortic diameter enlargement, preserved medial elastin fibers and smooth muscle cells, and attenuated mural macrophage infiltration, angiogenesis, and aortic metalloproteinase 2 and 9 expression after porcine pancreatic elastase infusion. MKEY initiated after porcine pancreatic elastase infusion also stabilized or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation after angiotensin II infusion in apolipoprotein E-deficient mice. MKEY suppresses AAA formation and progression in 2 complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.

Mechanism for the Increased Permeability in Endothelial Monolayers Induced by Elastase

PubMed Central

Ishii, Y.; Kitamura, S.

1994-01-01

The aim of this study was to investigate the mechanism for the increase in endothelial permeability induced by human neutrophil elastase (HNE). Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with HNE(0-30 μg/ml) for 1 h produced a concentration dependent increase in 125I-albumin clearance. The effect was reversible and was not due to cytolysis. Pretreatment of BPAEC with sodium tungstate, which depletes xanthine oxidase, or with oxypurinol, did not prevent HNE induced increased permeability. Heparin, which neutralizes the cationic charge of HNE, also had no protective effect. Pretreatment with heat inactivated HNE, which still had positive charge sites, did not result in increased endothelial permeability. Also, ONO-5046, a novel specific inhibitor of HNE, did prevent increased permeability. These results suggest that elastase increases endothelial permeability mainly through its proteolytic effects. PMID:18472917

Solar ultraviolet irradiation induces decorin degradation in human skin likely via neutrophil elastase.

PubMed

Li, Yong; Xia, Wei; Liu, Ying; Remmer, Henriette A; Voorhees, John; Fisher, Gary J

2013-01-01

Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.

Neutral endopeptidase determines the severity of pancreatitis-associated lung injury.

PubMed

Day, Amy Lightner; Wick, Elizabeth; Jordan, Thomas H; Jaffray, Colleen E; Bunnett, Nigel W; Grady, Eileen F; Kirkwood, Kimberly S

2005-09-01

Neutral endopeptidase (NEP) is a cell-surface metalloprotease that degrades proinflammatory peptides such as substance P, neurokinin A, and bradykinin. Inhibition of NEP exacerbates both experimental pancreatitis and the associated lung injury. It is unclear if worsened lung injury is the indirect result of more severe pancreatitis or if it is a direct effect of NEP inhibition in the lung. We used a model of pancreatitis-associated lung injury (PALI) to test the hypothesis that antagonism or genetic deletion of NEP augments PALI inflammation and pulmonary damage irregardless of the degree of pancreatitic inflammation. In NEP(+/+) mice, intraperitoneal injection of porcine pancreatic elastase (elastase, 0.085 U/g at t = 0 h and t = 1 h) caused a 7-fold increase in lung myeloperoxidase (MPO) activity and marked pulmonary edema, neutrophil infiltration, and hemorrhage at 4 h as compared to control animals. The pattern of lung injury induced by elastase mimicked that observed among a separate group of animals with PALI induced by cerulein but was not associated with pancreatitis. Both NEP(-/-) mice and NEP(+/+) mice pretreated with the NEP antagonist phosphoramidon (10 mg/kg s.c.) had significant elevations of lung MPO and worsened lung histology compared to NEP(+/+) mice given elastase alone. Antagonism of either the vanilloid receptor transient receptor vanilloid 1 or the substance P receptor NK1-R had no effect on elastase-mediated lung injury in NEP-deficient mice. NEP is an inhibitor of pancreatic elastase-induced lung injury, presumably via degradation of proinflammatory mediators.

Neutrophil elastase-mediated increase in airway temperature during inflammation.

PubMed

Schmidt, Annika; Belaaouaj, Azzaq; Bissinger, Rosi; Koller, Garrit; Malleret, Laurette; D'Orazio, Ciro; Facchinelli, Martino; Schulte-Hubbert, Bernhard; Molinaro, Antonio; Holst, Otto; Hammermann, Jutta; Schniederjans, Monika; Meyer, Keith C; Damkiaer, Soeren; Piacentini, Giorgio; Assael, Baroukh; Bruce, Kenneth; Häußler, Susanne; LiPuma, John J; Seelig, Joachim; Worlitzsch, Dieter; Döring, Gerd

2014-12-01

How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation.

PubMed

Edgington-Mitchell, Laura E; Barlow, Nicholas; Aurelio, Luigi; Samha, Aminath; Szabo, Monika; Graham, Bim; Bunnett, Nigel

2017-01-15

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Roles of oxygen radicals and elastase in citric acid-induced airway constriction of guinea-pigs

PubMed Central

Lai, Y -L; Chiou, W -Y; Lu, F J; Chiang, L Y

1999-01-01

Antioxidants attenuate noncholinergic airway constriction. To further investigate the relationship between tachykinin-mediated airway constriction and oxygen radicals, we explored citric acid-induced bronchial constriction in 48 young Hartley strain guinea-pigs, divided into six groups: control; citric acid; hexa(sulphobutyl)fullerenes+citric acid; hexa(sulphobutyl)fullerenes+phosphoramidon+citric acid; dimethylthiourea (DMTU)+citric acid; and DMTU+phosphoramidon+citric acid. Hexa(sulphobutyl)fullerenes and DMTU are scavengers of oxygen radicals while phosphoramidon is an inhibitor of the major degradation enzyme for tachykinins. Animals were anaesthetized, paralyzed, and artificially ventilated. Each animal was given 50 breaths of 4 ml saline or citric acid aerosol. We measured dynamic respiratory compliance (Crs), forced expiratory volume in 0.1 (FEV0.1), and maximal expiratory flow at 30% total lung capacity (V[dot above]max30) to evaluate the degree of airway constriction. Citric acid, but not saline, aerosol inhalation caused marked decreases in Crs, FEV0.1 and V[dot above]max30, indicating marked airway constriction. This constriction was significantly attenuated by either hexa(sulphobutyl)fullerenes or by DMTU. In addition, phosphoramidon significantly reversed the attenuating action of hexa(sulphobutyl)fullerenes, but not that of DMTU. Citric acid aerosol inhalation caused increases in both lucigenin- and t-butyl hydroperoxide-initiated chemiluminescence counts, indicating citric acid-induced increase in oxygen radicals and decrease in antioxidants in bronchoalveolar lavage fluid. These alterations were significantly suppressed by either hexa(sulphobutyl)fullerenes or DMTU. An elastase inhibitor eglin-c also significantly attenuated citric acid-induced airway constriction, indicating the contributing role of elastase in this type of constriction. We conclude that both oxygen radicals and elastase play an important role in tachykinin-mediated, citric acid-induced airway constriction. PMID:10188991

Antioxidant Vitamin C attenuates experimental abdominal aortic aneurysm development in an elastase-induced rat model.

PubMed

Shang, Tao; Liu, Zhao; Liu, Chang-jian

2014-05-01

We investigated the hypothesis that an antioxidant, Vitamin C, could attenuate abdominal aortic aneurysm (AAA) development in a rat model. An AAA model induced by intraluminal infusion was created in 36 male Sprague Dawley rats, which were randomly distributed into three groups: Sham (saline infused, placebo treated), Control (elastase infused, placebo treated), and Vitamin C (elastase infused, vitamin C treated). Vitamin C and placebo were intraperitoneally injected, initiating 1 wk before the infusion and continuing throughout the study. The aortic dilatation ratio was measured, and aortic tissues were further examined using biochemical and histologic techniques. Vitamin C attenuated the development of AAA, decreasing maximal aortic diameter by 25.8% (P < 0.05) and preserving elastin lamellae (P < 0.05). Vitamin C also decreased 8-hydroxyguanine (a marker of oxidative damage to DNA) and 8-isoprostane content (a marker of oxidative stress) in aortic tissues (P < 0.05, respectively). The proteins of matrix metalloproteinase (MMP)-2, MMP-9, and interleukin 6 were markedly downregulated (P < 0.05, respectively), accompanied with notably reduced messenger RNA expression of tumor necrosis factor-α, MMP-2/9, and interleukin 1β (P < 0.05, respectively). However, messenger RNA of tissue inhibitors of metalloproteinase-1 and tissue inhibitors of metalloproteinase-2 were both significantly upregulated in Vitamin C group. Vitamin C treatment had no significant effect on systolic blood pressure (P > 0.05). Vitamin C attenuated AAA development in an elastase-induced rat model via crucial protective effect, which was mediated by an increased level of antioxidant in cooperation with preserving elastin lamellae, inhibiting matrix-degrading proteinases and suppressing inflammatory responses. Copyright © 2014. Published by Elsevier Inc.

Protease Inhibition by Oleic Acid Transfer From Chronic Wound Dressings to Albumin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, J. V.; Howley, Phyllis; Davis, Rachel M.

High elastase and cathepsin G activities have been observed in chronic wounds. These levels can inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid (18:1) is a non-toxic elastase inhibitor with some potential for redressing the imbalance of elastase activity found in chronic wounds. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of 18:1 by albumin under conditions mimicking chronic wounds. 18:1-treated cotton was examined for its ability to bind and release the fatty acid in the presence of albumin. The mechanism of 18:1 uptake from cotton and binding bymore » albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin-18:1 complexes under liquid-liquid equilibrium conditions revealed fully saturated albumin-18:1 complexes with a 1:1 weight ratio of albumin:18:1. Cotton chromatography under liquid-solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of 18:1 per mole albumin. Cotton was contrasted with hydrogel, and hydrocolloid wound dressing for its comparative ability to lower elastase activity. Each dressing material evaluated was found to release 18:1 in the presence of albumin with significant inhibition of elastase activity. The 18:1-formulated wound dressings lowered elastase activity in a dose dependent manner in the order cotton gauze > hydrogel > hydrocolloid. In contrast the cationic serine protease Cathepsin G was inihibited by 18:1 within a narrow range of 18:1-cotton formulations. Four per cent Albumin solutions were most effective in binding cotton bound-18:1. However, 2% albumin was sufficient to transfer quantities of 18:1 necessary to achieve a significant elastase-lowering effect. Formulations with 128 mg 18:1/g cotton gauze had equivalent elastase lowering with 1 - 4% albumin. 18:1 bound to cotton wound dressings may have promise in the selective lowering of cationic serine protease activity useful in topical application for chronic inflammatory pathogenesis.« less

Screening for anti-inflammatory activity of 12 Arnica (Asteraceae) species assessed by inhibition of NF-kappaB and release of human neutrophil elastase.

PubMed

Ekenäs, Catarina; Zebrowska, Anna; Schuler, Barbara; Vrede, Tobias; Andreasen, Katarina; Backlund, Anders; Merfort, Irmgard; Bohlin, Lars

2008-12-01

Several species in the genus Arnica have been used in traditional medicine to treat inflammatory-related disorders. Extracts of twelve Arnica species and two species closely related to arnica ( Layia hieracioides and Madia sativa) were investigated for inhibition of human neutrophil elastase release and inhibition of transcription factor NF-kappaB. Statistical analyses reveal significant differences in inhibitory capacities between extracts. Sesquiterpene lactones of the helenanolide type, of which some are known inhibitors of human neutrophil elastase release and NF-kappaB, are present in large amounts in the very active extracts of A. montana and A. chamissonis. Furthermore, A. longifolia, which has previously not been investigated, shows a high activity similar to that of A. montana and A. chamissonis in both bioassays. Sesquiterpene lactones of the xanthalongin type are present in large amounts in A. longifolia and other active extracts and would be interesting to evaluate further. COX-2:cyclooxygenase 2 EMSA:electrophoretic mobility shift assay fMLP: N-formyl-methionyl-leucyl-phenylalanine HaCaT:human keratinocyte HNE:human neutrophil elastase IkappaB:inhibitory subunit of kappaB iNOS:inducible nitric oxide synthase NF-kappaB:nuclear factor kappaB PAF:platelet activating factor STL:sesquiterpene lactone TNF-alpha:tumor necrosis factor alpha.

Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging I: Reduced Skin Elasticity, Highly Associated with Enhanced Dermal Elastase Activity, Triggers Wrinkling and Sagging

PubMed Central

Imokawa, Genji; Ishida, Koichi

2015-01-01

The repetitive exposure of skin to ultraviolet B (UVB) preferentially elicits wrinkling while ultraviolet A (UVA) predominantly elicits sagging. In chronically UVB or UVA-exposed rat skin there is a similar tortuous deformation of elastic fibers together with decreased skin elasticity, whose magnitudes are greater in UVB-exposed skin than in UVA-exposed skin. Comparison of skin elasticity with the activity of matrix metalloproteinases (MMPs) in the dermis of ovariectomized rats after UVB or UVA irradiation demonstrates that skin elasticity is more significantly decreased in ovariectomized rats than in sham-operated rats, which is accompanied by a reciprocal increase in elastase activity but not in the activities of collagenases I or IV. Clinical studies using animal skin and human facial skin demonstrated that topical treatment with a specific inhibitor or an inhibitory extract of skin fibroblast-derived elastase distinctly attenuates UVB and sunlight-induced formation of wrinkling. Our results strongly indicated that the upregulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. PMID:25856675

Endostatin expression in a pancreatic cell line is modulated by a TNFα-dependent elastase

PubMed Central

Brammer, R D; Bramhall, S R; Eggo, M C

2005-01-01

Endostatin, an inhibitor of angiogenesis, is a 20 kDa fragment of the basement membrane protein, collagen XVIII. The formation of endostatin relies upon the action of proteases on collagen XVIII. TNFα, produced by activated macrophages, is a multifunctional proinflammatory cytokine with known effects on endothelial function. We postulated that TNFα may modulate the activities of proteases and thus regulate endostatin formation in pancreatic cells. Collagen XVIII/endostatin mRNA was expressed in one pancreatic cell line, SUIT-2, but not in BxPc-3. The 20 kDa endostatin was found in the cell-conditioned medium of SUIT-2 cells. Precursor forms only were found in the cells. Exogenous endostatin was degraded by cellular lysates of SUIT-2 cells. Elastase activity was found in cell extracts but not the cell-conditioned media of SUIT-2 cells. Incubation of SUIT-2 cells with TNFα increased intracellular elastase activity and also increased secretion of endostatin into the medium. We conclude that endostatin is released by SUIT-2 cells and that increases in intracellular elastase, induced by TNFα, are correlated with increased secretion. Endostatin is however susceptible to degradation by intracellular proteases and if tissue injury accompanies inflammation, endostatin may be degraded, allowing angiogenesis to occur. PMID:16234817

Activation of proteinase 3 contributes to Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance.

PubMed

Toonen, Erik J M; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine T N; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B

2016-05-24

Activation of inflammatory pathways is known to accompany development of obesity-induced non-alcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive pro-inflammatory mediators IL-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In the present study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human alpha-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3 deficient mice showed strongly reduced levels of lipids in the liver after fed a high fat diet. Moreover, these mice were resistant to high fat diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1(-/-) mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with alpha-1 antitrypsin during the last 10 days of a 16 week high fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential.

EI-2128-1, a novel interleukin-1beta converting enzyme inhibitor produced by Penicillium sp. E-2128.

PubMed

Koizumi, Fumito; Agatsuma, Tsutomu; Ando, Katsuhiko; Kondo, Hidemasa; Saitoh, Yutaka; Matsuda, Yuzuru; Nakanishi, Satoshi

2003-11-01

EI-2128-1, a novel interleukin-1beta converting enzyme (ICE) inhibitor, was isolated from the culture broths of Penicillium sp. E-2128. EI-2128-1 selectively inhibited human recombinant ICE activity with IC50 value of 0.59 microM, without inhibiting elastase and cathepsin B. EI-2128-1 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 value of 0.28 microM.

Freezing the Bioactive Conformation to Boost Potency: The Identification of BAY 85-8501, a Selective and Potent Inhibitor of Human Neutrophil Elastase for Pulmonary Diseases

PubMed Central

von Nussbaum, Franz; Li, Volkhart M-J; Allerheiligen, Swen; Anlauf, Sonja; Bärfacker, Lars; Bechem, Martin; Delbeck, Martina; Fitzgerald, Mary F; Gerisch, Michael; Gielen-Haertwig, Heike; Haning, Helmut; Karthaus, Dagmar; Lang, Dieter; Lustig, Klemens; Meibom, Daniel; Mittendorf, Joachim; Rosentreter, Ulrich; Schäfer, Martina; Schäfer, Stefan; Schamberger, Jens; Telan, Leila A; Tersteegen, Adrian

2015-01-01

Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNE inhibitors should reestablish the protease–anti-protease balance. By means of medicinal chemistry a novel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yielded orally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity, picomolar potency was achieved by locking the bioactive conformation of these inhibitors with a strategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY 85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in a rodent animal model related to ALI. BAY 85-8501 is currently being tested in clinical studies for the treatment of pulmonary diseases. PMID:26083237

Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI) expression.

PubMed

Wex, Thomas; Kuester, Doerthe; Schönberg, Cornelius; Schindele, Daniel; Treiber, Gerhard; Malfertheiner, Peter

2011-05-26

Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P < 0.05). Overall, no correlations between mucosal Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI) expression

PubMed Central

2011-01-01

Background Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. Methods The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. Results H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P < 0.05). Overall, no correlations between mucosal Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Conclusions Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis. PMID:21612671

Suppression of receptor-mediated Ca2+ mobilization and functional leukocyte responses by hyperforin.

PubMed

Feisst, Christian; Werz, Oliver

2004-04-15

We have recently identified hyperforin, a lipophilic constituent of the herb Hypericum perforatum (St. John's wort), as a dual inhibitor of the proinflammatory enzymes cyclooxygenase-1 and 5-lipoxygenase. The aim of the present study was to further elucidate antiinflammatory properties and respective targets of hyperforin. We found that hyperforin inhibited the generation of reactive oxygen species (ROS) as well as the release of leukocyte elastase (degranulation) in human isolated polymorphonuclear leukocytes (PMNL), challenged by the G protein-coupled receptor (GPCR) ligand N-formyl-methionyl-leucyl-phenylalanine (fMLP) with an IC 50 approximately equal 0.3 microM. When PMNL were stimulated with phorbol-12-myristate-13-acetate (PMA) or ionomycin, hyperforin (up to 10 microM) failed to inhibit ROS production and elastase release, respectively. Moreover, hyperforin blocked receptor-mediated Ca(2+) mobilization ( IC 50 approximately equal 0.4 and 4 microM, respectively) in PMNL and monocytic cells, and caused a rapid decline of the intracellular Ca(2+) concentration in resting cells. In contrast, the Ca(2+) influx induced by ionomycin or thapsigargin was not suppressed. Comparative studies with the specific phospholipase C inhibitor U-73122 and hyperforin revealed similarities between both compounds. Thus, U-73122 and hyperforin blocked fMLP- and PAF-induced Ca(2+) mobilization, ROS formation, and elastase release, but failed to suppress these responses when cells were stimulated by PMA or ionomycin. Also, both compounds rapidly decreased basal Ca(2+) levels in resting cells and led to a rapid decline of the Ca(2+) elevations evoked by fMLP or PAF. Our data suggest that hyperforin targets component(s) within G protein signaling cascades that regulate Ca(2+) homeostasis, coupled to proinflammatory leukocyte functions.

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure.

PubMed

Lang, R; Kocourek, A; Braun, M; Tschesche, H; Huber, R; Bode, W; Maskos, K

2001-09-28

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP. Copyright 2001 Academic Press.

Alpha 1-protease inhibitor moderates human neutrophil elastase-induced emphysema and secretory cell metaplasia in hamsters.

PubMed

Stone, P J; Lucey, E C; Virca, G D; Christensen, T G; Breuer, R; Snider, G L

1990-06-01

A study was undertaken to determine whether emphysema and airway secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be moderated by pretreatment with human alpha 1-protease inhibitor (API). API (4.9 mg) was given intratracheally to hamsters 1 h before 0.3 mg HNE. Eight weeks later, lung volumes and pressure-volume relationships were measured in the anaesthetized animals. Mean linear intercepts and secretory cell indices were measured in lung sections. API given 1 h before HNE moderated the development of bronchial secretory cell metaplasia. The severity of emphysema was reduced by 75%. Clearance studies indicated that 80% of the functional activity of instilled API could be lavaged from the lungs after 1 h, indicating a 4 h half-life in the lavageable compartment of the lungs. We calculate that for 50% protection from emphysema the molar ratio of lavageable API to HNE at the time of HNE instillation was 4.8 as compared with 0.78 for 50% inhibition of elastolytic activity in vitro, indicating that API is only 16% as efficient in vivo as compared with its in vitro HNE inhibitory effectiveness. Nevertheless, we conclude that human API given intratracheally is efficacious against HNE-induced emphysema and secretory cell metaplasia.

Increased galectin-3 levels are associated with abdominal aortic aneurysm progression and inhibition of galectin-3 decreases elastase-induced AAA development.

PubMed

Fernandez-García, Carlos-Ernesto; Tarin, Carlos; Roldan-Montero, Raquel; Martinez-Lopez, Diego; Torres-Fonseca, Monica; Lindhot, Jes S; Vega de Ceniga, Melina; Egido, Jesus; Lopez-Andres, Natalia; Blanco-Colio, Luis-Miguel; Martín-Ventura, Jose-Luis

2017-11-15

Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients ( n =225) compared with the control group ( n =100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

PubMed

Sanda, A; Uchida, A; Itagaki, T; Kobayashi, H; Inokuchi, N; Koyama, T; Iwama, M; Ohgi, K; Irie, M

2001-12-01

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

PubMed

Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

2018-04-10

Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix-interacting protein. Copyright © 2018 Tseng et al.

Ilex kaushue and Its Bioactive Component 3,5-Dicaffeoylquinic Acid Protected Mice from Lipopolysaccharide-Induced Acute Lung Injury

PubMed Central

Chen, Yu-Li; Hwang, Tsong-Long; Yu, Huang-Ping; Fang, Jia-You; Chong, Kowit Yu; Chang, Yao-Wen; Chen, Chun-Yu; Yang, Hsuan-Wu; Chang, Wen-Yi; Hsieh, Pei-Wen

2016-01-01

Acute lung injury (ALI) is a severe respiratory disease with high mortality rates worldwide. Recent reports suggest that human neutrophil elastase (HNE) plays a key role in the inflammatory response that is characteristic of ALI, which indicates that the development of HNE inhibitors could be an efficient treatment strategy. In the current study, an enzyme-based screening assay was used to identify effective HNE inhibitors from a number of traditional Chinese medicines (TCMs). Among them, a water extract of Ilex kaushue (IKWE) effectively inhibited HNE activity (IC50, 11.37 ± 1.59 μg/mL). Using bioactivity-guided fractionation, one new compound and 23 known compounds were identified. Compound 6 (identified as 3,5-dicaffeoylquinic acid; 3,5-DCQA) exerted the most potent and selective inhibitory effect on HNE activity (IC50, 1.86 ± 0.06 μM). In a cell-based assay, 3,5-DCQA not only directly reduced superoxide generation and elastase activity but also attenuated the Src family kinase (SRKs)/Vav signaling pathway in N-formyl-L-Met-L-Leu-L-Phe (fMLF)-stimulated human neutrophils. In an animal disease model, both 3,5-DCQA and standardized IKWE protected against lipopolysaccharide-induced ALI in mice, which provides support for their potential as candidates in the development of new therapeutic agents for neutrophilic inflammatory diseases. PMID:27681838

Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors.

PubMed

Cathcart, George R; Gilmore, Brendan F; Greer, Brett; Harriott, Pat; Walker, Brian

2009-11-01

We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide containing dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC 3.4.24.26). The aim of the study was to derive an inhibitor profile for LasB with regard to mapping the S'1 binding site of the enzyme. Consequently, a focussed library of 160 members has been synthesised, using standard Fmoc-solid phase methods (on a Rink-amide resin), in which a subset of amino acids including examples of those with basic (Lys, Arg), aromatic (Phe, Trp), large aliphatic (Val, Leu) and acidic (Asp, Glu) side-chains populated the P'2 position of the inhibitor sequence and all 20 natural amino acids were incorporated, in turn, at the P'1 position. The study has revealed a preference for aromatic and/or large aliphatic amino acids at P'1 and a distinct bias against acidic residues at P'2. Ten inhibitor sequences were discovered that exhibited sub to low micromolar Ki values.

Elastase kinetics and structural features of colorimetric and fluorescent peptides on cellulose nanocrystals

USDA-ARS?s Scientific Manuscript database

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases with destructive proteolytic activity. Because of this activity, there is considerable interest in elastase sensors. Herein we report the synthesis, characterization, and kinetic profiles of tri- and tetrapept...

Role of LTB4 in the pathogenesis of elastase-induced murine pulmonary emphysema

PubMed Central

Paige, Mikell; Hanna, Halim; Kim, Su H.; Burdick, Marie D.; Strieter, Robert M.

2010-01-01

Exaggerated levels of the leukotriene B4 (LTB4) frequently coexist at sites of inflammation and tissue remodeling. Therefore, we hypothesize that the LTB4 pathway plays an important role in the pathogenesis of neutrophilic inflammation that contributes to pulmonary emphysema. In this study, significant levels of LTB4 were detected in human lung tissues with emphysema compared with lungs without emphysema (9,497 ± 2,839 vs. 4,142 ± 1,173 pg/ml, n = 9 vs. 10, P = 0.04). To further determine the biological role of LTB4 in the pathogenesis of emphysema, we compared the lungs of wild-type (WT) and LTA4 hydrolase−/− mice (LTB4 deficient, LTA4H−/−) exposed to intranasal elastase or vehicle control. We found that intranasal elastase induced accumulation of LTB4 in the lungs and caused progressively worsening emphysema between 14 and 28 days after elastase exposure in WT mice but not in LTA4H−/− mice. Premortem physiology documented increased lung compliance in elastase-exposed WT mice compared with elastase-exposed LTA4H−/− mice as measured by Flexivent (0.058 ± 0.005 vs. 0.041 ± 0.002 ml/cmH2O pressure). Postmortem morphometry documented increased total lung volume and alveolar sizes in elastase-exposed WT mice compared with elastase-exposed LTA4H−/− mice as measured by volume displacement and alveolar chord length assessment. Furthermore, elastase-exposed LTA4H−/− mice were found to have significantly delayed influx of the CD45highCD11bhighLy6Ghigh leukocytes compatible with neutrophils compared with elastase-exposed WT mice. Mechanistic insights to these phenotypes were provided by demonstrating protection from elastase-induced murine emphysema with neutrophil depletion in the elastase-exposed WT mice and by demonstrating time-dependent modulation of cysteinyl leukotriene biosynthesis in the elastase-exposed LTA4H−/− mice compared with elastase-exposed WT mice. Together, these findings demonstrated that LTB4 played an important role in promoting the pathogenesis of pulmonary emphysema associated with neutrophilic pulmonary inflammation. PMID:20817777

Intravenous and intratracheal mesenchymal stromal cell injection in a mouse model of pulmonary emphysema.

PubMed

Tibboel, Jeroen; Keijzer, Richard; Reiss, Irwin; de Jongste, Johan C; Post, Martin

2014-06-01

The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

PubMed

Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

1999-05-01

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors

USDA-ARS?s Scientific Manuscript database

Both human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases that have been associated with destructive proteolytic activity when their levels are elevated in chronic diseases. Thus there is considerable interest in the development of elastase sensors. Nanocyrsta...

Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution.

PubMed

Edwards, J V; Yager, D R; Cohen, I K; Diegelmann, R F; Montante, S; Bertoniere, N; Bopp, A F

2001-01-01

Dressings for chronic human wounds have been aimed at protection, removal of exudate, and improved appearance. However since the time of ancient Greece wound care and dressing strategies have primarily relied on empiricism. Recent studies have shown that chronic wounds contain high levels of tissue and cytokine destroying proteases including collagenase and neutrophil elastase. Therefore we sought to develop an effective wound dressing that could absorb elastase through affinity sequestration. Cotton gauze was modified by oxidation, phosphorylation, and sulfonation to enhance elastase affinity by ionic or active site uptake. Type VII absorbent cotton gauze was oxidized to dialdehyde cotton which was subsequently converted in part to the bisulfite addition product. Gauze preparations were also phosphorylated and carboxymethylated. Modified cotton gauzes were compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were soaked in oxidized, sulfonated, and phosphorylated cotton gauze showed reduced elastase activity. The initial velocities (v(o)) and turnover rates of elastase showed significant decreases compared with solutions taken from untreated gauze. The reduction in enzyme activity with dialdehyde cotton gauze was confirmed in solution by determining elastase inhibition with dialdehyde starch. The dialdehyde cotton gauze also decreased elastase activity in human wound fluid in a dose response relation based on weight of gauze per volume of wound fluid. Absorbency, pH, air permeability and strength properties of the modified gauze were also compared with untreated cotton gauze. This report shows the effect of reducing elastase activity in solution with cotton containing aldehydic or negatively charged cellulose fibers that may be applicable to treatment modalities in chronic wounds.

Evaluation of fecal elastase and serum cholecystokinin in dogs with a false positive fecal elastase test.

PubMed

Steiner, J M; Rehfeld, J F; Pantchev, N

2010-01-01

An assay for the measurement of pancreatic elastase in dog feces has been introduced. The goal of this study was to evaluate the rate of false-positive fecal-elastase test results in dogs with suspected exocrine pancreatic insufficiency (EPI) and to assess serum cholecystokinin (CCK) concentrations in dogs with a false positive fecal elastase test result. Twenty-six fecal and serum samples from dogs suspected of EPI, for which samples had been submitted to a commercial laboratory (Vet Med Labor) for analysis. Prospective study. Serum trypsin-like immunoreactivity (TLI) was measured in 26 dogs with a decreased fecal elastase concentration of <10 microg/g feces. Serum CCK concentrations were measured in 21 of these dogs. Of 26 dogs with a decreased fecal elastase concentration, 6 (23%) had serum TLI concentrations within or above the reference range. Serum CCK concentrations were significantly higher in dogs with a true positive fecal elastase test result (median: 1.1 pmol/L; range: 0.1-3.3 pmol/L) than in those with a false positive fecal elastase test result (median: 0.1 pmol/L; range: 0.1-0.9 pmol/L; P value = .0163). The rate of false positive fecal elastase test results was high in this group of dogs, suggesting that diagnosis of EPI must be confirmed by other means. The decreased CCK concentration in dogs with a false positive fecal elastase test result could suggest that false positive results are because of decreased stimulation of exocrine pancreatic function caused by other conditions.

Anti-inflammatory effects of secondary metabolites of marine Pseudomonas sp. in human neutrophils are through inhibiting P38 MAPK, JNK, and calcium pathways.

PubMed

Yang, Shun-Chin; Sung, Ping-Jyun; Lin, Chwan-Fwu; Kuo, Jimmy; Chen, Chun-Yu; Hwang, Tsong-Long

2014-01-01

Activated neutrophils play a significant role in the pathogenesis of many inflammatory diseases. The metabolites of marine microorganisms are increasingly employed as sources for developing new drugs; however, very few marine drugs have been studied in human neutrophils. Herein, we showed that secondary metabolites of marine Pseudomonas sp. (N11) significantly inhibited superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils, with IC50 values of 0.67±0.38 µg/ml and 0.84±0.12 µg/ml, respectively. In cell-free systems, neither superoxide anion-scavenging effect nor inhibition of elastase activity was associated with the suppressive effects of N11. N11 inhibited the phosphorylation of p38 MAP kinase and JNK, but not Erk and Akt, in FMLP-induced human neutrophils. Also, N11 dose-dependently attenuated the transient elevation of intracellular calcium concentration in activated neutrophils. In contrast, N11 failed to alter phorbol myristate acetate-induced superoxide anion generation, and the inhibitory effects of N11 were not reversed by protein kinase A inhibitor. In conclusion, the anti-inflammatory effects of N11 on superoxide anion generation and elastase release in activated human neutrophils are through inhibiting p38 MAP kinase, JNK, and calcium pathways. Our results suggest that N11 has the potential to be developed to treat neutrophil-mediated inflammatory diseases.

All-trans retinoic acid results in irregular repair of septa and fails to inhibit proinflammatory macrophages.

PubMed

Seifart, C; Muyal, J P; Plagens, A; Yildirim, A Ö; Kohse, K; Grau, V; Sandu, S; Reinke, C; Tschernig, T; Vogelmeier, C; Fehrenbach, H

2011-08-01

All-trans retinoic acid (ATRA) is controversially discussed in emphysema therapy. We re-evaluated ATRA in the elastase model and hypothesised that beneficial effects should be reflected by increased alveolar surface area, elastin expression and downregulation of inflammatory mediators and matrix metalloproteinases (MMPs). Emphysema was induced by porcine pancreatic elastase versus saline in Sprague-Dawley rats. On days 26-37, rats received daily intraperitoneal injections with ATRA (500 μg · kg(-1) body weight) versus olive oil. Lungs were removed at day 38. Rat alveolar epithelial L2 cells were incubated with/without elastase followed by ATRA- or vehicle-treatment, respectively. ATRA only partially ameliorated structural defects. Alveolar walls exhibited irregular architecture: increased arithmetic mean thickness, reduction in surface coverage by alveolar epithelial cells type II. ATRA only partially restored reduced soluble elastin. It tended to increase the ratio of ED1(+):ED2(+) macrophages. Bronchoalveolar lavage (BAL) cells exhibited a proinflammatory state and high expression of interleukin-1β, cytokine-induced neutrophil chemoattractant-1, tumour necrosis factor-α, nuclear factor-κB, MMP-2, MMP-9, MMP-12, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in emphysema, with ATRA exerting only few effects. MMP-7 was highly induced by ATRA in healthy but not in emphysematous lungs. ATRA reduced both MMP-2 and TIMP-1 activity in BAL fluid of emphysematous lungs. ATRA-therapy may bear the risk of unwanted side-effects on alveolar septal architecture in emphysematous lungs.

High-density lipoproteins potentiate α1-antitrypsin therapy in elastase-induced pulmonary emphysema.

PubMed

Moreno, Juan-Antonio; Ortega-Gomez, Almudena; Rubio-Navarro, Alfonso; Louedec, Liliane; Ho-Tin-Noé, Benoit; Caligiuri, Giuseppina; Nicoletti, Antonino; Levoye, Angelique; Plantier, Laurent; Meilhac, Olivier

2014-10-01

Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1-3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 μm versus 30.7 ± 4.5 μm; P < 0.001), whereas injection of HDL or AAT alone only showed a moderate, nonsignificant protective effect (28.2 ± 4.2 μm versus 30.7 ± 5 μm [P = 0.23] and 27.3 ± 5.66 μm versus 30.71 ± 4.96 μm [P = 0.18], respectively). Indeed, protection by HDL-AAT was significantly higher than that observed with HDL or AAT (P = 0.006 and P = 0.048, respectively). This protective effect was associated (at 6, 24, and 72 h) with: (1) a reduction in neutrophil and macrophage number in the bronchoalveolar lavage fluid; (2) decreased concentrations of IL-6, monocyte chemoattractant protein-1, and TNF-α in both bronchoalveolar lavage fluid and plasma; (3) a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-9 activities; and (4) a reduction in the degradation of fibronectin, a marker of tissue damage. In addition, HDL-AAT reduced acute cigarette smoke-induced inflammatory response. Intravenous HDL-AAT treatment afforded a better protection against elastase-induced pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency.

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

PubMed Central

Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard

2018-01-01

Background & aims The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Method Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Results Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Conclusion Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS. PMID:29529042

Characterization of elastase-deficient clinical isolates of Pseudomonas aeruginosa.

PubMed Central

Hamood, A N; Griswold, J; Colmer, J

1996-01-01

Elastase production in Pseudomonas aeruginosa is regulated by the lasR, lasI, rhlR, and rhlI genes. Recently, we have analyzed several clinical isolates of P. aeruginosa for the production of elastase and other extracellular virulence factors. Four of these isolates (CIT1, CIW5, CIW7, and CIW8) produced no elastolytic activity. We have characterized these isolates with respect to their elastase-deficient phenotype. Elastase was detected by immunoblotting experiments using elastase-specific antiserum. We also determined the presence of IasB and IasR mRNAs by Northern (RNA) blot hybridization experiments using lasB and lasR internal probes, respectively. None of the four elastase-deficient strains produced either the elastase protein or the lasB mRNA. Complementation experiments (using plasmids carrying either the lasB or the lasR gene) were conducted to determine if the isolates carry defective lasB or lasR genes. The presence of either a lasB or a lasR plasmid in CIW7 and CIW8 resulted in the production of very low levels of elastase and lasB mRNA. Neither elastase nor lasB mRNA was detected in CIT1 and CIW5 carrying the lasB plasmid. The presence of the lasR plasmid in CIT1 and CIW5 resulted in the production of lasB mRNA and elastase protein in CIW5 only. All elastase-deficient strains produced detectable levels of lasR mRNA which were enhanced in the presence of the lasR plasmid. The Pseudomonas autoinducer (which is encoded by lasI) was also produced by all strains. CIT1 produced both hemolysin and alkaline protease but was defective in pyocyanin production. These results suggest that (i) CIT1 may contain a defect in a lasB-regulatory gene, (ii) CIW5 carries a defect within lasR, and (iii) the defect in isolates CIW7 and CIW8 affects the efficiency of lasB transcription. PMID:8757847

Tissue factor pathway inhibitor 2 is found in skin and its C-terminal region encodes for antibacterial activity.

PubMed

Papareddy, Praveen; Kalle, Martina; Sørensen, Ole E; Lundqvist, Katarina; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2012-01-01

Tissue factor pathway inhibitor 2 (TFPI-2) is a matrix-associated serine protease inhibitor with an enigmatic function in vivo. Here, we describe that TFPI-2 is present in fibrin of wounds and also expressed in skin, where it is up-regulated upon wounding. Neutrophil elastase cleaved TFPI-2, and a C-terminal fragment was found to bind to bacteria. Similarly, a prototypic peptide representing this C-terminal part, EDC34, bound to bacteria and bacterial lipopolysaccharide, and induced bacterial permeabilization. The peptide also induced leakage in artificial liposomes, and displayed a random coil conformation upon interactions with liposomes as well as lipopolysaccharide. EDC34 was antibacterial against both Gram-negative and Gram-positive bacteria in physiological buffer conditions. The results demonstrate that the C-terminus of TFPI-2 encodes for antimicrobial activity, and may be released during wounding.

Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

PubMed Central

Saffarzadeh, Mona; Juenemann, Christiane; Queisser, Markus A.; Lochnit, Guenter; Barreto, Guillermo; Galuska, Sebastian P.; Lohmeyer, Juergen; Preissner, Klaus T.

2012-01-01

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction. PMID:22389696

Elastase, alpha2-macroglobulin and alkaline phosphatase in crevicular fluid from implants with and without periimplantitis.

PubMed

Plagnat, Dominique; Giannopoulou, Catherine; Carrel, Anne; Bernard, Jean-Pierre; Mombelli, Andrea; Belser, Urs C

2002-06-01

The aim of this investigation was to determine the presence of selected enzymes and enzyme inhibitors in crevicular fluid collected from implants with and without clinical, radiographic and microbiological signs of periimplantitis. Eleven implants with symptoms of periimplantitis in eight patients (four men and four women) were compared to eleven implants in seven subjects (one man and six women) without periimplantitis. Periimplant crevicular fluid (PICF) was collected at the mesial and distal sites of each implant. Alkaline phosphatase activity (ALP) was measured by using p-nitrophenyl-phosphate as substrate, elastase activity (EA) by the use of a low molecular weight fluorogenic substrate, and the inhibitor alpha2-macroglobulin (alpha2M) by ELISA. ALP, EA and alpha2M were detected in the majority of samples in both groups. In comparison to the clinically healthy implants, total amounts of each of these substances were significantly higher in PICF collected around implants with periimplantitis. The mean total amounts of EA, alpha2M and ALP in the healthy group were: EA: 1.8 ng, alpha2M: 3.1 ng, ALP: 24.1 U, and in the periimplantitis group EA: 23.1 ng, alpha2M: 25.2 ng and ALP: 142.3 U. In addition, all three mediators were correlated with the clinical parameters. The results confirm the similarity of the inflammatory response of tissues surrounding implants and natural teeth, and suggest that ALP and EA could be promising markers of bone loss around dental implants.

Pharmacokinetics and Tolerability of Oral Sildenafil in Adults with Cystic Fibrosis Lung Disease

PubMed Central

Taylor-Cousar, JL; Wiley, C; Felton, LA; St Clair, C; Jones, M; Curran-Everett, D; Poch, K; Nichols, DP; Solomon, GM; Saavedra, MT; Accurso, FJ; Nick, JA

2014-01-01

Rationale Airway inflammation is central to cystic fibrosis (CF) pathophysiology. Pre-clinical models have shown that phosphodiesterase inhibitors (PDEi) like sildenafil have anti-inflammatory activity. PDEi have not been studied in CF subjects. Objectives We evaluated the pharmacokinetics, tolerability, and safety of sildenafil in subjects with CF. Sputum biomarkers were used to explore efficacy. Methods An open-label pilot study of oral sildenafil administration was conducted in adults with mild to moderate CF lung disease. Subjects received oral sildenafil 20 or 40 mg p.o. t.i.d. for 6 weeks. Measurements and Main Results Twenty subjects completed the study. Estimated elimination rate constants were statistically different in subjects with CF compared to previously published non-CF subjects. Side effects were generally mild. There were no drug-related serious adverse events. Sputum neutrophil elastase activity decreased. Conclusions Subjects with CF may eliminate sildenafil at a faster rate than non-CF subjects. Sildenafil administration was safe in subjects with CF, and decreased sputum elastase activity. Sildenafil warrants further study as an anti-inflammatory in CF. PMID:25466700

Granulocyte elastase, beta-thromboglobulin, and C3d during acetate or bicarbonate hemodialysis with Hemophan compared to a cellulose acetate membrane.

PubMed

Stegmayr, B G; Esbensen, K; Gutierrez, A; Lundberg, L; Nielsen, B; Stroemsaeter, C E; Wehle, B

1992-01-01

Twenty-two patients were dialysed in a cross-over design using Hemophan or cellulose acetate membranes. The dialysate buffer was acetate (n = 12) or bicarbonate (n = 10). Blood was sampled at 0, 15, 60 and 180 min and mean values were adjusted for changes in total protein in each sample. At 15 min during dialysis a decrease in leukocytes and platelets occurred with both membranes, irrespective of the buffer (Wilcoxon, p less than 0.006). During dialysis, increases were found in granulocyte elastase inhibitor complex (E- alpha 1-PI), beta-thromboglobulin and C3d. beta 2-microglobulin was not significantly changed in blood after dialysis with Hemophan or cellulose acetate membranes with bicarbonate buffer. Side effects were more pronounced at 180 min during dialysis with bicarbonate in patients using cellulose acetate than with Hemophan (p = 0.021, n = 8). Hemophan seemed to be more favourable than cellulose acetate membranes in regard to leukopenia and E- alpha 1-PI. The dialysate buffer may also alter membrane biocompatibility.

Effect of Elastin Digestion on the Quasi-static Tensile Response of Medial Collateral Ligament

PubMed Central

Henninger, Heath B.; Underwood, Clayton J.; Romney, Steven J.; Davis, Grant L.; Weiss, Jeffrey A.

2014-01-01

Elastin is a structural protein that provides resilience to biological tissues. We examined the contributions of elastin to the quasi-static tensile response of porcine medial collateral ligament through targeted disruption of the elastin network with pancreatic elastase. Elastase concentration and treatment time were varied to determine a dose response. Whereas elastin content decreased with increasing elastase concentration and treatment time, the change in peak stress after cyclic loading reached a plateau above 1 U/ml elastase and 6 hr treatment. For specimens treated with 2 U/ml elastase for 6 hr, elastin content decreased approximately 35%. Mean peak tissue strain after cyclic loading (4.8%, p≥0.300), modulus (275 MPa, p≥0.114) and hysteresis (20%, p≥0.553) were unaffected by elastase digestion, but stress decreased significantly after treatment (up to 2 MPa, p≤0.049). Elastin degradation had no effect on failure properties, but tissue lengthened under the same pre-stress. Stiffness in the linear region was unaffected by elastase digestion, suggesting that enzyme treatment did not disrupt collagen. These results demonstrate that elastin primarily functions in the toe region of the stress-strain curve, yet contributes load support in the linear region. The increase in length after elastase digestion suggests that elastin may pre-stress and stabilize collagen crimp in ligaments. PMID:23553827

Biotechnological traps for the reduction of inflammation due to cardiopulmonary bypass operations.

PubMed

Grano, Valentina; Salamino, Franca; Melloni, Edon; Minafra, Roberto; Regola, Eliana; Diano, Nadia; Nicolucci, Carla; Attanasio, Angelina; Nappi, Gianantonio; Cotrufo, Maurizio; Maresca, Lucio; De Santo, Natale Gaspare; Mita, Damiano Gustavo

2006-07-01

Cardiopulmonary bypass induces a systemic inflammatory response (SIR), characterized by the activation of cellular and humoral elements, with concomitant release of neutrophil elastase and matrix-metallo proteinases. In the present study, the protease release during extracorporeal circulation in 28 patients undergoing cardiac surgical operations was monitored using casein zymography. A peak in protease activity was found in all patients at the end of cardiopulmonary bypass. Plasma samples of patients were allowed to interact with different traps obtained by immobilizing different protease inhibitors on specific carriers. alpha1-Antitrypsin, Bovine Pancreatic Trypsin Inhibitor, Elastatinal or Leupeptin were used as inhibitors and were covalently immobilized by diazotization or by condensation. A reduction in the proteolytic activity of the plasma samples was observed after interaction with the different traps. The most efficient traps, i.e. the ones displaying greatest power to inhibit protease activity, were those obtained by immobilizing Bovine Pancreatic Trypsin Inhibitor and Leupeptin. The biocompatibility of traps was also tested. Results show that protease activity in blood can be decreased by our protease traps.

Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants

PubMed Central

Thring, Tamsyn SA; Hili, Pauline; Naughton, Declan P

2009-01-01

Background Owing to their roles in tissue remodelling in health and disease, several studies have reported investigations on plant extracts as inhibitors of proteinases and as anti-oxidants. Methods The anti-ageing and anti-oxidant properties of 23 plant extracts (from 21 plant species) were assessed as anti-elastase and anti-collagenase activities and in selected anti-oxidant assays along with phenolic content. Results Anti-elastase activities were observed for nine of the extracts with inhibitory activity in the following order: white tea (~89%), cleavers (~58%), burdock root (~51%), bladderwrack (~50%), anise and angelica (~32%). Anti-collagenase activities were exhibited by sixteen plants of which the highest activity was seen in white tea (~87%), green tea (~47%), rose tincture (~41%), and lavender (~31%). Nine plant extracts had activities against both elastase (E) and collagenase (C) and were ranked in the order of white tea (E:89%, C:87%) > bladderwrack (E:50%, C:25%) > cleavers (E:58%, C:7%) > rose tincture (E:22%, C:41%) > green tea (E:10%: C:47%) > rose aqueous (E: 24%, C:26%) > angelica (E:32%, C:17%) > anise (E:32%, C:6%) > pomegranate (E:15%, C:11%). Total phenolic content varied between 0.05 and 0.26 mg gallic acid equivalents (GAE)/mL with the exception of white tea (0.77 mg GAE/mL). For anti-oxidant assessment, the Trolox equivalent anti-oxidant capacity (TEAC) assay revealed activity for all extracts. White tea had the highest activity equivalent to ~21 μM Trolox for a 6.25 μg aliquot. In addition, seven extracts exhibited activities = 10 μM Trolox with witch hazel (6.25 μg = 13 μM Trolox) and rose aqueous (6.25 μg = 10 μM Trolox) showing very high activities at low concentrations. A high activity for white tea was also found in the superoxide dismutase (SOD) assay in which it exhibited ~88% inhibition of reduction of nitroblue tetrazolium. High activities were also observed for green tea (86.41%), rose tincture (82.77%), witch hazel (82.05%) and rose aqueous (73.86%). Conclusion From a panel of twenty three plant extracts, some one dozen exhibit high or satisfactory anti-collagenase or anti-elastase activities, with nine having inhibitory activity against both enzymes. These included white tea which was found to have very high phenolic content, along with high TEAC and SOD activities. PMID:19653897

Biodistribution and pharmacokinetics of the (99m)Tc labeled human elastase inhibitor, elafin, in rats.

PubMed

Kaschwich, Mark; Lützen, Ulf; Zhao, Yi; Tjiong, Angelina; Marx, Marlies; Haenisch, Sierk; Wiedow, Oliver; Preuss, Stefanie; Culman, Juraj; Zuhayra, Maaz

2016-04-01

Elafin is a potent reversible inhibitor of the pro-inflammatory proteases leukocyte elastase and protease 3. It is currently in clinical development for the use in postoperative inflammatory diseases. We investigated the pharmacokinetics of (99m)Tc-labeled elafin ((99m)Tc-Elafin) in blood and individual organs in rat after bolus intravenous injection using the single photon emission tomography (SPECT). (99m)Tc-Elafin predominantly accumulated in the kidney reaching a maximum of 8.5% ± 0.1% of the injected dose per gram (ID/g) at 5 min post injection (p.i) and decreased only slowly during 24 h. In contrast, the initially high radio activity recorded in the other organs rapidly decreased parallel to the radioactivity detected in blood. The blood kinetics fits to a two compartment kinetics model. The radio activity in the dissected kidney was 4.98 ± 1.24%ID/g 24 h p.i, while in other organs, including the brain, no accumulation of (99m)Tc-Elafin was detected. At this time point 30% of the detected radioactivity in the kidney was identified to be not metabolized (99m)Tc-Elafin. In conclusion, the blood and organ-specific kinetic data provide a basis for planning of adequate dosing regimens and the high accumulation of intact elafin in the kidney favors clinical developments targeting inflammatory kidney diseases, such as chronic allograft nephropathy after kidney transplantation. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Proton MRI as a noninvasive tool to assess elastase-induced lung damage in spontaneously breathing rats.

PubMed

Quintana, Harry Karmouty; Cannet, Catherine; Zurbruegg, Stefan; Blé, François-Xavier; Fozard, John R; Page, Clive P; Beckmann, Nicolau

2006-12-01

Elastase-induced changes in lung morphology and function were detected in spontaneously breathing rats using conventional proton MRI at 4.7 T. A single dose of porcine pancreatic elastase (75 U/100 g body weight) or vehicle (saline) was administered intratracheally (i.t.) to male Brown Norway (BN) rats. MRI fluid signals were detected in the lungs 24 hr after administration of elastase and resolved within 2 weeks. These results correlated with perivascular edema and cellular infiltration observed histologically. Reductions in MRI signal intensity of the lung parenchyma, and increases in lung volume were detected as early as 2 weeks following elastase administration and remained uniform throughout the study, which lasted 8 weeks. Observations were consistent with air trapping resulting from emphysema detected histologically. In a separate experiment, animals were treated daily intraperitoneally (i.p.) with all-trans-retinoic acid (ATRA; 500 microg/kg body weight) or its vehicle (triglyceride oil) starting on day 21 after elastase administration and continuing for 12 days. Under these conditions, ATRA did not elicit a reversal of elastase-induced lung damage as measured by MRI and histology. The present approach complements other validated applications of proton MRI in experimental lung research as a method for assessing drugs in rat models of respiratory diseases.

Activation of cellular immune response in acute pancreatitis.

PubMed Central

Mora, A; Pérez-Mateo, M; Viedma, J A; Carballo, F; Sánchez-Payá, J; Liras, G

1997-01-01

BACKGROUND: Inflammatory mediators have recently been implicated as potential markers of severity in acute pancreatitis. AIMS: To determine the value of neopterin and polymorphonuclear (PMN) elastase as markers of activation of cellular immunity and as early predictors of disease severity. PATIENTS: Fifty two non-consecutive patients classified according to their clinical outcome into mild (n = 26) and severe pancreatitis (n = 26). METHODS: Neopterin in serum and the PMN elastase/A1PI complex in plasma were measured during the first three days of hospital stay. RESULTS: Within three days after the onset of acute pancreatitis, PMN elastase was significantly higher in the severe pancreatitis group. Patients with severe disease also showed significantly higher values of neopterin on days 1 and 2 but not on day 3 compared with patients with mild disease. There was a significant correlation between PMN elastase and neopterin values on days 1 and 2. PMN elastase on day 1 predicted disease severity with a sensitivity of 76.7% and a specificity of 91.6%. Neopterin did not surpass PMN elastase in the probability of predicting disease severity. CONCLUSIONS: These data show that activation of cellular immunity is implicated in the pathogenesis of acute pancreatitis and may be a main contributory factor to disease severity. Neopterin was not superior to PMN elastase in the prediction of severity. PMID:9245935

Center for Macromolecular Crystallography, University of Alabama in Birmingham

NASA Technical Reports Server (NTRS)

Navia, Manuel A.

1991-01-01

Porcine pancreatic elastase (PPE) crystals grown under microgravity conditions on mission STS-26 of the Space Shuttle Discovery were shown to diffract to considerably higher resolution than the best PPE crystals grown by us on the ground. We have now independently refined both the microgravity and ground-based data. Preliminary results of these refinements are summarized. These results show nearly a doubling of experimental diffraction data for this structure, exceeding 1.3 A resolution. Improved phase information derived from the refined structure of PPE based on this microgravity data has allowed us to interpret previously-uninterpretable electron density obtained from ground-based crystals of a complex of PPE with a chemically-reactive inhibitor. Intermediate stages in the enzyme-inhibitor reaction mechanism in the crystal can now be directly observed. Further refinement of PPE structures is in progress.

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

PubMed Central

Pfundt, R; van Ruissen, F; van Vlijmen-Willems, I M; Alkemade, H A; Zeeuwen, P L; Jap, P H; Dijkman, H; Fransen, J; Croes, H; van Erp, P E; Schalkwijk, J

1996-01-01

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases. PMID:8823304

Identification and characterization of α1 -antitrypsin in fibrin clots.

PubMed

Talens, S; Malfliet, J J M C; van Hal, P Th W; Leebeek, F W G; Rijken, D C

2013-07-01

Preliminary studies indicated that α1 -antitrypsin (A1AT) is the most abundant protein that is non-covalently bound to fibrin clots prepared from plasma. The aim of this study was to identify and characterize fibrin(ogen)-bound A1AT. Plasma clots were prepared and extensively washed with saline. Clot-bound A1AT could only be extracted using denaturing agents such as urea, thiourea or SDS, pointing to an apparently strong association. Purified fibrinogen, but still containing A1AT as a contaminant, was gel filtered, which showed that the A1AT was bound to fibrinogen. A specific ELISA detected the presence of A1AT-fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)-Sepharose chromatography indicated that A1AT purified from plasma contained a small fraction of fibrin(ogen)-binding A1AT. To study the inhibitory activity of fibrin(ogen)-bound A1AT, both fibrinogen containing A1AT and washed plasma clots were incubated with increasing amounts of elastase. SDS-PAGE and Western blotting showed under both conditions the generation of the A1AT-elastase complex as well as cleaved A1AT. The inhibitory activity of fibrin(ogen)-bound A1AT was also demonstrated by measuring elastase-induced lysis of fibrin clots. Fibrin clots contain strongly bound A1AT, which is functionally active as a serine protease inhibitor (serpin). This A1AT might play a role in the local regulation of proteases involved in coagulation or fibrinolysis and represent a novel link between the inflammatory and hemostatic systems. © 2013 International Society on Thrombosis and Haemostasis.

Pathogenesis of aortic dilatation in mucopolysaccharidosis VII mice may involve complement activation

PubMed Central

Baldo, Guilherme; Wu, Susan; Howe, Ruth A.; Ramamoothy, Meera; Knutsen, Russell H.; Fang, Jiali; Mecham, Robert P.; Liu, Yuli; Wu, Xiaobo; Atkinson, John P.; Ponder, Katherine P.

2012-01-01

Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme β-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding β-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases. PMID:21944884

Elastin Degradation by Cathepsin V Requires Two Exosites*

PubMed Central

Du, Xin; Chen, Nelson L. H.; Wong, Andre; Craik, Charles S.; Brömme, Dieter

2013-01-01

Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions. PMID:24121514

The elastase-PK101 structure: Mechanism of an ultrasensitive activity-based probe revealed

DOE PAGES

Lechtenberg, Bernhard C.; Robinson, Howard R.; Kasperkiewicz, Paulina; ...

2015-01-22

Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle( O-Bzl) function in the P4 position of PK101 reveals and leverages an “exo-pocket” on HNEmore » as a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly–Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling.« less

Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

PubMed

Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

2016-10-01

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cotton Study: Albumin Binding and its Effect on Elastase Activity in the Chronic Non-Healing Wound

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro, N.; Goheen, S.

Cotton, as it is used in wound dressings is composed of nearly pure cellulose. During the wound-healing process, cotton is exposed to various blood components including water, salts, cells, and blood proteins. Albumin is the most prominent protein in blood. Elastase is an enzyme secreted by white blood cells and takes an active role in tissue reconstruction. In the chronic non-healing wound, elastase is often over-expressed such that this enzyme digests tissue and growth factors, and interferes with the normal healing process. Our goal is to design a cotton wound dressing that will sequester elastase or assist in reducing elastasemore » activity in the presence of other blood proteins such as albumin. The ability of cotton and various cotton derivatives to sequester elastase and albumin has been studied by examining the adsorption of these two proteins separately. We undertook the present work to confirm the binding of albumin to cotton and to quantify the activity of elastase in the presence of various derivatives of cotton. We previously observed a slight increase in elastase activity when exposed to cotton. We also observed a continuous accumulation of albumin on cotton using high-performance liquid chromatography methods. In the present study, we used an open-column-absorption technique coupled with a colorimetric protein assay to confirm losses of albumin to cotton. We have also confirmed increased elastase activity after exposure to cotton. The results are discussed in relation to the porosity of cotton and the use of cotton for treating chronic non-healing wounds.« less

Detection of Human Neutrophil Elastase with Fluorescent Peptide Sensors Conjugated to Nanocellulosic Solid Supports Targeting Wound Care Diagnostics

USDA-ARS?s Scientific Manuscript database

Human neutrophil elastase (HNE) is a biomarker for chronic wounds and a therapeutic target for certain diseases. An unchecked influx of neutrophils, which contain about one pictogram of elastase per neutrophil, is responsible for degrading growth factors and collagen formation, indefinitely delaying...

The effect of primary hyperparathyroidism on pancreatic exocrine function.

PubMed

Sisman, P; Avci, M; Akkurt, A; Sahin, A B; Gul, O O; Ersoy, C; Erturk, E

2018-03-01

Elastase-1 is a proteolytic enzyme secreted by pancreatic acinar cells, and measurements of the concentration this enzyme are used to evaluate pancreatic exocrine function. We aimed to determine whether pancreatic exocrine function declines due to chronic hypercalcemia by measuring fecal elastase levels. 75 patients with primary hyperparathyroidism (18 men and 47 women) and 30 healthy subjects (11 men and 19 women) participated in this study. Renal function tests, lipid parameters, bone mineral density, and serum calcium, phosphorus, vitamin D, parathormone, glucose, and thyroid stimulating hormone levels as well as fecal elastase concentrations, were determined in these patients and controls. The mean fecal elastase level was 335.3 ± 181.4 μg/g in the PHPT group and 317.4 ± 157.3 μg/g in the control group. There was no significant difference in fecal elastase levels between the two groups (p = 0.5). Chronic hypercalcemia in primary hyperparathyroidism did not decrease the fecal elastase level, which is an indirect indicator of chronic pancreatitis; therefore, chronic hypercalcemia in PHPT may not cause chronic pancreatitis.

Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

PubMed Central

Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

2011-01-01

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

PubMed

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

PubMed

Duan, Jianhui; Xiaokaiti, Yilixiati; Fan, Shengjun; Pan, Yan; Li, Xin; Li, Xuejun

2017-05-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120 µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60 µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an in silico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66 kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120 µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

PubMed

Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

2016-07-15

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

Nitric oxide-dependent neutrophil recruitment: role in nasal secretion.

PubMed

Cardell, L O; Agustí, C; Nadel, J A

2000-12-01

Leukotriene B4 (LTB4), an inflammatory mediator, is a potent chemoattractant for neutrophils that plays an important role in nasal secretion via release of elastase. Nitric oxide (NO) is an important modulator of leucocyte-endothelial cell interactions, endogenously produced in large quantities in the paranasal sinuses. To examine the role of NO in LTB4-stimulated nasal secretion. A newly-developed method for isolating and superfusing a nasal segment in dogs was used. Instillation of LTB4 into the nasal segment caused a time-dependent increase in the volume of airway fluid and in the recruitment of neutrophils. N(G)-nitro-L-arginine-methylester (L-NAME), an inhibitor of NO synthase, prevented LTB4-induced neutrophil recruitment and nasal secretion. These studies show that NO modulates LTB4-induced neutrophil recruitment and subsequent fluid secretion in the nose, and they suggest a therapeutic role for NO inhibitors in modulating neutrophil-dependent nasal secretion.

Identification and activity of a lower eukaryotic serine proteinase inhibitor (serpin) from Cyanea capillata: analysis of a jellyfish serpin, jellypin.

PubMed

Cole, Elisabeth B; Miller, David; Rometo, David; Greenberg, Robert M; Brömme, Dieter; Cataltepe, Sule; Pak, Stephen C; Mills, David R; Silverman, Gary A; Luke, Cliff J

2004-09-21

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved approximately 1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, approximately 1000 MYA.

A Drug-Repositioning Screening Identifies Pentetic Acid as a Potential Therapeutic Agent for Suppressing the Elastase-Mediated Virulence of Pseudomonas aeruginosa

PubMed Central

Gi, Mia; Jeong, Junhui; Lee, Keehoon; Lee, Kang-Mu; Toyofuku, Masanori; Yong, Dong Eun

2014-01-01

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 μM DTPA. Supplementation with Zn2+ or Mn2+ ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection. PMID:25246397

A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

PubMed

Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

2015-11-10

In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

Non-surgical periodontal therapy decreases serum elastase levels in aggressive but not in chronic periodontitis.

PubMed

Eickholz, Peter; Siegelin, Yasemin; Scharf, Susanne; Schacher, Beate; Oremek, Gerhard M; Sauer-Eppel, Hildegund; Schubert, Ralf; Wohlfeil, Martin

2013-04-01

Assessment of the effect of non-surgical periodontal therapy (SRP) on serum inflammatory parameters in patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. Overall, 31 ChP and 29 AgP were examined clinically prior to and 12 weeks after SRP (subgingival scaling of all pockets within 2 days) with systemic antibiotics for patients positive for Aggregatibacter actinomycetemcomitans (14 AgP, 9 ChP). Blood was sampled prior to, one day, 6, and 12 weeks after the first SRP visit. Serum elastase, C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts were assessed. At baseline, serum elastase, CRP, and LBP were significantly (p < 0.01) higher in AgP than ChP. Serum elastase, CRP, LBP, and IL-6 were significantly (p < 0.001) elevated one day after scaling in both groups. Both groups showed significant clinical improvement (p < 0.001). A significant difference was observed regarding change of serum elastase 12 weeks after SRP between AgP and ChP (p = 0.015). Multiple regression analysis revealed AgP, African origin, and bleeding on probing to be associated with more pronounced elastase reduction. CRP reduction was associated with African origin, systemic antibiotics, and baseline probing pocket depth. SRP results in serum elastase reduction in AgP but not in ChP. © 2013 John Wiley & Sons A/S.

Elastase-Sensitive Elastomeric Scaffolds with Variable Anisotropy for Soft Tissue Engineering

PubMed Central

Guan, Jianjun; Fujimoto, Kazuro L.; Wagner, William R.

2010-01-01

Purpose To develop elastase-sensitive polyurethane scaffolds that would be applicable to the engineering of mechanically active soft tissues. Methods A polyurethane containing an elastase-sensitive peptide sequence was processed into scaffolds by thermally induced phase separation. Processing conditions were manipulated to alter scaffold properties and anisotropy. The scaffold’s mechanical properties, degradation, and cytocompatibility using muscle-derived stem cells were characterized. Scaffold in vivo degradation was evaluated by subcutaneous implantation. Results When heat transfer was multidirectional, scaffolds had randomly oriented pores. Imposition of a heat transfer gradient resulted in oriented pores. Both scaffolds were flexible and relatively strong with mechanical properties dependent upon fabrication conditions such as solvent type, polymer concentration and quenching temperature. Oriented scaffolds exhibited anisotropic mechanical properties with greater tensile strength in the orientation direction. These scaffolds also supported muscle-derived stem cell growth more effectively than random scaffolds. The scaffolds expressed over 40% weight loss after 56 days in elastase containing buffer. Elastase-sensitive scaffolds were complete degraded after 8 weeks subcutaneous implantation in rats, markedly faster than similar polyurethanes that did not contain the peptide sequence. Conclusion The elastase-sensitive polyurethane scaffolds showed promise for application in soft tissue engineering where controlling scaffold mechanical properties and pore architecture are desirable. PMID:18509596

Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice.

PubMed

Braster, Quinte; Silvestre Roig, Carlos; Hartwig, Helene; Beckers, Linda; den Toom, Myrthe; Döring, Yvonne; Daemen, Mat J; Lutgens, Esther; Soehnlein, Oliver

2016-01-01

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver.

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

PubMed Central

Kessenbrock, Kai; Fröhlich, Leopold; Sixt, Michael; Lämmermann, Tim; Pfister, Heiko; Bateman, Andrew; Belaaouaj, Azzaq; Ring, Johannes; Ollert, Markus; Fässler, Reinhard; Jenne, Dieter E.

2008-01-01

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents. PMID:18568075

Therapeutic utility and medicinal chemistry of cathepsin C inhibitors.

PubMed

Guay, Daniel; Beaulieu, Christian; Percival, M David

2010-01-01

The lysosomal cysteine protease cathepsin C (Cat C), also known as dipeptidyl peptidase I, activates a number of granule-associated serine proteases with pro-inflammatory and immune functions by removal of their inhibitory N-terminal dipeptides. Thus, Cat C is a therapeutic target for the treatment of a number of inflammatory and autoimmune diseases. Cathepsin C null mice and humans with Cat C loss of function mutations (Papillon-Lefèvre syndrome) show deficiencies in disease-relevant proteases including neutrophil elastase, cathepsin G, chymases and granzymes and the Cat C mice are protected in a number of disease models. Several methodologies have been recently reported for assessing the effects of Cat C inhibitors on serine protease activities in cellular assays and prolonged treatment of rats with a reversible, selective Cat C inhibitor reduced the activity of three leukocyte serine proteases. Nearly all potent and selective Cat C inhibitors described are based on the preferred dipeptide substrates bearing either irreversible (e.g. diazomethylketone, acyloxymethyl ketone, o-acyl hydroxamic acid and vinyl sulfone) or reversible (e.g. semicarbazide, nitrile and cyanamide) electrophilic warheads. While potent and highly selective, the best inhibitors described to date still have poor stability and/or rodent pharmacokinetics, likely resulting from their peptidic nature. The lack of selective compounds with appropriate rodent pharmacokinetic properties has hampered the assessment of the effects of Cat C inhibitors on the activation of disease-relevant proteases in vivo and the full evaluation of the therapeutic utility of Cat C inhibitors.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

PubMed

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy. Georg Thieme Verlag KG Stuttgart · New York.

A Modified Method for Creating Elastase-Induced Aneurysms by Ligation of Common Carotid Arteries in Rabbits and Its Effect on Surrounding Arteries.

PubMed

Kainth, Daraspreet; Salazar, Pascal; Safinia, Cyrus; Chow, Ricky; Bachour, Ornina; Andalib, Sasan; McKinney, Alexander M; Divani, Afshin A

2017-01-01

Rabbit models of intracranial aneurysms are frequently used in pre-clinical settings. This study aimed to demonstrate an alternative, extravascular method for creating elastase-induced aneurysms, and how ligation of the right common carotid arteries (RCCA) can impact flow redistribution into left CCA (LCCA). Elastase-induced aneurysms in 18 New Zealand rabbits (4.14 ± 0.314 kg) were created by applying 3-5 U of concentrated elastase solution to the exterior of the right and left CCA roots (RCCA and LCCA). After the induction of the aneurysm, the aneurysm was either kept intact to the rest of the corresponding CCA, severed from the rest of the CCA to allow for a free standing aneurysm, or was anchored to nearby tissue to influence the angle and orientation of the aneurysm with respect to the parent vessel. Ultrasound studies were performed before and after creation of aneurysms to collect blood flow measurements inside the aneurysm pouch and surrounding arteries. Prior to sacrificing the animals, computed tomography angiography studies were performed. Harvested aneurysmal tissues were used for histological analysis. Elastase-induced aneurysms were successfully created by the extravascular approach. Histological studies showed that the biological response was similar to human cerebral aneurysms and previously published elastase-induced rabbit aneurysm models. Ultrasound measurements indicated that after the RCCA was ligated, blood flow significantly increased in the LCCA at one-month follow-up. An alternate method for creating elastase-induced aneurysms has been demonstrated. The novel aspects of our method allow for ligation of one or both common carotid arteries to create a single or bilateral aneurysm with an ability to control the orientation of the induced aneurysm.

A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient.

PubMed

Estrellas, P S; Alionte, L G; Hobden, J A

2000-03-01

Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

PubMed

Rival, S; Besson, C; Saulnier, J; Wallach, J

1999-02-01

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

PubMed

Nomura, Kazuaki; Obata, Kazufumi; Keira, Takashi; Miyata, Ryo; Hirakawa, Satoshi; Takano, Ken-ichi; Kohno, Takayuki; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2014-02-18

Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells

PubMed Central

2014-01-01

Background Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. Methods To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. Results PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. Conclusions PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis. PMID:24548792

Perioperative elafin for ischaemia-reperfusion injury during coronary artery bypass graft surgery: a randomised-controlled trial

PubMed Central

Alam, S R; Lewis, S C; Zamvar, V; Pessotto, R; Dweck, M R; Krishan, A; Goodman, K; Oatey, K; Harkess, R; Milne, L; Thomas, S; Mills, N M; Moore, C; Semple, S; Wiedow, O; Stirrat, C; Mirsadraee, S; Newby, D E; Henriksen, P A

2015-01-01

Background Elafin is a potent endogenous neutrophil elastase inhibitor that protects against myocardial inflammation and injury in preclinical models of ischaemic-reperfusion injury. We investigated whether elafin could inhibit myocardial ischaemia-reperfusion injury induced during coronary artery bypass graft (CABG) surgery. Methods and results In a randomised double-blind placebo-controlled parallel group clinical trial, 87 patients undergoing CABG surgery were randomised 1:1 to intravenous elafin 200 mg or saline placebo administered after induction of anaesthesia and prior to sternotomy. Myocardial injury was measured as cardiac troponin I release over 48 h (area under the curve (AUC)) and myocardial infarction identified with MRI. Postischaemic inflammation was measured by plasma markers including AUC high-sensitive C reactive protein (hs-CRP) and myeloperoxidase (MPO). Elafin infusion was safe and resulted in >3000-fold increase in plasma elafin concentrations and >50% inhibition of elastase activity in the first 24 h. This did not reduce myocardial injury over 48 h (ratio of geometric means (elafin/placebo) of AUC troponin I 0.74 (95% CI 0.47 to 1.15, p=0.18)) although post hoc analysis of the high-sensitive assay revealed lower troponin I concentrations at 6 h in elafin-treated patients (median 2.4 vs 4.1 μg/L, p=0.035). Elafin had no effect on myocardial infarction (elafin, 7/34 vs placebo, 5/35 patients) or on markers of inflammation: mean differences for AUC hs-CRP of 499 mg/L/48 h (95% CI −207 to 1205, p=0.16), and AUC MPO of 238 ng/mL/48 h (95% CI −235 to 711, p=0.320). Conclusions There was no strong evidence that neutrophil elastase inhibition with a single-dose elafin treatment reduced myocardial injury and inflammation following CABG-induced ischaemia-reperfusion injury. Trial registration number (EudraCT 2010-019527-58, ISRCTN82061264). PMID:26310261

Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva

PubMed Central

Sun, Xiuli; Salih, Erdjan; Oppenheim, Frank G.; Helmerhorst, Eva J.

2009-01-01

Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50–75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer. PMID:20011683

Isolated immune-related pancreatic exocrine insufficiency associated with pembrolizumab therapy.

PubMed

Prasanna, Thiru; McNeil, Catriona M; Nielsen, Theresa; Parkin, David

2018-03-01

We report a case of isolated immune-related pancreatic exocrine insufficiency in a patient treated with pembrolizumab for metastatic melanoma. This patient presented with explosive diarrhea and was treated with high dose corticosteroids for possible immune-related colitis. However, biopsies from colon and duodenum did not show any histological evidence of colitis/enteritis. Serum amylase and lipase were not elevated. There was no evidence of pancreatitis or pancreatic metastases on imaging. Significantly lower fecal elastase test on two occasions confirmed the diagnosis of pancreatic exocrine insufficiency. He was treated with pancreatic enzyme supplementation with complete resolution of diarrhea. This case reinforces the importance of awareness and anticipation of unusual immune-related adverse events related to checkpoint inhibitors.

Human neutrophil elastase and collagenase sequestration with phosphorylated cotton wound dressings.

PubMed

Edwards, J Vincent; Howley, Phyllis S

2007-11-01

The design and preparation of wound dressings that redress the protease imbalance in chronic wounds is an important goal of wound healing and medical materials science. Chronic wounds contain high levels of tissue and cytokine-destroying proteases including matrix metalloprotease and neutrophil elastase. Thus, the lowering of excessive protease levels in the wound environment by wound dressing sequestration prevents the breakdown of extracellular matrix proteins and growth factors necessary for wound healing. Phosphorylated cotton wound dressings were prepared to target sequestration of proteases from chronic wound exudate through a cationic uptake binding mechanism involving salt bridge formation of the positively charged amino acid side chains of proteases with the phosphate counterions of the wound dressing fiber. Dressings were prepared by applying sodium hexametaphosphate and diammonium phosphate in separate formulations to cotton gauze by pad/dry/cure methods. Phosphorylated cotton dressings were assessed for their ability to lower elastase and collagenase activity. The phosphorylated cotton dressings lowered elastase and collagenase activity 40-80% more effectively than the untreated cotton wound dressings under conditions that mimic chronic wound exudate. Efficacy of the phosphorylated cotton was found to be related to the level of phosphorylation and a lower pH due to protonated phosphate at the surface of the dressing. The capacity of the modified gauze to sequester continued elastase secretions similar to that found in a chronic wound over a 24-h period was retained within a 80% retention of elastase sequestration and was dose-dependent. Copyright (c) 2007 Wiley Periodicals, Inc.

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

PubMed Central

Stapels, Daphne A. C.; Ramyar, Kasra X.; Bischoff, Markus; von Köckritz-Blickwede, Maren; Milder, Fin J.; Ruyken, Maartje; Eisenbeis, Janina; McWhorter, William J.; Herrmann, Mathias; van Kessel, Kok P. M.; Geisbrecht, Brian V.; Rooijakkers, Suzan H. M.

2014-01-01

Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity. PMID:25161283

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

PubMed

Bakele, Martina; Lotz-Havla, Amelie S; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C; Gersting, Soeren W; Hartl, Dominik

2014-07-25

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

An Interactive Network of Elastase, Secretases, and PAR-2 Protein Regulates CXCR1 Receptor Surface Expression on Neutrophils*

PubMed Central

Bakele, Martina; Lotz-Havla, Amelie S.; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C.; Gersting, Soeren W.; Hartl, Dominik

2014-01-01

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis. PMID:24914212

Digestive proteolytic and amylolytic activities of Helicoverpa armigera in response to feeding on different soybean cultivars.

PubMed

Naseri, Bahram; Fathipour, Yaghoub; Moharramipour, Saeid; Hosseininaveh, Vahid; Gatehouse, Angharad M R

2010-12-01

Digestive proteolytic and amylolytic activities of the larvae of Helicoverpa armigera (Hübner) fed either on artificial diet or on different soybean cultivars (356, M4, M7, M9, Clark, Sahar, JK, BP, Williams, L17, Zane, Gorgan3 and DPX) and response of the larvae to feeding on some soybean-based protease inhibitors were studied. The highest general and specific proteolytic activities were in artificial-diet-fed larvae. Although the highest general proteolytic activity was in the larvae fed on L17, M4 and Sahar cultivars, the lowest tryptic activity was on L17 and Sahar, which may be due to the presence of some serine protease inhibitors in these two cultivars, resulting in hyperproduction of chymotrypsin- and elastase-like enzymes in response to the inhibition of these enzymes. The highest amylolytic activity was on M4, and the lowest was on Williams and DPX. General proteolytic activity of SKTI-fed larvae was the highest compared with SBBI- and STI-fed larvae. The findings demonstrated that the cultivars L17 and Sahar were partially resistant to this pest, probably because of some secondary chemicals or proteinaceous protease inhibitors of these cultivars.

Isolation, cDNA cloning, and structure-based functional characterization of oryctin, a hemolymph protein from the coconut rhinoceros beetle, Oryctes rhinoceros, as a novel serine protease inhibitor.

PubMed

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-09-24

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.

Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor*

PubMed Central

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-01-01

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant 13C,15N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with Ki values of 3.9 × 10−10 m, 6.2 × 10−10 m, 1.4 × 10−9 m, and 1.2 × 10−8 m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections. PMID:20630859

Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue

PubMed Central

Into, T; Inomata, M; Kanno, Y; Matsuyama, T; Machigashira, M; Izumi, Y; Imamura, T; Nakashima, M; Noguchi, T; Matsushita, K

2006-01-01

Chronic periodontitis is correlated with Porphyromonas gingivalis infection. In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil-derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P. gingivalis infection. The addition of vesicles of P. gingivalis decreased the amount of SLPI in the media of primary human gingival keratinocytes compared to untreated cultures. We therefore investigated how arginine-specific gingipains (Rgps) affect the functions of SLPI, because Rgps are the major virulence factors in the vesicles and cleave a wide range of in-host proteins. We found that Rgps digest SLPI in vitro, suppressing the release of SLPI. Rgps proteolysis of SLPI disrupted SLPI functions, which normally suppresses neutrophil elastase and neutralizes pro-inflammatory effects of bacterial cell wall compounds in cultured human gingival fibroblasts. The protease inhibitory action of SLPI was not exerted towards Rgps. These results suggest that Rgps reduce the protective effects of SLPI on neutrophil proteases and bacterial proinflammatory compounds, by which disease in gingival tissue may be accelerated at the sites with P. gingivalis infection. PMID:16907925

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

PubMed

Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

2011-08-15

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader. Copyright © 2011 Elsevier Inc. All rights reserved.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

PubMed Central

Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

2012-01-01

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

PubMed

Ruffin, Manon; Bilodeau, Claudia; Maillé, Émilie; LaFayette, Shantelle L; McKay, Geoffrey A; Trinh, Nguyen Thu Ngan; Beaudoin, Trevor; Desrosiers, Martin-Yvon; Rousseau, Simon; Nguyen, Dao; Brochiero, Emmanuelle

2016-09-01

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

Increased systemic elastase and C-reactive protein in aggressive periodontitis (CLOI-D-00160R2).

PubMed

Wohlfeil, Martin; Scharf, Susanne; Siegelin, Yasemin; Schacher, Beate; Oremek, Gerhard M; Sauer-Eppel, Hildegund; Schubert, Ralf; Eickholz, Peter

2012-08-01

The inflammatory mediators, serum elastase and C-reactive protein (CRP), are associated with an increased risk for coronary heart disease. Thus, the aim of this study is to compare systemic inflammatory mediators in periodontally healthy controls (C), patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. C [periodontal pocket probing depth (PPD)  <3.6 or <5 mm without bleeding (BOP), BOP < 10%], ChP (PDD ≥ 3.6 mm and probing attachment loss ≥5 mm at >30% of sites; age >35 years), and AgP (clinically healthy; PDD ≥ 3.6 mm at >30% of sites, bone loss ≥50% at ≥2 teeth; age ≤35 years) were examined clinically, and the body mass index was assessed. Blood was sampled for assessment of serum levels of elastase, CRP, lipopolysaccharide binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts. Thirty C, 31 ChP, and 29 AgP were analyzed. Elastase, CRP, LBP, and IL-6 levels were elevated in AgP compared to C (p < 0.013), whereas leukocyte counts and IL-8 were similar. Multiple regression analysis identified AgP (p < 0.001) and education level (p < 0.001) to explain 47% of the variation of elastase. AgP (p = 0.003), African origin (p = 0.006), female sex (p = 0.002), and BMI (p < 0.001) explained 39% of the variation of CRP. Serum elastase and CRP are significantly elevated in AgP compared to C. AgP patients exhibit a stronger systemic inflammatory burden than C patients.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

PubMed

Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

2016-10-01

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Antibacterial activity of antileukoprotease.

PubMed Central

Hiemstra, P S; Maassen, R J; Stolk, J; Heinzel-Wieland, R; Steffens, G J; Dijkman, J H

1996-01-01

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection. PMID:8890201

Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

PubMed

Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

2015-05-01

A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

PubMed

Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

2015-07-01

Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. © 2014 Wiley Periodicals, Inc.

Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

PubMed

Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

2012-12-01

This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Elastolytic activity of sputum and its relation to purulence and to lung function in patients with bronchiectasis.

PubMed Central

Stockley, R A; Hill, S L; Morrison, H M; Starkie, C M

1984-01-01

Sputum samples from 34 patients with bronchiectasis were assessed subjectively and the results related to objective measurements of elastase activity and albumin content. The results suggest that the macroscopic appearance of the sample is related to the elastase content. 88.7% of the purulent samples but none of the mucoid samples showing elastase activity. The macroscopic appearance was also associated with changes in protein transudation into the secretions. The median sputum: serum albumin concentration ratio was 0.71 X 10(-2) (range 0.22-4.7) in the mucoid samples but was greater in purulent samples (p less than 0.005), with a median value of 1.52 X 10(-2) (range 0.55-12.72), suggesting that purulence in the stable state was associated with low grade pulmonary inflammation or epithelial damage or both. Abnormalities of air flow were found in 24 of the patients (70.6%) but there was a significantly higher ratio of residual volume to total lung capacity (p less than 0.025) in patients who regularly produced purulent sputum (mean (SD) RV/TLC 44.4% (9.0%] than in those with mucoid or mucopurulent secretions (38.0% (9.9%]. A similar difference was found between those who produced elastase positive secretions and those who produced elastase negative ones. PMID:6565423

Trehalose does not affect the functions of human neutrophils in vitro.

PubMed

Tanaka, Koji; Kawamura, Mikio; Otake, Kohei; Toiyama, Yuji; Okugawa, Yoshinaga; Inoue, Yasuhiro; Uchida, Keiichi; Araki, Toshimitsu; Mohri, Yasuhiko; Kusunoki, Masato

2014-02-01

Trehalose, naturally occurring disaccharide, has been reported to prevent postoperative abdominal adhesions in animal models. We investigated whether trehalose affects the function of human polymorphonuclear neutrophils (PMNs) in vitro to assess the feasibility of its clinical application as an anti-adhesive barrier. Human PMNs were obtained from 17 healthy volunteers. Escherichia coli and Staphylococcus aureus were used for the bacterial infection model, whereas lipopolysaccharide (LPS) and interleukin (IL)-1β were used for inflammation induction model. The PMN phagocytosis rates of bacteria and apoptosis/necrosis were assessed on trehalose, maltose, and control media. Cytokines; namely, tumor necrosis factor-α, IL-1α, IL-1Ra, IL-6, and IL-8; and PMN-elastase were measured on each medium in both models. There were no significant differences in the phagocytosis rates, apoptosis/necrosis rates, or levels of all cytokines or PMN-elastase among the three media in the bacterial infection model. There were also no significant differences in the levels of all cytokines and PMN-elastase among the three media in the IL-1β inflammation induction model. PMN-elastase was lower in trehalose and maltose medium after LPS stimulation, at 3 and 24 h. Our results suggest that trehalose does not affect the cellular function, cytokine production, or release of PMN-elastase of human PMNs in an in vitro bacterial infection model.

Sulfated caffeic acid dehydropolymer attenuates elastase and cigarette smoke extract-induced emphysema in rats: sustained activity and a need of pulmonary delivery.

PubMed

Saluja, Bhawana; Li, Hua; Desai, Umesh R; Voelkel, Norbert F; Sakagami, Masahiro

2014-08-01

Although emphysema destroys alveolar structures progressively and causes death eventually, no drug has been discovered to prevent, intervene, and/or resolve this life-threatening disease. We recently reported that sulfated caffeic acid dehydropolymer CDSO3 is a novel potent triple-action inhibitor of elastolysis, oxidation, and inflammation in vitro, and therefore, a potential anti-emphysema agent. However, the in vivo therapeutic potency, duration and mode of actions, and effective route remain to be demonstrated. Emphysema was induced in rats with human sputum elastase (HSE) combined with cigarette smoke extract (CSE). CDSO3 at 5, 30, or 100 μg/kg was dosed to the lung or injected subcutaneously at 2, 6, or 24 h before or 1 or 24 h or 1 week after the HSE/CSE instillation. At 1 h or 48 h or on day 21-22 or day 28, lungs were examined for airway-to-blood injurious barrier damage; their elastolytic, oxidative, and inflammatory activities; lung luminal leukocytes infiltration; functional treadmill exercise endurance; and/or morphological airspace enlargement. CDSO3, when dosed to the lung at 30 or 100 μg/kg, but not via systemic subcutaneous injection, significantly (43-93 %) attenuated HSE/CSE-induced (1) barrier damage measured by luminal hemorrhage and protein leak; (2) elastolytic, oxidative, and inflammatory activities measured with elastase, reduced glutathione, and TNFα levels, respectively; (3) luminal neutrophil infiltration and tissue myeloperoxidase activity; (4) functional impairment of exercise endurance; and (5) airspace enlargement, in both preventive and interventional dosing protocols. Notably, the effects were shown to last for 24 h at the greater 100-μg/kg dose, and the 1-week-delayed administration was also capable of attenuating the development of emphysema. CDSO3 is a novel, potent, long-acting, nonpeptidic macromolecule that inhibits HSE/CSE-induced elastolysis, oxidation, and inflammation in the lung and thereby attenuates the development of emphysema in rats, in both preventive and interventional manners, when administered locally to the lung.

Effect of dialyzer geometry on granulocyte and complement activation.

PubMed

Schaefer, R M; Heidland, A; Hörl, W H

1987-01-01

During hemodialysis with cuprophan membranes, the complement system as well as leukocytes become activated. In order to clarify the role of dialyzer geometry, the effect of hollow-fiber versus flat-sheet dialyzers and of different surface areas on C3a generation and leukocyte degranulation was investigated. Plasma levels of leukocyte elastase in complex with alpha 1-proteinase inhibitor were significantly increased after 1 h (+55%) and 3 h (+62%) of hemodialysis with flat-sheet dialyzers as compared to hollow-fiber devices. In addition, plasma levels of lactoferrin, released from the specific granules of leukocytes during activation, were significantly higher (+42%) 3 h after the onset of dialysis treatment with flat-sheet than with hollow-fiber dialyzers. With respect to surface area, larger dialyzers tended to cause more release of leukocyte elastase as compared to dialyzers with smaller surface areas, irrespectively of the configuration of the dialyzer used. On the other hand, activation of the complement system, as measured by the generation of C3a-desarg, did not differ with both types of configurations. The same held true for leukopenia, which was almost identical for hollow-fiber and flat-sheet dialyzers. From these findings two lines of evidence emerge: First, not only the type of membrane material used in a dialyzer may influence its biocompatibility, but the geometry of the extracorporeal device also determines the degree of compatibility. Hence, the extent of leukocyte activation correlated with both configuration of the dialyzer and surface area of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

PubMed

Price, Joseph A

2015-09-01

Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Increasing transcriptome response of serpins during the ontogenetic stages in the salmon louse Caligus rogercresseyi (Copepoda: Caligidae).

PubMed

Maldonado-Aguayo, W; Gallardo-Escárate, C

2014-06-01

Serine protease inhibitors, or serpins, target serine proteases, and are important regulators of intra- and extracellular proteolysis. For parasite survival, parasite-derived protease inhibitors have been suggested to play essential roles in evading the host's immune system and protecting against exogenous host proteases. The aim of this work was to identify serpins via high throughput transcriptome sequencing and elucidate their potential functions during the lifecycle of the salmon louse Caligus rogercresseyi. Eleven putative, partial serpin sequences in the C. rogercresseyi transcriptome were identified and denoted as Cr-serpins 1 to 11. Comparative analysis of the deduced serpin-like amino acid sequences revealed a highly conserved reactive center loop region. Interestingly, P1 residues suggest putative functions involved with the trypsin/subtilisin, elastase, or subtilisin inhibitors, which evidenced increasing gene expression profiles from the copepodid to adult stage in C. rogercresseyi. Concerning this, Cr-serpin 10 was mainly expressed in the copepodid stage, while Cr-serpins 3, 4, 5, and 11 were mostly expressed in chalimus and adult stages. These results suggest that serpins could be involved in evading the immune response of the host fish. The identification of these serpins furthers the understanding of the immune system in this important ectoparasite species. Copyright © 2014 Elsevier B.V. All rights reserved.

The biochemical and functional food properties of the bowman-birk inhibitor.

PubMed

Losso, Jack N

2008-01-01

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. In 1999, FDA allowed a health claim on food labels stating that a daily diet containing 25 grams of soy protein, also low in saturated fat and cholesterol, may reduce the risk of heart disease [corrected] This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.

Saliva and wound healing.

PubMed

Brand, Henk S; Ligtenberg, Antoon J M; Veerman, Enno C I

2014-01-01

Oral wounds heal faster and with less scar formation than skin wounds. One of the key factors involved is saliva, which promotes wound healing in several ways. Saliva creates a humid environment, thus improving the survival and functioning of inflammatory cells that are crucial for wound healing. In addition, saliva contains several proteins which play a role in the different stages of wound healing. Saliva contains substantial amounts of tissue factor, which dramatically accelerates blood clotting. Subsequently, epidermal growth factor in saliva promotes the proliferation of epithelial cells. Secretory leucocyte protease inhibitor inhibits the tissue-degrading activity of enzymes like elastase and trypsin. Absence of this protease inhibitor delays oral wound healing. Salivary histatins in vitro promote wound closure by enhancing cell spreading and cell migration, but do not stimulate cell proliferation. A synthetic cyclic variant of histatin exhibits a 1,000-fold higher activity than linear histatin, which makes this cyclic variant a promising agent for the development of a new wound healing medication. Conclusively, recognition of the many roles salivary proteins play in wound healing makes saliva a promising source for the development of new drugs involved in tissue regeneration.

Inhibitory effects of constituents of Morinda citrifolia seeds on elastase and tyrosinase.

PubMed

Masuda, Megumi; Murata, Kazuya; Fukuhama, Akiko; Naruto, Shunsuke; Fujita, Tadashi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2009-07-01

A 50% ethanolic extract (MCS-ext) from seeds of Morinda citrifolia ("noni" seeds) showed more potent in vitro inhibition of elastase and tyrosinase, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than extracts of M. citrifolia leaves or flesh. Activity-guided fractionation of MCS-ext using in vitro assays led to the isolation of ursolic acid as an active constituent of elastase inhibitory activity. 3,3'-Bisdemethylpinoresinol, americanin A, and quercetin were isolated as active constituents having both tyrosinase inhibitory and radical scavenging activities. Americanin A and quercetin also showed superoxide dismutase (SOD)-like activity. These active compounds were isolated from noni seeds for the first time.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent

PubMed Central

Wang, Xintong; Zachman, Angela L.; Chun, Young Wook; Shen, Fang-Wen; Hwang, Yu-Shik; Sung, Hak-Joon

2014-01-01

Background Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. Methods Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. Results We demonstrated poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule-1 (VCAM) along with decreased nitric oxide production, indicating ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced release of elastase or elastase-like protease, which further accelerated polymer degradation. Conclusions This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis. PMID:24820736

Cotton and Protein Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.

The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, itmore » may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.« less

Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

PubMed

Galdino, Anna Clara M; Viganor, Lívia; Ziccardi, Mariangela; Nunes, Ana Paula F; Dos Santos, Kátia R N; Branquinha, Marta H; Santos, André L S

2017-12-01

Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Elastase-Induced Parenchymal Disruption and Airway Hyper Responsiveness in Mouse Precision Cut Lung Slices: Toward an Ex vivo COPD Model.

PubMed

Van Dijk, Eline M; Culha, Sule; Menzen, Mark H; Bidan, Cécile M; Gosens, Reinoud

2016-01-01

Background: COPD is a progressive lung disease characterized by emphysema and enhanced bronchoconstriction. Current treatments focused on bronchodilation can delay disease progression to some extent, but recovery or normalization of loss of lung function is impossible. Therefore, novel therapeutic targets are needed. The importance of the parenchyma in airway narrowing is increasingly recognized. In COPD, the parenchyma and extracellular matrix are altered, possibly affecting airway mechanics and enhancing bronchoconstriction. Our aim was to set up a comprehensive ex vivo Precision Cut Lung Slice (PCLS) model with a pathophysiology resembling that of COPD and integrate multiple readouts in order to study the relationship between parenchyma, airway functionality, and lung repair processes. Methods: Lungs of C57Bl/6J mice were sliced and treated ex vivo with elastase (2.5 μg/ml) or H 2 O 2 (200 μM) for 16 h. Following treatment, parenchymal structure, airway narrowing, and gene expression levels of alveolar Type I and II cell repair were assessed. Results: Following elastase, but not H 2 O 2 treatment, slices showed a significant increase in mean linear intercept (Lmi), reflective of emphysema. Only elastase-treated slices showed disorganization of elastin and collagen fibers. In addition, elastase treatment lowered both alveolar Type I and II marker expression, whereas H 2 O 2 stimulation lowered alveolar Type I marker expression only. Furthermore, elastase-treated slices showed enhanced methacholine-induced airway narrowing as reflected by increased pEC50 (5.87 at basal vs. 6.50 after elastase treatment) and Emax values (47.96 vs. 67.30%), and impaired chloroquine-induced airway opening. The increase in pEC50 correlated with an increase in mean Lmi. Conclusion: Using this model, we show that structural disruption of elastin fibers leads to impaired alveolar repair, disruption of the parenchymal compartment, and altered airway biomechanics, enhancing airway contraction. This finding may have implications for COPD, as the amount of elastin fiber and parenchymal tissue disruption is associated with disease severity. Therefore, we suggest that PCLS can be used to model certain aspects of COPD pathophysiology and that the parenchymal tissue damage observed in COPD contributes to lung function decline by disrupting airway biomechanics. Targeting the parenchymal compartment may therefore be a promising therapeutic target in the treatment of COPD.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds

PubMed Central

Machado, Richele J. A.; Monteiro, Norberto K. V.; Migliolo, Ludovico; Silva, Osmar N.; Pinto, Michele F. S.; Oliveira, Adeliana S.; Franco, Octávio L.; Kiyota, Sumika; Bemquerer, Marcelo P.; Uchoa, Adriana F.; Morais, Ana H. A.; Santos, Elizeu A.

2013-01-01

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30–60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10−8 mol.L−1 and constant inhibition (Ki) of 1.0×10−8 mol.L−1, by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation. PMID:23737945

[Case of Legionella pneumonia caused by a household humidifier].

PubMed

Endo, Keiichi; Ito, Kazuhisa

2009-05-01

A 60-year-old-man with diabetes mellitus was admitted to our hospital because of pyrexia of up to 40 degrees and dyspnea. Chest radiograph revealed ground glass infiltrates in both lung fields. Legionella pneumonia was diagnosed as a consequence of the urinary examination performed for Legionella antigen detection; further, we initiated intravenous pazufloxacin administration. Despite an antibiotic, sivelestat sodium, and mechanical ventilation, the condition of the patient worsened and he died of respiratory failure 5 days following admission. Pulse-field gel electrophoresis (PFGE) was performed to investigate the route of infection: this technique was applied to patient sputum samples and water from the humidifier in the room of the patient. The same DNA pattern as Legionella pneumophilla serogroup 1 was identified via analysis with PFGE.

Morbidity and mortality associated with creation of elastase-induced saccular aneurysms in a rabbit model

PubMed Central

Lewis, Debra A; Ding, Yong Hong; Dai, Daying; Kadirvel, Ramanathan; Danielson, Mark A; Cloft, Harry J; Kallmes, David F

2008-01-01

Background and Purpose Elastase-induced aneurysms in rabbits have been proposed as a useful preclinical tool for device development. The object of this study is to report rates of morbidity and mortality associated with creation and embolization of the elastase-induced rabbit aneurysm, and to assess the impact of operator experience on these rates. Methods Elastase-induced model aneurysms were created in New Zealand White rabbits (n=700). One neuroradiologist/investigator, naïve to the aneurysm creation procedure at the outset of the experiments, performed all surgeries. All morbidity and deaths related to aneurysm creation (n=700) and embolization procedures (n=529) were categorized into acute and chronic deaths. Data were analyzed with single regression analysis and ANOVA. To assess the impact of increasing operator experience, the number of animals was broken into 50 animal increments. Results There were 121 (17%) deaths among 700 subjects. Among 700 aneurysm creation procedures, 59 deaths (8.4%) were noted. Among 529 aneurysm embolization procedures, 43 deaths (8.1%) were noted. Nineteen additional deaths (2.7% of 700 subjects) were unrelated to procedures. Simple regression indicated mortality associated with procedures diminished with increasing operator experience (R2=0.38; p=0.0180) and that for each 50 rabbit increment mortality is reduced on average by 0.6 percent. Conclusions Mortality rates of approximately 8% are associated with both experimental aneurysm creation and with embolization in the rabbit, elastase-induced aneurysm model. Increasing operator experience is inversely correlated with mortality and the age of the rabbit is positively associated with morbidity. PMID:19001536

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

PubMed

Oki, Hiroshi; Yazawa, Takuya; Baba, Yasuko; Kanegae, Yumi; Sato, Hanako; Sakamoto, Seiko; Goto, Takahisa; Saito, Izumu; Kurahashi, Kiyoyasu

2017-07-01

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 10 9 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Exocrine and endocrine pancreatic function in 21 patients suffering from autoimmune pancreatitis before and after steroid treatment.

PubMed

Frulloni, Luca; Scattolini, Chiara; Katsotourchi, Anna Maria; Amodio, Antonio; Gabbrielli, Armando; Zamboni, Giuseppe; Benini, Luigi; Vantini, Italo

2010-01-01

Autoimmune pancreatitis (AIP) responds rapidly and dramatically to steroid therapy. The aim of this study was to evaluate pancreatic exocrine and endocrine function in patients suffering from AIP both before and after steroid therapy. Fecal elastase 1 and diabetes were evaluated before steroid therapy and within 1 month of its suspension in 21 patients (13 males and 8 females, mean age 43 +/- 16.5 years) diagnosed as having AIP between 2006 and 2008. At clinical onset, fecal elastase 1 was 107 +/- 126 microg/g stool. Thirteen patients (62%) showed severe pancreatic insufficiency (<100 microg/g stool), 4 (19%) had mild insufficiency (100-200 microg/g stool), while 4 (19%) had normal pancreatic function (>200 microg/g stool). Before steroids, diabetes was diagnosed in 5 patients (24%), all of whom had very low levels of fecal elastase 1 (<19 microg/g stool). Following steroids, fecal elastase 1 increased in all patients (237 +/- 193 microg/g stool) and observed levels were significantly higher than those seen before steroids (p = 0.001). Patients suffering from AIP display exocrine and/or endocrine pancreatic insufficiency at clinical onset. These insufficiencies improve after steroid therapy. Copyright 2010 S. Karger AG, Basel.

Mechanistic Insight into the Elastin Degradation Process by the Metalloprotease Myroilysin from the Deep-Sea Bacterium Myroides profundi D25

PubMed Central

Yang, Jie; Zhao, Hui-Lin; Tang, Bai-Lu; Chen, Xiu-Lan; Su, Hai-Nan; Zhang, Xi-Ying; Song, Xiao-Yan; Zhou, Bai-Cheng; Xie, Bin-Bin; Weiss, Anthony S.; Zhang, Yu-Zhong

2015-01-01

Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1′ positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin. PMID:25793427

The anti-dermatophyte activity of Zataria multiflora essential oils.

PubMed

Mahboubi, M; HeidaryTabar, R; Mahdizadeh, E

2017-06-01

Dermtophytes are a group of pathogenic fungi and the major cause of dermatophytosis in humans and animals. Fighting dermatophytes by natural essential oils is one important issue in new researches. In this investigation, we evaluated the anti-dermatophyte activities of three samples of Z. multiflora essential oils against dermatophytes along with analysis of chemical compositions of the essential oils and their anti-elastase activities on elastase production in dermatophytes. Carvacrol (1.5-34.4%), thymol (25.8-41.2%), carvacrol methyl ether (1.9-28.3%) and p-cymene (2.3-8.3%) were the main components of Z. multiflora essential oils. Z. multiflora essential oils (100ppm) inhibited the mycelium growth of dermatophytes (6±1.7-47.0±1.4%) and had the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values of 0.03-0.25μl/ml against dermatophytes. Essential oils inhibited elastase produced in dermatophytes and pure porcine elastase. Z. multiflora essential oils can be used as natural anti-dermatophyte agent for fighting dermatophytes in further preclinical and clinical studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Periodontal status and neutrophilic enzyme levels in gingival crevicular fluid during pregnancy and postpartum.

PubMed

Gürsoy, Mervi; Könönen, Eija; Gürsoy, Ulvi K; Tervahartiala, Taina; Pajukanta, Riitta; Sorsa, Timo

2010-12-01

Pregnancy induces or enhances susceptibility to gingivitis; however, the presence and role of neutrophilic enzymes in pregnancy-related gingivitis are not well known. The present study demonstrates the relationship between neutrophilic enzymes in gingival crevicular fluid (GCF) and periodontal status during pregnancy and postpartum. At baseline, 30 periodontally healthy pregnant women (Pr group) and 24 non-pregnant women (N-Pr group) as their controls participated in the study. The Pr group was examined once per each trimester and twice during postpartum and the N-Pr group three times (on successive months). During each visit, GCF samples were collected from all first molars, and clinical measurements (visible plaque index, bleeding on probing [BOP], probing depth [PD], and clinical attachment level) were recorded. The samples were analyzed for matrix metalloproteinase (MMP)-8, polymorphonuclear neutrophil (PMN) elastase, myeloperoxidase (MPO), and tissue inhibitor of matrix metalloproteinase (TIMP)-1. Their levels were compared to the periodontal status at the collection site. In the Pr group, BOP and PD scores significantly increased between the first and second trimester, indicating pregnancy gingivitis. This increased inflammation was not reflected by the enzymes examined in GCF; the amounts of PMN elastase decreased continuously during the follow-up period, and those of MPO and MMP-8 did not increase until delivery, whereas TIMP-1 amounts remained stable throughout the follow-up period. In the N-Pr group, all parameters remained steady. Despite an increased susceptibility to gingivitis during mid-pregnancy, the host response does not seem to activate its own degradative enzymes.

Longitudinal study of salivary proteinases during pregnancy and postpartum.

PubMed

Gürsoy, M; Könönen, E; Tervahartiala, T; Gürsoy, U K; Pajukanta, R; Sorsa, T

2010-08-01

Matrix metalloproteinases (MMPs) and their regulators are connected to periodontal inflammation and destruction. However, the presence and role of the salivary MMPs in pregnancy-related gingivitis are not well known. Our longitudinal study aimed to monitor salivary proteinase levels and possible changes, and relate them to periodontal status during pregnancy and postpartum. Salivary samples were collected from 30 periodontally healthy pregnant women five times (once during each trimester, 4-6 wk after delivery and after lactation) and, as their controls, from 24 non-pregnant women three times (during successive months). Periodontal examination included visible plaque index, bleeding on probing, probing pocket depth and clinical attachment level measurements. Matrix metalloproteinase-8 levels were measured by immunofluorometric assay, and MMP-2 and MMP-9 levels and molecular forms by gelatin zymography. Salivary elastase, myeloperoxidase and tissue inhibitor of matrix metalloproteinase-1 levels were measured by ELISA. Elastase concentrations maintained stable during the follow-up, while myeloperoxidase concentrations increased significantly after delivery. During pregnancy, MMP-8 concentrations were significantly lower than postpartum concentrations, being lowest during the second trimester and highest after delivery, and varying inversely to pregnancy gingivitis, observed as elevated percentages of bleeding on probing and probing pocket depth during the second and third trimester. In pregnant women, the highest MMP-2 and MMP-9 levels were found in saliva after lactation. In the control group, both clinical and enzymological findings remained stable during the follow-up period. Our results suggest that hormonal changes during pregnancy induce or enhance susceptibility to gingivitis, while salivary proteinase and myeloperoxidase levels are reduced.

Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

PubMed

Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M; Neves-Ferreira, Ana G C; Sánchez, Elda E; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

2016-01-01

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed. Copyright © 2015 Elsevier Inc. All rights reserved.

Gastroprotective and antielastase effects of protein inhibitors from Erythrina velutina seeds in an experimental ulcer model.

PubMed

Oliveira de Lima, Vanessa Cristina; de Araújo Machado, Richele Janaína; Vieira Monteiro, Norberto Kássio; de Lyra, Ibson Lucas; da Silva Camillo, Christina; Coelho Serquiz, Alexandre; Silva de Oliveira, Adeliana; da Silva Rufino, Fabíola Patrícia; Leal Lima Maciel, Bruna; Ferreira Uchôa, Adriana; Antunes Dos Santos, Elizeu; de Araújo Morais, Ana Heloneida

2017-04-01

Trypsin and chymotrypsin inhibitors from Erythrina velutina seeds have been previously isolated by our group. In previous studies using a sepsis model, we demonstrated the antitumor and anti-inflammatory action of these compounds. This study aimed to evaluate the gastroprotective and antielastase effects of protein inhibitors from E. velutina seeds in an experimental stress-induced ulcer model. Two protein isolates from E. velutina seeds, with antitrypsin (PIAT) and antichymotrypsin (PIAQ) activities, were tested. Both protein isolates showed a high affinity and inhibitory effect against human neutrophil elastase, with 84% and 85% inhibition, respectively. Gastric ulcer was induced using ethanol (99%) in 6 groups of animals (female Wistar rats, n = 6). Before ulcer induction, these animals were treated for 5 days with one of the following: (1) PIAT (0.2 mg·kg -1 ), (2) PIAT (0.4 mg·kg -1 ), (3) PIAQ (0.035 mg·kg -1 ), (4) ranitidine hydrochloride (50 mg·kg -1 ), (5) saline solution (0.9%), or (6) no intervention (sham). Both PIAT and PIAQ protected gastric mucosa, preventing hemorrhagic lesions, edema, and mucus loss. No histologic toxic effects of PIAT or PIAQ were seen in liver and pancreatic cells. Our results show that protein isolates from E. velutina seeds have potential gastroprotective effects, placing these compounds as natural candidates for gastric ulcer prevention.

Crystal Structures of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

PubMed Central

Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

2010-01-01

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 Å resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network, and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH’s catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 Å) than previously reported. The higher resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggest a functional convergence between the amidase signature enzymes and serine proteases. PMID:20493882

Crystal structure of fatty acid amide hydrolase bound to the carbamate inhibitor URB597: discovery of a deacylating water molecule and insight into enzyme inactivation.

PubMed

Mileni, Mauro; Kamtekar, Satwik; Wood, David C; Benson, Timothy E; Cravatt, Benjamin F; Stevens, Raymond C

2010-07-23

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 A resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 A) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (4). 6-month repeated dose intravenous toxicity study in rats with 1-month recovery test].

PubMed

Yamaguchi, K; Aze, Y; Shimizu, K; Shichino, Y; Oku, H; Mori, H; Shinomiya, K; Ueda, H; Suzuki, Y; Oida, H; Nishibata, K; Tanaka, M; Yanagizawa, Y; Nanba, T; Nishiyama, K; Yonezawa, H; Fujita, T

1998-07-01

A 6-month repeated dose toxicity study with 1-month recovery test of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in Sprague-Dawley (SD) rats. The rats of both sexes were administered ONO-5046.Na intravenously at a daily dose of 0 (vehicle control), 18.75, 37.5 or 75 mg/kg. ONO-5046.Na did not affect clinical signs, body weight, food consumption, opthalmology, urinalysis, hematology, blood chemistry, organ weight, necropsy or histopathology at any dose. These results indicate that the NOAEL of ONO-5046.Na in rats is 75 mg/kg/day for both sexes in this study.

Extracellular proteases as targets for drug development

PubMed Central

Cudic, Mare

2015-01-01

Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV), cysteine proteases (cathepsin B), and renin system are discussed herein. PMID:19689354

Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica, Manilkara zapota and silymarin: an in vitro comparative study for anti-aging applications.

PubMed

Pientaweeratch, Sirinya; Panapisal, Vipaporn; Tansirikongkol, Anyarporn

2016-09-01

Context Phyllanthus emblica L. (Euphorbiaceae) (amla), Manilkara zapota L.P. Royen (Sapotaceae) (sapota) and silymarin are reported to contain antioxidant effects. However, information on other biological activities relating to the anti-aging properties is limited. Objective To compare in vitro antioxidants, anti-collagenase (MMP-1 and MMP-2) and anti-elastase properties as well as the phenolic and flavonoid contents of amla, sapota and silymarin as potential anti-aging ingredients. Materials and methods The ethanol amla and sapota fruit extracts were prepared by three cycles of maceration with 24 h duration each. The total phenolic (TPC) and flavonoid (TFC) contents were determined. The antioxidant capacity was evaluated by DPPH and ABTS assays. The effects of MMP-1, MMP-2 and elastase inhibitions were determined by using the EnzChek® assay kits (Molecular-Probes, Eugene, OR). Results Amla exhibited the highest in TPC (362.43 ± 11.2 mg GAE/g) while silymarin showed the highest in TFC (21.04 ± 0.67 mg QE/g). Results of antioxidant activity by DPPH and ABTS methods showed that amla possessed the most potent capacity with IC50 values of 1.70 ± 0.07 and 4.45 ± 0.10 μg/mL, respectively. Highest inhibitions against MMP-1, MMP-2 and elastase were detected for sapota with IC50 values of 89.61 ± 0.96, 86.47 ± 3.04 and 35.73 ± 0.61 μg/mL, respectively. Discussion and conclusion Test extracts offered anti-aging properties in different mechanisms. Amla showed the highest phenolic content and antioxidant property with moderate anti-collagenase. Silymarin exhibited measurable flavonoid content with anti-elastase effect. Sapota showed the highest collagenase and elastase inhibitions with moderate antioxidant effect. Thus, extracts might be added as a mixture to gain the overall anti-aging effects.

[The inhibitory effect of elastase on calcium increase in brain and spinal cord of rabbits with atherosclerosis induced by cholesterol-rich diet].

PubMed

Yasui, M; Yano, I; Yoshida, H; Yoshimasu, F; Ota, K; Oshima, A

1989-08-01

The aim of present experiment was to investigate the decalcified effects of exogenous elastase in liver, kidney and central nervous system (CNS) of rabbits with atherosclerosis experimentally induced by the modified procedure of Kritchevsky et al. Twenty five male rabbits, weighing approximately 2 kg, were divided into 6 groups. Animals were fed for 3 months with standard diet (group A), standard diet containing 1.5% cholesterol (group B) and 1.5% cholesterol-rich diet plus intraperitoneal (ip) daily administration of elastase 450 EL. U/kg (group C). Another groups were kept for 6 months with standard diet (group D), standard diet containing 0.67% cholesterol (group E) and 0.67% cholesterol-rich diet plus same dose of elastase (group F). The rabbits treated with cholesterol-rich diet were confirmed to be induced atherosclerosis biochemically as well as histologically. All groups were maintained under these conditions for experimental periods and allowed tap water. After 3 and 6 months, blood collected by cardiocentesis using ether anesthesia and then sacrificed to remove CNS and internal organs. Blood had stood for 1 hour at room temperature. Serum was separated by centrifugation at 3,000 rpm for 10 min to determine total cholesterol, triglyceride, phospholipids, HDL-cholesterol, and so on. Calcium contents in the cerebral frontal lobe, cerebellum, pons, spinal cord, liver and kidney were measured by neutron activation analysis method. In this experiment the amelioration of atherosclerosis by ip administration of elastase was ascertained. In rabbits given cholesterol-rich diet, calcium content in CNS tissues was higher than that of another tissues and paralleled to a rise of serum cholesterol level.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

PubMed

Smith, Karen; Rajendran, Ranjith; Kerr, Stephen; Lappin, David F; Mackay, William G; Williams, Craig; Ramage, Gordon

2015-09-01

In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Four Surgical Modifications to the Classic Elastase Perfusion Aneurysm Model Enable Haemodynamic Alterations and Extended Elastase Perfusion.

PubMed

Busch, Albert; Chernogubova, Ekaterina; Jin, Hong; Meurer, Felix; Eckstein, Hans-Henning; Kim, Mia; Maegdefessel, Lars

2018-04-24

Abdominal aortic aneurysm (AAA) is an individual and socioeconomic burden in today's ageing society. Treatment relies on surgical exclusion of the dilated aorta by open or endovascular repair. For research purposes, animal models are necessary and the elastase induced aneurysm model closely mimics end stage human aneurysm disease. To improve the translational value of this model, four modifications to the classic elastase perfusion procedure (PPE) in relation to human aneurysm morphology were conducted. In ten week old male C57BL/6J wild type mice the PPE procedure was modified in four ways using two different techniques. Flow alteration was simulated by partial ligation of the common iliac artery or the distal aorta. Additionally, careful exploration of the abdominal aortic branches allowed PPE induction at the suprarenal and iliac level. Molecular biology, ultrasound, and immunohistochemistry were used to evaluate these pilot results. Two aortic outflow obstructions simulating distal aortic or iliac stenosis significantly increase murine AAA diameter (p = .046), and affect local vascular wall remodelling. Suprarenal aortic dissection allows a juxtarenal aneurysm to be induced, similar to the angiotensin II induced aneurysm model. A separate investigation for canonical activation of transforming growth factor β in the two embryonically distinct juxtarenal and infrarenal segments showed no distinct difference. Creating an aortoiliac bifurcated aneurysm completes the mimicry of human aneurysm morphology. The alteration of the classic PPE aneurysm by outflow modulation and further elastase perfusion to the juxtarenal and aortoiliac segment modifies morphology and diameter, and thus increases the translational value in future research. Copyright © 2018 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.

Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

NASA Astrophysics Data System (ADS)

Annapoorani, Angusamy; Umamageswaran, Venugopal; Parameswari, Radhakrishnan; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2012-09-01

Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.

Investigation of Pseudomonas aeruginosa quorum-sensing signaling system for identifying multiple inhibitors using molecular docking and structural analysis methodology.

PubMed

Soheili, Vahid; Bazzaz, Bibi Sedigheh Fazly; Abdollahpour, Nooshin; Hadizadeh, Farzin

2015-12-01

Pseudomonas aeruginosa is an opportunistic human pathogen and a common Gram-negative bacterium in hospital-acquired infections. It causes death in many burn victims, cystic-fibrosis and neutropenic-cancer patients. It is known that P. aeruginosa biofilm maturation and production of cell-associated and extracellular virulence factors such as pyocyanin, elastase and rhamnolipids are under the control of a quorum-sensing (QS) system. Among several proteins involved in the Pseudomonas QS mechanism, LasR and PqsE play an important role in its cascade signaling system. They can cause increases in QS factors, biofilm maturation, and the production of virulence factors. Therefore, inhibition of these proteins can reduce the pathogenicity of P. aeruginosa. According to the structure of corresponding auto-inducers bound to these proteins, in silico calculations were performed with some non-steroidal anti-inflammatory drugs (NSAIDs) to estimate possible interactions and find the co-inhibitors of LasR and PqsE. The results showed that oxicams (Piroxicam and Meloxicam) can interact well with active sites of both proteins with the Ki of 119.43 nM and 4.0 μM for Meloxicam and 201.39 nM and 4.88 μM against LasR and PqsE, respectively. These findings suggested that Piroxicam and Meloxicam can be used as potential inhibitors for control of the P. aeruginosa QS signaling system and biofilm formation, and may be used in the design of multiple inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

PubMed Central

Weng, Lixing; Zhang, Yuqian; Yang, Yuxiang; Wang, Lianhui

2014-01-01

Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation. PMID:24736783

Nutritional status, fecal elastase-1, and 13C-labeled mixed triglyceride breath test in the long-term after pancreaticoduodenectomy.

PubMed

Muniz, Cinara Knychala; dos Santos, José Sebastião; Pfrimer, Karina; Ferrioli, Eduardo; Kemp, Rafael; Marchini, Júlio Sérgio; Cunha, Selma Freire

2014-04-01

This study aimed to compare the body composition, dietary intake and serum levels of vitamins and minerals, and exocrine pancreatic function in patients late after pancreaticoduodenectomy (PD) and healthy subjects. Fifteen patients (PD group) who had undergone PD over 1 year before the study and 15 health volunteers (control group) were included in the study. All volunteers underwent dietary intake evaluation, body composition, laboratory data, exocrine pancreatic function by elastase-1, and carbon (C )-labeled triglycerides in breath tests. The PD group subjects also underwent upper gastrointestinal endoscopy and small intestinal bacterial overgrowth analysis. Nutrient intake was adequate, and there were no differences in body mass index and mineral serum levels between the groups. The PD group showed lower serum levels of retinol, α-tocopherol, and ascorbic acid. Small intestinal bacterial overgrowth occurred in 39% of the patients. Fecal elastase-1 was lower in the PD group. The PD group had a higher C peak time; the cumulative label C recovery in 7 hours was similar in both groups. Fecal elastase-1 decreased, and the excretion of C in breath was similar to healthy controls. Although the data point toward an adaptation in the absorptive capacity of fats, A, C, and E hypovitaminosis indicate that some absorptive insufficiency persists late after PD.

Post-fertilization effect of bilateral primary testicular damage induced by unilateral cryptorchidism in the rat model.

PubMed

Tsounapi, P; Honda, M; Dimitriadis, F; Shimizu, S; Hikita, K; Muraoka, K; Sejima, T; Saito, M; Tomita, S; Sofikitis, N; Takenaka, A

2016-03-01

Cryptorchidism, a common anomaly of the male genitalia, affects 2-4% of male infants. The post-fertilization effects of unilateral cryptorchidism model in the rat and the effects of antioxidant treatment were investigated. Six-week-old male Wistar rats were randomly separated into four groups. Unilateral cryptorchidism was induced in the right testis of three groups. One group was treated with saline intraperitoneally (i.p.) (Crypto), one group was treated with taurine (500 mg/kg, i.p.; Tau), and another group was treated with sivelestat (15 mg/kg i.p.; Siv). The control group was treated with saline i.p. The treatment was daily for 8 weeks. Five days before sacrifice, mating studies were performed. Body, testicular, and epididymal weights were recorded. Malondialdehyde (MDA) levels in the seminal vesicular fluid (SVF) were measured. Testicular levels of MDA and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were determined bilaterally. TUNEL assay was used to examine DNA fragmentation bilaterally. Histological examination and the Johnsen score were used to evaluate morphological testicular alterations. The Crypto group demonstrated significantly lower right testicular and epididymal weights, significantly increased SVF-MDA levels, testicular MDA and 8-OHdG levels, and the apoptotic score bilaterally compared to the controls. Furthermore, histological evaluation revealed significantly reduced spermatogenesis and mild injury to the cryptorchid testes compared to the control. Treatment with both taurine and sivelestat significantly reduced SVF-MDA levels, testicular MDA, 8-OHdG, and apoptosis bilaterally compared to the Crypto group. Antioxidant treatment was unable to ameliorate spermatogenesis. Newborns delivered by females that mated with Crypto-males had significantly lower body weight compared with the respective animals from the control, Tau and Siv groups. The present study demonstrated that unilateral cryptorchidism-induced testicular damage can significantly affect the contralateral testis as well having further deleterious post-fertilization effect on the development of newborns. Treatment with antioxidants can partially improve the testicular damage bilaterally with beneficial effects for the newborns. © 2016 American Society of Andrology and European Academy of Andrology.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum

PubMed Central

Kautto, Liisa; Nevalainen, Helena

2017-01-01

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases. PMID:28060882

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

PubMed

Han, Zhiping; Kautto, Liisa; Nevalainen, Helena

2017-01-01

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

Elevated Neutrophil Elastase in Tears of Ocular Graft-Versus-Host Disease Patients.

PubMed

Arafat, Samer N; Robert, Marie-Claude; Abud, Tulio; Spurr-Michaud, Sandra; Amparo, Francisco; Dohlman, Claes H; Dana, Reza; Gipson, Ilene K

2017-04-01

To investigate the levels of neutrophil elastase (NE), matrix metalloproteinases (MMPs), and myeloperoxidase (MPO) in tear washes of patients with ocular graft-vs-host disease (oGVHD). Case-control study. Based on established criteria, oGVHD patients (n = 14; 28 eyes) and age-/sex-matched healthy controls (n = 14; 28 eyes) were enrolled. Tear washes were collected and analyzed for NE using a single-analyte enzyme-linked immunosorbent assay (ELISA). MMPs (1, 2, 3, 7, 8, 9, 12), MPO, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were analyzed using multianalyte bead-based ELISA assays. Total MMP activity was measured using a fluorimetric assay. Correlation studies were performed between NE, MMP-8, MMP-9, and MPO within study groups. NE, MMP-8, MMP-9, and MPO levels were elevated in oGVHD tears when compared with controls (P < .0001). NE was the most elevated analyte. MMP activity was higher and TIMP-1 levels were lower in oGVHD than in control (P < .0001). In oGVHD, NE significantly correlated with MMP-8 (r = 0.92), MMP-9 (r = 0.90), and MPO (r = 0.79) (P < .0001). MMP-8 correlated with MMP-9 (r = 0.96, P < .0001), and MPO (r = 0.60, P = .001). MMP-9 correlated with MPO (r = 0.55, P = .002). In controls, NE, MMP-9, and MPO significantly correlated with each other (P < .0001). The marked increase in NE in oGVHD tears that correlated strongly with elevated MMP-8, MMP-9, and MPO suggests a common neutrophilic source and provides evidence of neutrophil activity on the ocular surface of oGVHD patients. Copyright © 2017 Elsevier Inc. All rights reserved.

AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema.

PubMed

Cheng, Xiao-Yu; Li, Yang-Yang; Huang, Cheng; Li, Jun; Yao, Hong-Wei

2017-04-04

Current drug therapy fails to reduce lung destruction of chronic obstructive pulmonary disease (COPD). AMP-activated protein kinase (AMPK) has emerged as an important integrator of signals that control energy balance and lipid metabolism. However, there are no studies regarding the role of AMPK in reducing inflammatory responses and cellular senescence during the development of emphysema. Therefore, we hypothesize that AMPK reduces inflammatroy responses, senescence, and lung injury. To test this hypothesis, human bronchial epithelial cells (BEAS-2B) and small airway epithelial cells (SAECs) were treated with cigarette smoke extract (CSE) in the presence of a specific AMPK activator (AICAR, 1 mM) and inhibitor (Compound C, 5 μM). Elastase injection was performed to induce mouse emphysema, and these mice were treated with a specific AMPK activator metformin as well as Compound C. AICAR reduced, whereas Compound C increased CSE-induced increase in IL-8 and IL-6 release and expression of genes involved in cellular senescence. Knockdown of AMPKα1/α2 increased expression of pro-senescent genes (e.g., p16, p21, and p66shc) in BEAS-2B cells. Prophylactic administration of an AMPK activator metformin (50 and 250 mg/kg) reduced while Compound C (4 and 20 mg/kg) aggravated elastase-induced airspace enlargement, inflammatory responses and cellular senescence in mice. This is in agreement with therapeutic effect of metformin (50 mg/kg) on airspace enlargement. Furthermore, metformin prophylactically protected against but Compound C further reduced mitochondrial proteins SOD2 and SIRT3 in emphysematous lungs. In conclusion, AMPK reduces abnormal inflammatory responses and cellular senescence, which implicates as a potential therapeutic target for COPD/emphysema.

Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937.

PubMed

Lindmark, A; Persson, A M; Olsson, I

1990-12-01

The processing of the neutral proteases cathepsin G and neutrophil elastase, normally synthesized in myeloid precursor cells and stored in azurophil granules, were investigated by biosynthetic labeling with 14C-leucine of the monoblastic cell line U-937. The proteases were precipitated with specific antibodies and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. The transfer to lysosomes of newly synthesized proteases was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenates in a Percoll gradient. The presence of a closely spaced polypeptide band-doublet at intermediate gradient density suggested cleavage of the specific aminoterminal pro dipeptide extension before storage in lysosomes. The molecular heterogeneity observed for cathepsin G and neutrophil elastase seemed to be due to modifications occurring after sorting into lysosomes, most likely because of C-terminal processing. Modifications of the secreted enzymes were not detectable by SDS-PAGE. In contrast to other lysosomal enzymes, no phosphorylation was demonstrated. Newly synthesized cathepsin G and neutrophil elastase rapidly became resistant to endoglycosidase H, indicating transport through the medial and trans cisternae of the Golgi complex and conversion to "complex" oligosaccharide side chains. This conversion was inhibited by an agent swainsonine, but translocation from the Golgi complex and secretion were unaffected. The processing described may play a role in activation of the proteases.

Effect of conformational mobility and hydrogen-bonding interactions on the selectivity of some guanidinoaryl-substituted mechanism-based inhibitors of trypsin-like serine proteases.

PubMed

Rai, R; Katzenellenbogen, J A

1992-11-13

Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

PubMed

Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

2016-12-01

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PubMed Central

Blalock, Timothy D.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Gipson, Ilene K.

2008-01-01

Purpose Three membrane-associated mucins (MAMs)—MUC1, MUC4 and MUC16—are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. Methods An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to MUC16’s cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in release/shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor (TNF), TNF-α-converting enzyme, and matrix metalloproteinases-7 and –9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. Results MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced release of MUC16 but not MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16—78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Conclusions Results suggest that extracellular domains of MUC1, 4, and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and TNF present in higher amounts in dry eye patients’ tears may cause MAM release—allowing rose bengal staining. PMID:18436821

Novel inhibitors of the Pseudomonas aeruginosa virulence factor LasB: a potential therapeutic approach for the attenuation of virulence mechanisms in pseudomonal infection.

PubMed

Cathcart, George R A; Quinn, Derek; Greer, Brett; Harriott, Pat; Lynas, John F; Gilmore, Brendan F; Walker, Brian

2011-06-01

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed "second-generation" antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (K(i) = 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

Regulator of calcineurin 1 controls growth plasticity of adult pancreas.

PubMed

Gurda, Grzegorz T; Crozier, Stephen J; Ji, Baoan; Ernst, Stephen A; Logsdon, Craig D; Rothermel, Beverly A; Williams, John A

2010-08-01

Growth of exocrine pancreas is regulated by gastrointestinal hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates Nuclear Factor of Activated T cells (NFATs), but the genetic underpinnings and feedback mechanisms that regulate this response are not known. Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI stimulation was performed on Affymetrix 430A chips, and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with "flox-on" Rcan1 mice. CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1-8 hours course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice. We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator, and firmly establish that CN-NFAT signaling is required for this response. Copyright (c) 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Pharmacological inhibition of cathepsin K: A promising novel approach for postmenopausal osteoporosis therapy.

PubMed

Mukherjee, Kakoli; Chattopadhyay, Naibedya

2016-10-01

Osteoporosis is a metabolic bone disease that is characterized by heightened state of bone resorption accompanied by diminished bone formation, leading to a reduction of bone mineral density (BMD) and deterioration of bone quality, thus increasing the risk of developing fractures. Molecular insight into bone biology identified cathepsin K (CatK) as a novel therapeutic target. CatK is a lysosomal cysteine protease secreted by activated osteoclasts during bone resorption, whose primary substrate is type I collagen, the major component of organic bone matrix. Available anti-resorptive drugs affect osteoclast survival and influence both resorption and formation of bone. CatK inhibitors are distinct from the existing anti-resorptives as they only target the resorption process itself without impairing osteoclast differentiation and do not interfere with bone formation. An inhibitor of CatK, odanacatib, robustly increased both trabecular and cortical BMD in postmenopausal osteoporosis patients. The phase III fracture prevention trial with odanacatib ended early due to good efficacy and a favorable benefit/risk profile, thus, enhancing the opportunity for CatK as a pharmacological target for osteoporosis. So far, all the inhibitors that reached to the stage of clinical trial targeted active site of CatK to abrogate the entire proteolytic activity of the enzyme in addition to the desired blockage of excessive elastin and collagen degradation, and could thus pose safety concerns with long term use. Identification of selective exosite inhibitors that inhibit CatK's elastase and/or collagenase activity but do not affect the hydrolysis of other physiologically relevant substrates of CatK would be an improved strategy to inhibit this enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

Neutrophil elastase mediates acute pathogenesis and is a determinant of long-term behavioral recovery after traumatic injury to the immature brain.

PubMed

Semple, Bridgette D; Trivedi, Alpa; Gimlin, Kayleen; Noble-Haeusslein, Linda J

2015-02-01

While neutrophil elastase (NE), released by activated neutrophils, is a key mediator of secondary pathogenesis in adult models of brain ischemia and spinal cord injury, no studies to date have examined this protease in the context of the injured immature brain, where there is notable vulnerability resulting from inadequate antioxidant reserves and prolonged exposure to infiltrating neutrophils. We thus reasoned that NE may be a key determinant of secondary pathogenesis, and as such, adversely influence long-term neurological recovery. To address this hypothesis, wild-type (WT) and NE knockout (KO) mice were subjected to a controlled cortical impact at post-natal day 21, approximating a toddler-aged child. To determine if NE is required for neutrophil infiltration into the injured brain, and whether this protease contributes to vasogenic edema, we quantified neutrophil numbers and measured water content in the brains of each of these genotypes. While leukocyte trafficking was indistinguishable between genotypes, vasogenic edema was markedly attenuated in the NE KO. To determine if early pathogenesis is dependent on NE, indices of cell death (TUNEL and activated caspase-3) were quantified across genotypes. NE KO mice showed a reduction in these markers of cell death in the injured hippocampus, which corresponded to greater preservation of neuronal integrity as well as reduced expression of heme oxygenase-1, a marker of oxidative stress. WT mice, treated with a competitive inhibitor of NE at 2, 6 and 12h post-injury, likewise showed a reduction in cell death and oxidative stress compared to vehicle-treated controls. We next examined the long-term behavioral and structural consequences of NE deficiency. NE KO mice showed an improvement in long-term spatial memory retention and amelioration of injury-induced hyperactivity. However, volumetric and stereological analyses found comparable tissue loss in the injured cortex and hippocampus independent of genotype. Further, WT mice treated acutely with the NE inhibitor showed no long-term behavioral or structural improvements. Together, these findings validate the central role of NE in both acute pathogenesis and chronic functional recovery, and support future exploration of the therapeutic window, taking into account the prolonged period of neutrophil trafficking into the injured immature brain. Copyright © 2014 Elsevier Inc. All rights reserved.

Neutrophil elastase mediates acute pathogenesis and is a determinant of long-term behavioral recovery after traumatic injury to the immature brain

PubMed Central

Semple, Bridgette D; Trivedi, Alpa; Gimlin, Kayleen; Noble-Haeusslein, Linda J

2014-01-01

While neutrophil elastase (NE), released by activated neutrophils, is a key mediator of secondary pathogenesis in adult models of brain ischemia and spinal cord injury, no studies to date have examined this protease in the context of the injured immature brain, where there is notable vulnerability resulting from inadequate antioxidant reserves and prolonged exposure to infiltrating neutrophils. We thus reasoned that NE may be a key determinant of secondary pathogenesis, and as such, adversely influence long-term neurological recovery. To address this hypothesis, wild-type (WT) and NE knockout (KO) mice were subjected to a controlled cortical impact at post-natal day 21, approximating a toddler-aged child. To determine if NE is required for neutrophil infiltration into the injured brain, and whether this protease contributes to vasogenic edema, we quantified neutrophil numbers and measured water content in the brains of each of these genotypes. While leukocyte trafficking was indistinguishable between genotypes, vasogenic edema was markedly attenuated in the NE KO. To determine if early pathogenesis is dependent on NE, indices of cell death (TUNEL and activated caspase-3) were quantified across genotypes. NE KO mice showed a reduction in these markers of cell death in the injured hippocampus, which corresponded to greater preservation of neuronal integrity as well as reduced expression of heme oxygenase-1, a marker of oxidative stress. WT mice, treated with a competitive inhibitor of NE at 2, 6 and 12 h post-injury, likewise showed a reduction in cell death and oxidative stress compared to vehicle-treated controls. We next examined the long-term behavioral and structural consequences of NE deficiency. NE KO mice showed an improvement in long-term spatial memory retention and amelioration of injury-induced hyperactivity. However, volumetric and stereological analyses found comparable tissue loss in the injured cortex and hippocampus independent of genotype. Further, WT mice treated acutely with the NE inhibitor showed no long-term behavioral or structural improvements. Together, these findings validate the central role of NE in both acute pathogenesis and chronic functional recovery, and support future exploration of the therapeutic window, taking into account the prolonged period of neutrophil trafficking into the injured immature brain. PMID:25497734

Moderate Aerobic Training Improves Cardiorespiratory Parameters in Elastase-Induced Emphysema

PubMed Central

Henriques, Isabela; Lopes-Pacheco, Miquéias; Padilha, Gisele A.; Marques, Patrícia S.; Magalhães, Raquel F.; Antunes, Mariana A.; Morales, Marcelo M.; Rocha, Nazareth N.; Silva, Pedro L.; Xisto, Débora G.; Rocco, Patricia R. M.

2016-01-01

Aim: We investigated the therapeutic effects of aerobic training on lung mechanics, inflammation, morphometry and biological markers associated with inflammation, and endothelial cell damage, as well as cardiac function in a model of elastase-induced emphysema. Methods: Eighty-four BALB/c mice were randomly allocated to receive saline (control, C) or 0.1 IU porcine pancreatic elastase (emphysema, ELA) intratracheally once weekly for 4 weeks. After the end of administration period, once cardiorespiratory impairment associated with emphysema was confirmed, each group was further randomized into sedentary (S) and trained (T) subgroups. Trained mice ran on a motorized treadmill, at moderate intensity, 30 min/day, 3 times/week for 4 weeks. Results: Four weeks after the first instillation, ELA animals, compared to C, showed: (1) reduced static lung elastance (Est,L) and levels of vascular endothelial growth factor (VEGF) in lung tissue, (2) increased elastic and collagen fiber content, dynamic elastance (E, in vitro), alveolar hyperinflation, and levels of interleukin-1β and tumor necrosis factor (TNF)-α, and (3) increased right ventricular diastolic area (RVA). Four weeks after aerobic training, ELA-T group, compared to ELA-S, was associated with reduced lung hyperinflation, elastic and collagen fiber content, TNF-α levels, and RVA, as well as increased Est,L, E, and levels of VEGF. Conclusion: Four weeks of regular and moderate intensity aerobic training modulated lung inflammation and remodeling, thus improving pulmonary function, and reduced RVA and pulmonary arterial hypertension in this animal model of elastase-induced emphysema. PMID:27536247

Structurally Related Monoterpenes p-Cymene, Carvacrol and Thymol Isolated from Essential Oil from Leaves of Lippia sidoides Cham. (Verbenaceae) Protect Mice against Elastase-Induced Emphysema.

PubMed

Games, Ellen; Guerreiro, Marina; Santana, Fernanda R; Pinheiro, Nathalia M; de Oliveira, Emerson A; Lopes, Fernanda D T Q S; Olivo, Clarice R; Tibério, Iolanda F L C; Martins, Mílton A; Lago, João Henrique G; Prado, Carla M

2016-10-20

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow obstruction and inflammation. Natural products, such as monoterpenes, displayed anti-inflammatory and anti-oxidant activities and can be used as a source of new compounds to COPD treatment. Our aim was to evaluate, in an elastase-induced pulmonary emphysema in mice, the effects of and underlying mechanisms of three related natural monoterpenes ( p -cymene, carvacrol and thymol) isolated from essential oil from leaves Lippia sidoides Cham. (Verbenaceae). Mices received porcine pancreatic elastase (PPE) and were treated with p -cymene, carvacrol, thymol or vehicle 30 min later and again on 7th, 14th and 28th days. Lung inflammatory profile and histological sections were evaluated. In the elastase-instilled animals, the tested monoterpenes reduced alveolar enlargement, macrophages and the levels of IL-1β, IL-6, IL-8 and IL-17 in bronchoalveolar lavage fluid (BALF), and collagen fibers, MMP-9 and p-65-NF-κB-positive cells in lung parenchyma ( p < 0.05). All treatments attenuated levels of 8-iso-PGF2α but only thymol was able to reduced exhaled nitric oxide ( p < 0.05). Monoterpenes p -cymene, carvacrol and thymol reduced lung emphysema and inflammation in mice. No significant differences among the three monoterpenes treatments were found, suggesting that the presence of hydroxyl group in the molecular structure of thymol and carvacrol do not play a central role in the anti-inflammatory effects.

NETosis Delays Diabetic Wound Healing in Mice and Humans.

PubMed

Fadini, Gian Paolo; Menegazzo, Lisa; Rigato, Mauro; Scattolini, Valentina; Poncina, Nicol; Bruttocao, Andrea; Ciciliot, Stefano; Mammano, Fabio; Ciubotaru, Catalin Dacian; Brocco, Enrico; Marescotti, Maria Cristina; Cappellari, Roberta; Arrigoni, Giorgio; Millioni, Renato; Vigili de Kreutzenberg, Saula; Albiero, Mattia; Avogaro, Angelo

2016-04-01

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

In vitro determination of the anti-aging potential of four southern African medicinal plants

PubMed Central

2013-01-01

Background Aging is an inevitable process for all living organisms. During this process reactive oxygen species generation is increased which leads to the activation of hyaluronidase, collagenase and elastase, which can further contribute to skin aging. Four southern African medicinal plants; Clerodendrum glabrum, Schotia brachypetala, Psychotria capensis and Peltophorum africanum, were investigated to assess their anti-aging properties. Methods Anti-elastase, anti-collagenase and anti-hyaluronidase activities of twenty-eight samples, consisting of methanol and ethyl acetate extracts of the four plants, were determined using spectrophotometric methods. Radical scavenging activity was determined by the ability of the plant extracts to scavenge the ABTS•+ radical. Results The majority of the samples in the anti-elastase assay and nine in the anti-collagenase assay showed more than 80% inhibition. The ethyl acetate extract of S. brachypetala bark and leaves of P. capensis inhibited elastase activity by more than 90%. The methanol extract of S. brachypetala bark contained the highest anti-hyaluronidase activity (75.13 ± 7.49%) whilst the ethyl acetate extract of P. africanum bark exhibited the highest antioxidant activity (IC50: 1.99 ± 0.23 μg/ml). Conclusion The free radical scavenging activity and enzyme inhibitory activity of the plant extracts investigated suggests that they can help restore skin elasticity and thereby slow the wrinkling process. P. africanum was the plant with the most promising activity and will be subjected to further testing and isolation of the active compound/s. PMID:24188320

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

PubMed Central

Horwitz, Marshall S.; Jenne, Dieter E.; Gauthier, Francis

2010-01-01

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies. PMID:21079042

Granulocyte elastase activity and PGE2 levels in gingival crevicular fluid in relation to the presence of subgingival periodontopathogens in subjects with untreated adult periodontitis.

PubMed

Jin, L J; Söder, P O; Leung, W K; Corbet, E F; Samaranayake, L P; Söder, B; Davies, W I

1999-08-01

This study aimed to determine the association between the levels of granulocyte elastase and prostaglandin E2 (PGE2) in GCE and the concomitant presence of periodontopathogens in untreated adult periodontitis (AP). GCF and subgingival plaque were sampled by paper strips and paper points respectively, from various periodontal sites in 16 AP subjects. Granulocyte elastase activity in GCF was analyzed with a low molecular weight substrate specific for granulocyte elastase, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA, mAbs/min/site) was calculated. PGE2 levels in GCF were determined by radioimmunoassay. 5 species-specific DNA probes were used to detect the presence of A. actinomyceterncomitans (A.a., ATCC 43718), B. forsythus (B.f, ATCC 43037), P. gingivalis (P.g., ATCC 33277), P. intermedia (P.i., ATCC 33563), and T. denticola (T.d., ATCC 35405), with a sensitivity of 10(3) cells/paper point. No A.a. was detectable from all sites sampled. The predominant combination of species detected was B.f., P.g., P.i. & T.d. and it was significantly higher at periodontitis sites (68%) than at healthy (7%) or gingivitis sites (29%) (p<0.05). Overall, MR-EA values were strongly correlated with PGE2 levels (r=0.655, p<0.001), especially at these periodontitis sites co-infected by B.f., P.g., P.i. & T.d. (r=0.722, p<0.001). The periodontitis sites co-infected by the 4 species were observable from 15 subjects. These sites were sub-grouped into 8 subjects with a high MR-EA and 7 subjects with a low MR-EA. The PGE2 levels in the high MR-EA group were significantly higher than in the low MR-EA group (p<0.05). No significant differences in clinical or bacterial data were found between the two groups. While within the high MR-EA group, similar results were found between the paired periodontitis sites in each subject with highest and lowest MR-EA values. This study shows that the local host response to bacterial challenge in untreated periodontal pockets is diverse in terms of the intensity of inflammatory response measured by granulocyte elastase and PGE2 levels in GCE A more thorough evaluation of the risk for active periodontal disease may involve the combined approaches to the test of the dynamic bacteria-host relations.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly).

PubMed

Gomes, Carlos E M; Barbosa, Aulus E A D; Macedo, Leonardo L P; Pitanga, Joelma C M; Moura, Fabiano T; Oliveira, Adeliana S; Moura, Raniere M; Queiroz, Alexandre F S; Macedo, Francisco P; Andrade, Lúcia B S; Vidal, Márcia S; Sales, Mauricio P

2005-12-01

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.

Comparison of monoclonal and polyclonal ELISAs for fecal elastase in patients with cystic fibrosis and pancreatic insufficiency.

PubMed

Borowitz, Drucy; Lin, Rong; Baker, Susan S

2007-02-01

Two enzyme-linked immunosorbent assay methodologies are used to detect pancreatic insufficiency: monoclonal and polyclonal. We sought to compare these assays in patients with cystic fibrosis and to correlate these with the coefficient of fat absorption (CFA). As part of a larger study, subjects had stool elastase measured by both methods while taking exogenous enzymes. Subjects subsequently stopped enzymes and had a fecal fat balance study performed; the CFA was then calculated. One hundred twenty-four subjects participated in this substudy. The median values for the monoclonal and polyclonal assays were 0.3 and 22.75 microg/g, respectively. The correlation coefficient between the 2 tests was 0.86 (P < 0.0001). The correlation coefficients between CFA and the monoclonal and polyclonal assays were 0.24 (P = 0.006) and 0.10 (P = 0.27), respectively. If the criterion standard definition of pancreatic insufficiency was set at a CFA < 90% and the test definition of pancreatic insufficiency was set at <100 microg/g, then the monoclonal and polyclonal assay positive predictive values were 97.6% (120 of 123) and 97.4% (111 of 114), respectively. The positive predictive value of both monoclonal and polyclonal fecal elastase in patients with cystic fibrosis is extremely good; however, correlation of either test with CFA was poor. The median value for the polyclonal elastase assay is higher than for the monoclonal assay, which could potentially lead to lower sensitivity of the polyclonal assay at lower cutpoints for the monoclonal assay is used.

Expression of IL-17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients.

PubMed

Żbikowska-Gotz, Magdalena; Pałgan, Krzysztof; Gawrońska-Ukleja, Ewa; Kuźmiński, Andrzej; Przybyszewski, Michał; Socha, Ewa; Bartuzi, Zbigniew

2016-03-01

Lymphocytes Th17 and other types of immune system cells produce IL17. By induction of cytokines and chemokines, the IL17 cytokine is involved in mechanisms of allergic reaction with participation of neutrophil granulocytes. It affects activation, recruitment, and migration of neutrophils to the tissues, regulating inflammatory reaction intensity. Excited neutrophils secrete inter alia elastase and reactive oxygen species (ROS)--significant mediators of inflammation process responsible for tissues damage.The aim of the study was to evaluate the concentrations of serum interleukin 17A, serum neutrophil elastase, and ROS production by neutrophils in patients with food allergy.The study included 30 patients with food allergy diagnosed based on interview, clinical symptoms, positive SPT, placebo controlled double-blind oral provocation trial, and the presence of asIgE in blood serum against selected food allergens using fluoro-immuno-enzymatic method FEIA UNICap 100. The control group consisted of 10 healthy volunteers. The concentrations of IL17A were determined in all patients using ELISA method with eBioscience kits, and elastase using BenderMed Systems kits. Chemiluminescence of non-stimulated neutrophils was evaluated using luminol-dependent kinetic method for 40 min on Luminoskan (Labsystems luminometer).The results of serum IL-17A concentrations and the values of chemiluminescence obtained by non-activated neutrophils, as well as elastase concentrations, were higher in patients with food allergic hypersensitivity compared to healthy volunteers.This study demonstrates a significance of IL-17A and activated neutrophil granulocytes in the course of diseases with food allergic hypersensitivity. © The Author(s) 2015.

Chronic fatigue syndrome: exercise performance related to immune dysfunction.

PubMed

Nijs, Jo; Meeus, Mira; McGregor, Neil R; Meeusen, Romain; de Schutter, Guy; van Hoof, Elke; de Meirleir, Kenny

2005-10-01

To date, the exact cause of abnormal exercise response in chronic fatigue syndrome (CFS) remains to be revealed, but evidence addressing intracellular immune deregulation in CFS is growing. Therefore, the aim of this cross-sectional study was to examine the interactions between several intracellular immune variables and exercise performance in CFS patients. After venous blood sampling, subjects (16 CFS patients) performed a maximal exercise stress test on a bicycle ergometer with continuous monitoring of cardiorespiratory variables. The following immune variables were assessed: the ratio of 37 kDa Ribonuclease (RNase) L to the 83 kDa native RNase L (using a radiolabeled ligand/receptor assay), RNase L enzymatic activity (enzymatic assay), protein kinase R activity assay (comparison Western blot), elastase activity (enzymatic-colorimetric assay), the percent of monocytes, and nitric oxide determination (for monocytes and lymphocytes; flow cytometry, live cell assay). Forward stepwise multiple regression analysis revealed 1) that elastase activity was the only factor related to the reduction in oxygen uptake at a respiratory exchange ratio (RER) of 1.0 (regression model: R = 0.53, F (1,14) = 15.5, P < 0.002; elastase activity P < 0.002); 2) that the protein kinase R activity was the principle factor related to the reduction in workload at RER = 1.0; and 3) that elastase activity was the principle factor related to the reduction in percent of target heart rate achieved. These data provide evidence for an association between intracellular immune deregulation and exercise performance in patients with CFS. To establish a causal relationship, further study of these interactions using a prospective longitudinal design is required.

Designing cellulosic and nanocellulosic sensors for interface with a protease sequestrant wound-dressing prototype: Implications of material selection for dressing and protease sensor design.

PubMed

Fontenot, Krystal R; Edwards, J Vincent; Haldane, David; Pircher, Nicole; Liebner, Falk; Condon, Brian D; Qureshi, Huzaifah; Yager, Dorne

2017-11-01

Interfacing nanocellulosic-based biosensors with chronic wound dressings for protease point of care diagnostics combines functional material properties of high specific surface area, appropriate surface charge, and hydrophilicity with biocompatibility to the wound environment. Combining a protease sensor with a dressing is consistent with the concept of an intelligent dressing, which has been a goal of wound-dressing design for more than a quarter century. We present here biosensors with a nanocellulosic transducer surface (nanocrystals, nanocellulose composites, and nanocellulosic aerogels) immobilized with a fluorescent elastase tripeptide or tetrapeptide biomolecule, which has selectivity and affinity for human neutrophil elastase present in chronic wound fluid. The specific surface area of the materials correlates with a greater loading of the elastase peptide substrate. Nitrogen adsorption and mercury intrusion studies revealed gas permeable systems with different porosities (28-98%) and pore sizes (2-50 nm, 210 µm) respectively, which influence water vapor transmission rates. A correlation between zeta potential values and the degree of protease sequestration imply that the greater the negative surface charge of the nanomaterials, the greater the sequestration of positively charged neutrophil proteases. The biosensors gave detection sensitivities of 0.015-0.13 units/ml, which are at detectable human neutrophil elastase levels present in chronic wound fluid. Thus, the physical and interactive biochemical properties of the nano-based biosensors are suitable for interfacing with protease sequestrant prototype wound dressings. A discussion of the relevance of protease sensors and cellulose nanomaterials to current chronic wound dressing design and technology is included.

[Position paper: Exocrine pancreatic insufficiency and diabetes mellitus].

PubMed

Weitgasser, Raimund; Abrahamian, Heidemarie; Clodi, Martin; Fortunat, Werner; Hammer, Heinz

2012-12-01

Exocrine pancreatic insufficiency in diabetic patients is frequent. Studies based on fecal elastase-1 measurement give prevalence rates of about 50 % in type 1 and 33 % in type 2 diabetic patients. Nevertheless, not all patients report typical symptoms like diarrhea, steatorrhea and weight loss. For indirect testing the determination of fecal elastase-1 has the highest sensitivity and specificity. This test should be performed at least in all symptomatic patients. For differential diagnosis celiac disease (with a prevalence of about 3-5 % of type 1 diabetic patients), autonomic neuropathy, but also diseases like irritable colon and gastrointestinal tumors have to be taken into account. Patients with symptoms and a fecal elastase-1 < 100 µg/g should be treated with pancreas enzymes in adequate daily doses administered at main meals. Treatment improves symptoms significantly, supply with fat soluble vitamins is normalised, risk for osteoporosis is reduced. An improvement of glucose metabolism is but not seen in all studies. A pancreatogenic diabetes, also termed as type 3c diabetes, has not primarily to be treated with insulin, often-at least initially-treatment with oral antidiabetic drugs is possible and sufficient.

Alendronate inhalation ameliorates elastase-induced pulmonary emphysema in mice by induction of apoptosis of alveolar macrophages.

PubMed

Ueno, Manabu; Maeno, Toshitaka; Nishimura, Satoshi; Ogata, Fusa; Masubuchi, Hiroaki; Hara, Kenichiro; Yamaguchi, Kouichi; Aoki, Fumiaki; Suga, Tatsuo; Nagai, Ryozo; Kurabayashi, Masahiko

2015-03-10

Alveolar macrophages play a crucial role in the pathogenesis of emphysema, for which there is currently no effective treatment. Bisphosphonates are widely used to treat osteoclast-mediated bone diseases. Here we show that delivery of the nitrogen-containing bisphosphonate alendronate via aerosol inhalation ameliorates elastase-induced emphysema in mice. Inhaled, but not orally ingested, alendronate inhibits airspace enlargement after elastase instillation, and induces apoptosis of macrophages in bronchoalveolar fluid via caspase-3- and mevalonate-dependent pathways. Cytometric analysis indicates that the F4/80(+)CD11b(high)CD11c(mild) population characterizing inflammatory macrophages, and the F4/80(+)CD11b(mild)CD11c(high) population defining resident alveolar macrophages take up substantial amounts of the bisphosphonate imaging agent OsteoSense680 after aerosol inhalation. We further show that alendronate inhibits macrophage migratory and phagocytotic activities and blunts the inflammatory response of alveolar macrophages by inhibiting nuclear factor-κB signalling. Given that the alendronate inhalation effectively induces apoptosis in both recruited and resident alveolar macrophages, we suggest this strategy may have therapeutic potential for the treatment of emphysema.

Cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema.

PubMed

Longhini-Dos-Santos, Nathalia; Barbosa-de-Oliveira, Valter Abraão; Kozma, Rodrigo Heras; Faria, Carolina Arruda de; Stessuk, Talita; Frei, Fernando; Ribeiro-Paes, João Tadeu

2013-04-01

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p < 0.05) were observed among the groups treated with BMMC and evaluated after 7, 14 and 21 days. Analysis of the mean linear intercept (Lm) values for the different groups allowed to observe that the group treated with BMMC and evaluated after 21 days showed the most significant result. The group that received no treatment showed a statistically significant difference when compared to other groups, except the group treated and evaluated after 21 days, evidencing the efficacy of cell therapy with BMMC in pulmonary emphysema.

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

PubMed

Massberg, Steffen; Grahl, Lenka; von Bruehl, Marie-Luise; Manukyan, Davit; Pfeiler, Susanne; Goosmann, Christian; Brinkmann, Volker; Lorenz, Michael; Bidzhekov, Kiril; Khandagale, Avinash B; Konrad, Ildiko; Kennerknecht, Elisabeth; Reges, Katja; Holdenrieder, Stefan; Braun, Siegmund; Reinhardt, Christoph; Spannagl, Michael; Preissner, Klaus T; Engelmann, Bernd

2010-08-01

Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

Prospective and randomised evaluation of the protease-modulating effect of oxidised regenerated cellulose/collagen matrix treatment in pressure sore ulcers.

PubMed

Kloeters, Oliver; Unglaub, Frank; de Laat, Erik; van Abeelen, Marjolijn; Ulrich, Dietmar

2016-12-01

In chronic wounds, excess levels and activity of proteases such as elastase and plasmin have been detected. Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) has been reported to ameliorate the wound microenvironment by binding and inactivating excess proteases in wound exudates. In this study, the levels and activity of elastase and plasmin in wound exudates of pressure sore ulcers were measured to determine the beneficial effect of ORC/collagen matrix treatment compared with control treatment with a foam dressing. A total of 33 patients with pressure sores were enrolled in the study and were followed up for 12 weeks after treatment. Ten control patients were treated with a foam hydropolymer dressing (TIELLE ® , Systagenix), and the remaining 23 patients were treated with ORC/collagen matrix plus the foam dressing (TIELLE ® , Systagenix) on top. Wound assessments were carried out over 12 weeks on a weekly basis, with dressing changes twice a week. Ulcers were photographed and wound exudates were collected on admission and at days 5, 14 and then every 14 days to provide a visual record of any changes in appearance of the ulcer and healing rate and for biochemical analysis of the wound. The levels and activity of elastase and plasmin were measured in wound exudates. Statistical analysis was performed using ANOVA and Bonferroni's post hoc test with P-values <0·05 considered to be significant. Compared with controls, ORC/collagen matrix-treated pressure sore wounds showed a significant faster healing rate, which positively correlated with a decreased activity of elastase and plasmin in wound exudates. No signs of infection or intolerance to the ORC/collagen matrix were observed. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Dynamic expression of chymotrypsin-like elastase 1 over the course of murine lung development

PubMed Central

Liu, Sheng; Young, Sarah Marie

2014-01-01

Postnatal lung development requires coordination of three processes (surface area expansion, microvascular growth, and matrix remodeling). Because normal elastin structure is important for lung morphogenesis, because physiological remodeling of lung elastin has never been defined, and because elastin remodeling is angiogenic, we sought to test the hypothesis that, during lung development, elastin is remodeled in a defined temporal-spatial pattern, that a novel protease is associated with this remodeling, and that angiogenesis is associated with elastin remodeling. By elastin in situ zymography, lung elastin remodeling increased 24-fold between embryonic day (E) 15.5 and postnatal day (PND) 14. Remodeling was restricted to major vessels and airways on PND1 with a sevenfold increase in alveolar wall elastin remodeling from PND1 to PND14. By inhibition assays and literature review, we identified chymotrypsin-like elastase 1 (CELA1) as a potential mediator of elastin remodeling. CELA1 mRNA levels increased 12-fold from E15.5 to PND9, and protein levels increased 3.4-fold from E18.5 to PND9. By costaining experiments, the temporal-spatial pattern of CELA1 expression matched that of elastin remodeling, and 58–85% of CELA1+ cells were <10 μm from an elastase signal. An association between elastin remodeling and angiogenesis was tested by similar methods. At PND7 and PND14, 60–95% of angiogenin+ cells were associated with elastin remodeling. Both elastase inhibition and CELA1 silencing impaired angiogenesis in vitro. Our data defines the temporal-spatial pattern of elastin remodeling during lung development, demonstrates an association of this remodeling with CELA1, and supports a role for elastin remodeling in regulating angiogenesis. PMID:24793170

Inhibition of host extracellular matrix destructive enzyme production and activity by a high-molecular-weight cranberry fraction.

PubMed

Bodet, C; Chandad, F; Grenier, D

2007-04-01

Periodontal diseases are a group of inflammatory disorders that are initiated by specific gram-negative bacteria and lead to connective tissue destruction. Proteolytic enzymes, including matrix metalloproteinases (MMPs) and elastase, produced by resident and inflammatory cells in response to periodontopathogens and their products, play a major role in gingival tissue destruction. The aim of this study was to investigate the effect of a high-molecular-weight fraction prepared from cranberry juice concentrate on MMP-3, MMP-9 and elastase activities, as well as on MMP production by human cells stimulated with lipopolysaccharide of Actinobacillus actinomycetemcomitans. MMP-3 and MMP-9 production by gingival fibroblasts and macrophages treated with the cranberry fraction and then stimulated with lipopolysaccharide was measured by enzyme-linked immunosorbent assay. MMP-3, MMP-9 and elastase activities in the presence of the cranberry fraction were evaluated using colorimetric or fluorogenic substrates. The changes in expression and phosphorylation state of fibroblast intracellular signaling proteins induced by A. actinomycetemcomitans lipopolysaccharide and the cranberry fraction were characterized by antibody microarrays. The lipopolysaccharide-induced MMP-3 and MMP-9 responses of fibroblasts and macrophages were inhibited in a dose-dependent manner by the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. MMP-3, MMP-9 and elastase activities were also efficiently inhibited by the cranberry fraction, even when it was used at low concentrations. These results suggest that cranberry compounds offer promising perspectives for the development of novel host-modulating strategies for an adjunctive treatment of periodontitis.

Attenuation of aortic aneurysms with stem cells from different genders.

PubMed

Davis, John P; Salmon, Morgan; Pope, Nicolas H; Lu, Guanyi; Su, Gang; Sharma, Ashish K; Ailawadi, Gorav; Upchurch, Gilbert R

2015-11-01

No medical therapies are yet available to slow abdominal aortic aneurysm (AAA) growth. This study sought to investigate the effect of different genders of bone marrow-derived mesenchymal stem cells (MSC) on AAA growth in a murine AAA model. Given the decreased rate of AAA in women, it is hypothesized that female MSC would attenuate AAA growth more so than male MSC. Aortas of 8-10-wk-old male C57Bl/6 mice were perfused with purified porcine pancreatic elastase to induce AAA formation. Bone marrow-derived MSC from male and female mice were dosed via tail vein injection (3 million cells per dose, 500 μL of volume per injection) on postaortic perfusion days 1, 3, and 5. Aortas were harvested after 14 d. Mean aortic dilation in the elastase group was 121 ± 5.2% (mean ± standard error of the mean), while male MSC inhibited AAA growth (87.8 ± 6.9%, P = 0.008) compared with that of elastase. Female MSC showed the most marked attenuation of AAA growth (75.2 ± 8.3% P = 0.0004). Proinflammatory cytokines tumor necrosis factor α, interleukin 1β, and monocyte chemotactic protein-1 (MCP-1) were only decreased in tissues treated with female MSC (P = 0.017, P = 0.001, and P < 0.0001, respectively, when compared with elastase). These data exhibit that female MSC more strongly attenuate AAA growth in the murine model. Furthermore, female MSC and male MSC inhibit proinflammatory cytokines at varying levels. The effects of MSC on aortic tissue offer a promising insight into biologic therapies for future medical treatment of AAAs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Evidence for inflammation and activation of cell-mediated immunity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): increased interleukin-1, tumor necrosis factor-α, PMN-elastase, lysozyme and neopterin.

PubMed

Maes, Michael; Twisk, Frank N M; Kubera, Marta; Ringel, Karl

2012-02-01

There is evidence that inflammatory pathways and cell-mediated immunity (CMI) play an important role in the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Activation of inflammatory and CMI pathways, including increased levels of cytokines, is known to induce fatigue and somatic symptoms. Given the broad spectrum inflammatory state in ME/CFS, the aim of this study was to examine whether inflammatory and CMI biomarkers are increased in individuals with ME/CFS. In this study we therefore measured plasma interleukin-(IL)1, tumor necrosis factor (TNF)α, and PMN-elastase, and serum neopterin and lysozyme in 107 patients with ME/CFS, 37 patients with chronic fatigue (CF), and 20 normal controls. The severity of ME/CFS was measured with the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale. Serum IL-1, TNFα, neopterin and lysozyme are significantly higher in patients with ME/CFS than in controls and CF patients. Plasma PMN-elastase is significantly higher in patients with ME/CFS than in controls and CF patients and higher in the latter than in controls. Increased IL-1 and TNFα are significantly correlated with fatigue, sadness, autonomic symptoms, and a flu-like malaise; neopterin is correlated with fatigue, autonomic symptoms, and a flu-like malaise; and increased PMN-elastase is correlated with concentration difficulties, failing memory and a subjective experience of infection. The findings show that ME/CFS is characterized by low-grade inflammation and activation of CMI. The results suggest that characteristic symptoms of ME/CFS, such as fatigue, autonomic symptoms and a flu-like malaise, may be caused by inflammatory mediators, e.g. IL-1 and TNFα. Copyright © 2011 Elsevier B.V. All rights reserved.

Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy

PubMed Central

Modi, Kshitij D.; Foster, Thomas H.

2013-01-01

Abstract. We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population. PMID:23897439

Enzymatic recontouring of auricular cartilage in a rabbit model.

PubMed

Massengill, Phillip L; Goco, Paulino E; Norlund, L Layne; Muir-Padilla, Jeanne

2005-01-01

To evaluate the effectiveness of contouring auricular cartilage in a rabbit model using biologically active enzymes injected subcutaneously. The first phase determined the most effective volume and concentration required to affect the cartilage. To accomplish this task, we used ex vivo rabbit ears from a slaughterhouse. In the second phase, we injected 1 mL of hyaluronidase (150 U per milliliter of isotonic sodium chloride solution [saline]), elastase (1 mg per milliliter of saline), or saline into the ears of live rabbits. The study took place at the Madigan Army Medical Center (Tacoma, Wash), and included 10 animals. In each rabbit, we injected the test compound in one ear and saline in the other ear (control). We injected hyaluronidase in 5 ears and elastase in 5 ears. After injection, the ears were contoured and splinted for 4 weeks. In the third phase, we changed the injection pathway in 5 animals. At 4 weeks, 4 (80%) of the 5 ears injected with hyaluronidase showed full response and 1 (20%) had a partial response. Of the 5 ears injected with elastase, 4 (80%) showed a full response while 1 (20%) demonstrated a partial response. There was a response in all 10 of the ears injected with a test compound. Of the 10 control ears, 3 (30%) showed a partial response. At 6 weeks, approximately 6 (30%) of the ears had maintained contour demonstrating a full response. The difference between the test ears and the control ears was statistically significant (P = .006). Compared with the control ears, the results were statistically significant for elastase (P = .004) and hyaluronidase (P = .02). Overall, both agents demonstrated a subjective and objective response compared with control ears. This study demonstrates that bioactive enzymes and splinting can be effective in correcting ear deformities in a rabbit model.

Adaptive changes of pancreatic protease secretion to a short-term vegan diet: influence of reduced intake and modification of protein.

PubMed

Walkowiak, Jaroslaw; Mądry, Edyta; Lisowska, Aleksandra; Szaflarska-Popławska, Anna; Grzymisławski, Marian; Stankowiak-Kulpa, Hanna; Przysławski, Juliusz

2012-01-01

In our previous study, we demonstrated that abstaining from meat, for 1 month, by healthy omnivores (lacto-ovovegetarian model) resulted in a statistical decrease in pancreatic secretion as measured by faecal elastase-1 output. However, no correlation between relative and non-relative changes of energy and nutrient consumption and pancreatic secretion was documented. Therefore, in the present study, we aimed to assess the changes of exocrine pancreatic secretion with a more restrictive dietetic modification, by applying a vegan diet. A total of twenty-one healthy omnivores (sixteen females and five males) participated in the prospective study lasting for 6 weeks. The nutrient intake and faecal output of pancreatic enzymes (elastase-1, chymotrypsin and lipase) were assessed twice during the study. Each assessment period lasted for 7 d: the first before the transition to the vegan diet (omnivore diet) and the second during the last week of the study (vegan diet). The dietary modification resulted in a significant decrease in faecal elastase-1 (P < 0·05) and chymotrypsin output (P < 0·04). The lipase excretion remained unchanged. The decrease in proteolytic enzymes was documented to be positively correlated with a decreased protein intake (P < 0·05). In addition, elastase-1 and chymotrypsin outputs were also related to the changes of protein type, plant v. animal (P < 0·04 and P < 0·03, respectively). It was concluded that significant reduction and modification of protein intake due to a short-term vegan diet resulted in an adaptation of pancreatic protease secretion in healthy volunteers.

Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

PubMed

Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

2016-01-01

Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

Oxidative and proteolysis-related parameters of skeletal muscle from hamsters with experimental pulmonary emphysema: a comparison between papain and elastase induction.

PubMed

Brunnquell, Cláudia R; Vieira, Nichelle A; Sábio, Laís R; Sczepanski, Felipe; Cecchini, Alessandra L; Cecchini, Rubens; Guarnier, Flávia A

2015-06-01

The objective of this study was to investigate whether emphysema induced by elastase or papain triggers the same effects on skeletal muscle, related to oxidative stress and proteolysis, in hamsters. For this purpose, we evaluated pulmonary lesions, body weight, muscle loss, oxidative stress (thiobarbituric acid-reactive substances, total and oxidized glutathiones, chemiluminescence stimulated by tert-butyl hydroperoxide and carbonyl proteins), chymotrypsin-like and calpain-like proteolytic activities and muscle fibre cross-sectional area in the gastrocnemius muscles of emphysemic hamsters. Two groups of animals received different intratracheal inductions of experimental emphysema: by 40 mg/ml papain (EP) or 5.2 IU/100 g animal (EE) elastase (n = 10 animals/group). The control group received intratracheal instillation of 300 μl sterile NaCl 0.9%. Compared with the control group, the EP group had reduced muscle weight (18.34%) and the EE group had increased muscle weight (8.37%). Additionally, tert-butyl hydroperoxide-initiated chemiluminescence, carbonylated proteins and chymotrypsin-like proteolytic activity were all elevated in the EP group compared to the CS group, while total glutathione was decreased compared to the EE group. The EE group showed more fibres with increased cross-sectional areas and increased calpain-like activity. Together, these data show that elastase and papain, when used to induce experimental models of emphysema, lead to different speeds and types of adaptation. These findings provide more information on choosing a suitable experimental model for studying skeletal muscle adaptations in emphysema. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

Effects of the association of diabetes and pulmonary emphysema on cardiac structure and function in rats.

PubMed

Di Petta, Antonio; Simas, Rafael; Ferreira, Clebson L; Capelozzi, Vera L; Salemi, Vera M C; Moreira, Luiz F P; Sannomiya, Paulina

2015-10-01

Chronic obstructive pulmonary disease is often associated with chronic comorbid conditions of cardiovascular disease, diabetes mellitus and hypertension. This study aimed to investigate the effects of the association of diabetes and pulmonary emphysema on cardiac structure and function in rats. Wistar rats were divided into control non-diabetic instilled with saline (CS) or elastase (CE), diabetic instilled with saline (DS) or elastase (DE), DE treated with insulin (DEI) groups and echocardiographic measurements, morphometric analyses of the heart and lungs, and survival analysis conducted 50 days after instillation. Diabetes mellitus was induced [alloxan, 42 mg/kg, intravenously (iv)] 10 days before the induction of emphysema (elastase, 0.25 IU/100 g). Rats were treated with NPH insulin (4 IU before elastase plus 2 IU/day, 50 days). Both CE and DE exhibited similar increases in mean alveolar diameter, which are positively correlated with increases in right ventricular (RV) wall thickness (P = 0.0022), cavity area (P = 0.0001) and cardiomyocyte thickness (P = 0.0001). Diabetic saline group demonstrated a reduction in left ventricular (LV) wall, interventricular (IV) septum, cardiomyocyte thickness and an increase in cavity area, associated with a reduction in LV fractional shortening (P < 0.05), and an increase in LViv relaxation time (P < 0.05). Survival rate decreased from 80% in DS group to 40% in DE group. In conclusion, alloxan diabetes did not affect RV hypertrophy secondary to chronic emphysema, even in the presence of insulin. Diabetes per se induced left ventricular dysfunction, which was less evident in the presence of RV hypertrophy. Survival rate was substantially reduced as a consequence, at least in part, of the coexistence of RV hypertrophy and diabetic cardiomyopathy. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.

1978-01-01

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1 to 1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contast, secretion of lysozyme was not affectedmore » by glucocorticoids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1 to 10 nM) similar to those that half-saturated the specific glucocorticoid receptors. At high concentrations of dexamethasone (100 to 1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (>95%) than the secretion of elastase (60 to 80%).Progesterone alone had no effect on secretion, but blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1 to 100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone.« less

Creation of Abdominal Aortic Aneurysms in Sheep by Extrapolation of Rodent Models: Is It Feasible?

PubMed

Verbrugghe, Peter; Verhoeven, Jelle; Clijsters, Marnick; Vervoort, Dominique; Coudyzer, Walter; Verbeken, Eric; Meuris, Bart; Herijgers, Paul

2018-06-07

Abdominal aortic aneurysms (AAAs) are a potentially deathly disease, needing surgical or endovascular treatment. To evaluate potentially new diagnostic tools and treatments, a large animal model, which resembles not only the morphological characteristics but also the pathophysiological background, would be useful. Rodent animal aneurysm models were extrapolated to sheep. Four groups were created: intraluminal infusion with an elastase-collagenase solution (n = 4), infusion with elastase-collagenase solution combined with proximal stenosis (n = 7), aortic xenograft (n = 3), and elastase-collagenase-treated xenograft (n = 4). At fixed time intervals (6, 12, and 24 weeks), computer tomography and autopsy with histological evaluation were performed. The described models had a high perioperative mortality (45%), due to acute aortic thrombosis or fatale hemorrhage. A maximum aortic diameter increase of 30% was obtained in the protease-stenosis group. In the protease-treated groups, some histological features of human AAAs, such as inflammation, thinning of the media, and loss of elastin could be reproduced. In the xenotransplant groups, a pronounced inflammatory reaction was visible at the start. In all models, inflammation decreased and fibrosis occurred at long follow-up, 24 weeks postoperatively. None of the extrapolated small animal aneurysm models could produce an AAA in sheep with similar morphological features as the human disease. Some histological findings of human surgical specimens could be reproduced in the elastase-collagenase-treated groups. Long-term histological evaluation indicated stabilization and healing of the aortic wall months after the initial stimulus. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

[The comparison of selected virulence factors in Pseudomonas aeruginosa catheter isolates].

PubMed

Olejnízková, Katerina; Holá, Veronika

2012-05-01

Healthcare quality improvement brings about an increasing number of invasive diagnostic and therapeutic procedures and thus also an increasing number of high-risk patients prone to hospital infections. Pseudomonas aeruginosa is one of the most commonly isolated nosocomial species and the treatment of the infection is often long and problematic, with frequent recurrences. The pathogenesis of Pseudomonas infection is associated with a range of virulence factors. In the present study, 93 catheter isolates of Pseudomonas aeruginosa were screened for the biofilm formation, motility and secretion of selected extracellular products. A high rate of the strains tested were producers of hemolysins, LasB elastase, and pyoverdines (> 70%). The biofilm formation was detected in 80% of isolates and formation of aerated biofilm was present in 90% of isolates with a positive correlation found between the two types of biofilm formation (p = 0.00583; gamma = 0.551). All strains showed swarming motility, 95% of strains showed swimming motility, and 75% of strains showed twitching motility. Among the virulence factors studied, only pyocyanin and pyochelin were produced by a lower proportion of isolates (< 25%). A positive correlation was seen between the production of some extracellular molecules (pyochelin and pyocyanin, pyocyanin and LasB elastase, and LasB elastase and haemolysins), between biofilm formation and formation of aerated biofilm, and between formation of aerated biofilm and pigments (pyoverdine and pyocyanin) production. On the other hand, a negative correlation was found between biofilm production and LasB elastase production and between the production of biofilm under immersion and pigments (pyoverdine and pyocyanin) production. All correlations are significant at the level p = 0.05, with the correlation coefficient gamma > 0.50.

Exercise training attenuates neutrophil infiltration and elastase expression in adipose tissue of high-fat-diet-induced obese mice

PubMed Central

Kawanishi, Noriaki; Niihara, Hiroyuki; Mizokami, Tsubasa; Yada, Koichi; Suzuki, Katsuhiko

2015-01-01

The innate immune system is associated with the development of local inflammation. Neutrophils play an essential role in the development of the adipose tissue (AT) inflammation associated with obesity by producing elastase, which can promote the activation and infiltration of macrophages. Exercise training attenuates AT inflammation via suppression of macrophage infiltration. However, the mechanisms driving this phenomenon remains to be elucidated. Here, we evaluated the effects of exercise training on the infiltration of neutrophils and elastase expression in an obese mouse model. Four-week-old male C57BL/6J mice were randomly assigned to one of three groups that either received a normal diet (ND) plus sedentary activity (n = 15), a high-fat diet (HFD) plus sedentary activity (n = 15), or a HFD plus exercise training (n = 15). Mice were fed the ND or HFD from the age of 4 weeks until 20 weeks. Mice in the exercise group ran on a treadmill for 60 min/day, 5 days/week over the same experimental period. Mice fed with the HFD had increased content of macrophages in the AT and increased inflammatory cytokine mRNA levels, which were reduced by exercise training. Similarly, AT from the HFD sedentary mice contained more neutrophils than AT from the ND mice, and the amount of neutrophils in this tissue in HFD-fed mice was lowered by exercise training. The mRNA levels of neutrophil elastase in AT were lower in the HFD exercise-trained mice than those in the HFD sedentary mice. These results suggest that exercise training plays a critical role in reducing macrophage infiltration and AT inflammation by regulating the infiltration of neutrophils. PMID:26341995

Matrix metalloproteinase, hyaluronidase and elastase inhibitory potential of standardized extract of Centella asiatica.

PubMed

Nema, Neelesh Kumar; Maity, Niladri; Sarkar, Birendra Kumar; Mukherjee, Pulok Kumar

2013-09-01

Centella asiatica (L.) Urban (Apiaceae), a valuable herb described in Ayurveda, is used in the indigenous system of medicine as a tonic to treat skin diseases. Centella asiatica methanol extract and its ethyl acetate, n-butanol and aqueous fraction, were subjected for the evaluation of skin care potential through the in vitro hyaluronidase, elastase and matrix metalloproteinase-1 (MMP-1) inhibitory assay. The C. asiatica plant was extracted with methanol and fractionated with ethyl acetate, n-butanol and water. The enzymatic activities were evaluated using ursolic acid and oleanolic acid as standards. Isolate molecule asiaticoside was quantified in the crude extract and fractions through high-performance liquid chromatography (HPLC) and structural was characterized by liquid chromatography-mass spectroscopy (LC-MS) and ¹H nuclear magnetic resonance (NMR). Isolated compound was also evaluated for in vitro enzyme assays. Extract exhibited anti-hyaluronidase and anti-elastase activity with IC₅₀ of 19.27 ± 0.37 and 14.54 ± 0.39 µg/mL, respectively, as compared to ursolic acid. Centella asiatica n-butanol fraction (CAnB) and isolated compound showed significant hyaluronidase (IC₅₀ = 27.00 ± 0.43 and 18.63 ± 0.33 µg/mL) and elastase (IC₅₀ = 29.15 ± 0.31 and 19.45 ± 0.25 µg/mL) inhibitory activities, respectively, and also showed significant MMP-1 inhibition (p < 0.05 and p < 0.01). n-Butanol fraction was found to be most effective among the all fractions from which asiaticoside was isolated and further quantified by HPLC. This work concludes that the asiaticoside from C. asiatica may be a prospective agent for skin care.

Deficiency of FAM3D (Family With Sequence Similarity 3, Member D), A Novel Chemokine, Attenuates Neutrophil Recruitment and Ameliorates Abdominal Aortic Aneurysm Development.

PubMed

He, Li; Fu, Yi; Deng, Jingna; Shen, Yicong; Wang, Yingbao; Yu, Fang; Xie, Nan; Chen, Zhongjiang; Hong, Tianpei; Peng, Xinjian; Li, Qingqing; Zhou, Jing; Han, Jingyan; Wang, Ying; Xi, Jianzhong; Kong, Wei

2018-07-01

Chemokine-mediated neutrophil recruitment contributes to the pathogenesis of abdominal aortic aneurysm (AAA) and may serve as a promising therapeutic target. FAM3D (family with sequence similarity 3, member D) is a recently identified novel chemokine. Here, we aimed to explore the role of FAM3D in neutrophil recruitment and AAA development. FAM3D was markedly upregulated in human AAA tissues, as well as both elastase- and CaPO 4 -induced mouse aneurysmal aortas. FAM3D deficiency significantly attenuated the development of AAA in both mouse models. Flow cytometry analysis indicated that FAM3D -/- mice exhibited decreased neutrophil infiltration in the aorta during the early stage of AAA formation compared with their wild-type littermates. Moreover, application of FAM3D-neutralizing antibody 6D7 through intraperitoneal injection markedly ameliorated elastase-induced AAA formation and neutrophil infiltration. Further, in vitro coculture experiments with FAM3D-neutralizing antibody 6D7 and in vivo intravital microscopic analysis indicated that endothelial cell-derived FAM3D induced neutrophil recruitment. Mechanistically, FAM3D upregulated and activated Mac-1 (macrophage-1 antigen) in neutrophils, whereas inhibition of FPR1 (formyl peptide receptor 1) or FPR2 significantly blocked FAM3D-induced Mac-1 activation, indicating that the effect of FAM3D was dependent on both FPRs. Moreover, specific inhibitors of FPR signaling related to Gi protein or β-arrestin inhibited FAM3D-activated Mac-1 in vitro, whereas FAM3D deficiency decreased the activation of both FPR-Gi protein and β-arrestin signaling in neutrophils in vivo. FAM3D, as a dual agonist of FPR1 and FPR2, induced Mac-1-mediated neutrophil recruitment and aggravated AAA development through FPR-related Gi protein and β-arrestin signaling. © 2018 American Heart Association, Inc.

Bombesin receptor-activated protein regulates neutrophil elastase-induced mucin5AC hypersecretion in human bronchial epithelial cells.

PubMed

Xu, Qing; Chen, Ling-Xiu; Ran, Dan-Hua; Xie, Wen-Yue; Li, Qi; Zhou, Xiang-Dong

2017-08-15

Bombesin receptor-activated protein (BRAP) is highly expressed in human bronchial epithelial cells. Recent studies have shown that BRAP reduces oxidative stress, inhibits airway inflammation and suppresses nuclear factor kappaB (NF-κB) activity. Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. Neutrophil elastase (NE) is a potent inducer of mucin5AC (MUC5AC), which is considered the predominant mucin secreted by human airway epithelial cells. Here, we hypothesize that BRAP may regulate NE-induced MUC5AC hypersecretion in a bronchial epithelial cell line (HBE16). We also investigated the underlying mechanism involved in the process. In this study, we found that BRAP was present in HBE16 human bronchial epithelial cells and was significantly increased by NE. Next, we found that the up-regulation of BRAP by pEGFP-N1-BRAP caused a significant decrease in the increased levels of MUC5AC expression, NF-κB activity, and the phosphorylation of extracellular signal-regulated kinases (ERK) and epidermal growth factor receptor (EGFR) induced by NE. Meanwhile, there was a significant decrease in ROS, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels when BRAP was up-regulated by pEGFP-N1-BRAP. Moreover, when cells were transfected with pEGFP-N1-BRAP and pretreated with NF-κB, ERK or EGFR inhibitors before the NE stimulation, there were further decreased in MUC5AC expression, NF-κB activity, and the phosphorylation of ERK and EGFR. These results suggest that BRAP plays an important role in airway inflammation and its overexpression may regulate NE-induced MUC5AC hypersecretion in HBE16 cells via the EGFR/ERK/NF-κB signaling pathway. Copyright © 2017. Published by Elsevier Inc.

Bidirectional Increase in Permeability of Nuclear Envelope upon Poliovirus Infection and Accompanying Alterations of Nuclear Pores

PubMed Central

Belov, George A.; Lidsky, Peter V.; Mikitas, Olga V.; Egger, Denise; Lukyanov, Konstantin A.; Bienz, Kurt; Agol, Vadim I.

2004-01-01

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3×EGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-l-alanyl-l-alanyl-l-prolyl-l-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed. PMID:15331749

Ursolic Acid Inhibits Superoxide Production in Activated Neutrophils and Attenuates Trauma-Hemorrhage Shock-Induced Organ Injury in Rats

PubMed Central

Hwang, Tsong-Long; Shen, Hsin-I; Liu, Fu-Chao; Tsai, Hsin-I; Wu, Yang-Chang; Chang, Fang-Rong; Yu, Huang-Ping

2014-01-01

Neutrophil activation is associated with the development of organ injury after trauma–hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma–hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma–hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma–hemorrhagic shock-induced organ injury in rats. PMID:25360589

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

PubMed

Rochael, Natalia C; Guimarães-Costa, Anderson B; Nascimento, Michelle T C; DeSouza-Vieira, Thiago S; Oliveira, Matheus P; Garcia e Souza, Luiz F; Oliveira, Marcus F; Saraiva, Elvira M

2015-12-17

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

PubMed Central

Jones, S; Yu, B; Bainton, N J; Birdsall, M; Bycroft, B W; Chhabra, S R; Cox, A J; Golby, P; Reeves, P J; Stephens, S

1993-01-01

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes. Images PMID:8508773

Anti-inflammatory triterpenoids from the stems of Microtropis fokienensis.

PubMed

Chen, I-Hsiao; Du, Ying-Chi; Hwang, Tsong-Long; Chen, I-Fen; Lan, Yu-Hsuan; Yen, Hsin-Fu; Chang, Fang-Rong; Wu, Yang-Chang

2014-04-14

Three new ursane- and four new oleanane- type triterpenoids 1-7 were isolated, along with six known compounds 8-13, from the methanolic extract of Microtropis fokienensis. All structures were elucidated by mass and NMR spectroscopic methods. The isolates 4-10 and known compounds 14-17 that were previously isolated from this material were evaluated for anti-inflammatory activity based on effects against superoxide anion generation and elastase release by neutrophils in response to fMLP/CB. 11α,30-Dihydroxy-2,3-seco-olean-12-en-2,3-dioic anhydride (7) was the first triterpene anhydride from the genus of Microtropis to have the ring A expanded to a seven-membered ring; it showed significant anti-inflammatory activity against superoxide anion generation and elastase release. Unexpectedly, 30-hydroxy-2,3-seco-lup-20(29)-ene-2,3-dioic acid (17) showed the best effect against superoxide anion generation and elastase release with IC50 values of 0.06±0.01 and 1.03±0.35 µg/mL, respectively. Compound 17 had a dioic acid function, and compound 7 had an anhydride function modification in ring A; both showed promising activity in the target assays.

Physiological effects of formulation containing tannase-converted green tea extract on skin care: physical stability, collagenase, elastase, and tyrosinase activities.

PubMed

Hong, Yang-Hee; Jung, Eun Young; Noh, Dong Ouk; Suh, Hyung Joo

2014-03-01

Green tea contains numerous polyphenols, which have health-promoting effects. The purpose of this study was to evaluate the effect of tannase-converted green tea extract (TGE) formulation on the physical stability and activities of skin-related enzymes. Physical stability was evaluated by measuring the pH, precipitation, and colors at 25 ± 2 °C/ambient humidity and at 40 ± 2 °C/70% ± 5% relative humidity for 4 months. Activities of collagenase, elastase, and tyrosinase as skin-related enzymes were assessed on TGE formulation. The concentrations of epigallocatechin-3-gallate and epicatechin-3-gallate in green tea extract were greatly decreased to the extent of negligible level when treated with tannase. The formulation containing 5% tannase-converted green tea extract showed relatively stable pH, precipitation, and color features for 16 weeks. When TGE was added to the formulation, there was a significant increase in the inhibition of elastase and tyrosinase activities ( p  < 0.05) compared with the formulation containing 5% normal green tea extract. The TGE could be used in cosmetics as skin antiwrinkling or depigmenting agent.

Emphysema induced by elastase enhances acute inflammatory pulmonary response to intraperitoneal LPS in rats.

PubMed

da Fonseca, Lídia Maria Carneiro; Reboredo, Maycon Moura; Lucinda, Leda Marília Fonseca; Fazza, Thaís Fernanda; Rabelo, Maria Aparecida Esteves; Fonseca, Adenilson Souza; de Paoli, Flavia; Pinheiro, Bruno Valle

2016-12-01

Abnormalities in lungs caused by emphysema might alter their response to sepsis and the occurrence of acute lung injury (ALI). This study compared the extension of ALI in response to intraperitoneal lipopolysaccharide (LPS) injection in Wistar rats with and without emphysema induced by elastase. Adult male Wistar rats were randomized into four groups: control, emphysema without sepsis, normal lung with sepsis and emphysema with sepsis. Sepsis was induced, and 24 h later the rats were euthanised. The following analysis was performed: blood gas measurements, bronchoalveolar lavage (BAL), lung permeability and histology. Animals that received LPS showed significant increase in a lung injury scoring system, inflammatory cells in bronchoalveolar lavage (BAL) and IL-6, TNF-α and CXCL2 mRNA expression in lung tissue. Animals with emphysema and sepsis showed increased alveolocapillary membrane permeability, demonstrated by higher BAL/serum albumin ratio. In conclusion, the presence of emphysema induced by elastase increases the inflammatory response in the lungs to a systemic stimulus, represented in this model by the intraperitoneal injection of LPS. © 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

PubMed Central

Huntington, J. A.; Patston, P. A.; Gettins, P. G.

1995-01-01

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7613461

Protein matrices for wound dressings =

NASA Astrophysics Data System (ADS)

Vasconcelos, Andreia Joana Costa

Fibrous proteins such as silk fibroin (SF), keratin (K) and elastin (EL) are able to mimic the extracellular matrix (ECM) that allows their recognition under physiological conditions. The impressive mechanical properties, the environmental stability, in combination with their biocompatibility and control of morphology, provide an important basis to use these proteins in biomedical applications like protein-based wound dressings. Along time the concept of wound dressings has changed from the traditional dressings such as honey or natural fibres, used just to protect the wound from external factors, to the interactive dressings of the present. Wounds can be classified in acute that heal in the expected time frame, and chronic, which fail to heal because the orderly sequence of events is disrupted at one or more stages of the healing process. Moreover, chronic wound exudates contain high levels of tissue destructive proteolytic enzymes such as human neutrophil elastase (HNE) that need to be controlled for a proper healing. The aim of this work is to exploit the self-assemble properties of silk fibroin, keratin and elastin for the development of new protein materials to be used as wound dressings: i) evaluation of the blending effect on the physical and chemical properties of the materials; ii) development of materials with different morphologies; iii) assessment of the cytocompatibility of the protein matrices; iv) ultimately, study the ability of the developed protein matrices as wound dressings through the use of human chronic wound exudate; v) use of innovative short peptide sequences that allow to target the control of high levels of HNE found on chronic wounds. Chapter III reports the preparation of silk fibroin/keratin (SF/K) blend films by solvent casting evaporation. Two solvent systems, aqueous and acidic, were used for the preparation of films from fibroin and keratin extracted from the respective silk and wool fibres. The effect of solvent system used was studied by evaluating the physical-chemical properties of the resulting films. It was shown that SF and K are able to establish intermolecular interactions when mixed and, that the mechanical properties and the biological degradation can be tuned by the blend composition. In Chapter IV, SF/K films were further used to serve as a platform for the release of HNE inhibitors peptides. Bowman-Birk inhibitor (BBI) based peptide was incorporated onto the SF/K films that were consequently incubated with porcine pancreatic elastase (PPE) as a model for HNE, to monitor the decrease in activity. The results indicated that swelling properties, degradation and release rates are dependent on the amount of keratin present in the blend. Furthermore, no cytotoxicity was observed in the presence of mouse fibroblasts, which makes these SF/K films suitable candidates for interactive wound dressings with a specific goal - controlling high levels of HNE. The next step of the work, Chapter V, reports for the first time blends of silk fibroin with elastin (SF/EL) for the production of scaffolds. These were prepared by lyophilization technique and crosslinked with a natural and low toxic agent, genipin. The crosslink allows the control of the scaffolds morphology, such as pore size and porosity, which in turns, modulates the ex vivo degradation rates, by a human chronic wound exudate, and the release rates of model compounds. In addition, no cytotoxicity was observed for SF/EL samples, with and without genipin, by human skin fibroblasts. Thus, the high porosity observed for SF/EL scaffolds, allowing the growth and cellular attachment, together with their biocompatibility provide fitting characteristics for wound dressings. Chapter VI, describes the design of two elastase inhibitors peptides based on the reactive site-loop of the BBI protein in order to control the high levels HNE. To a known peptide sequence, modifications were made at both N- and C-terminal. Inhibition kinetics analysis indicated that these peptides are competitive inhibitors for HNE and PPE and, that the inhibitory potency can be regulated by the introduced modifications. Additionally, these peptides showed no toxicity with human skin fibroblasts and, were also effective in reducing the HNE activity found in a human chronic wound exudate, which allow them to be applied to those wounds. The motivation for this thesis was to combine the excellent properties of silk fibroin with other proteins. Blending allows modulating the physical-chemical properties of the resulting materials such as mechanical strength, swelling, morphology, degradation and release rates. Silk fibroin is widely characterized in the literature for the production of biomaterials, but this work is the first that successfully evaluates the blends silk fibroin/keratin (SF/K) and silk fibroin/elastin (SF/EL) for their application as wound dressings.

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age123

PubMed Central

Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn

2017-01-01

Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26–40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24–26 wk GA)-delivering mothers and 9 from late GA (LGA) (27–32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL−1 · d−1 and 0.454 μg · mL−1 · d−1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not vary substantially across GA and postnatal age. PMID:28424255

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

PubMed

Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C

2017-06-01

Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not vary substantially across GA and postnatal age. © 2017 American Society for Nutrition.

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan Haiyan; Korzh, Svitlana; Li Zhen

2006-05-15

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficientmore » in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.« less

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils.

PubMed

Lin, Yin-Ku; Leu, Yann-Lii; Huang, Tse-Hung; Wu, Yi-Hsiu; Chung, Pei-Jen; Su Pang, Jong-Hwei; Hwang, Tsong-Long

2009-08-17

Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. Superoxide anion (O2(.-)) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. QD significantly inhibited O2(.-) generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2(.-) generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Inflammatory response, neutrophil activation, and free radical production after acute myocardial infarction: effect of thrombolytic treatment.

PubMed Central

Bell, D; Jackson, M; Nicoll, J J; Millar, A; Dawes, J; Muir, A L

1990-01-01

Activated neutrophils releasing proteolytic enzymes and oxygen free radicals have been implicated in extending myocardial injury after myocardial infarction. Neutrophil elastase was used as a marker of neutrophil activation and the non-peroxide diene conjugate of linoleic acid was used as an indicator of free radical activity in 32 patients after acute myocardial infarction; 17 were treated by intravenous thrombolysis. Patients with acute myocardial infarction had higher plasma concentrations of neutrophil elastase and the non-peroxide diene conjugated isomer of linoleic acid than normal volunteers or patients with stable ischaemic heart disease. Patients treated by thrombolysis had an early peak of neutrophil elastase at eight hours while those who had not been treated by thrombolysis showed a later peak 40 hours after infarction. The plasma concentration of non-peroxide conjugated diene of linoleic acid was highest 16 hours after the infarction irrespective of treatment by thrombolysis. Quantitative imaging with single photon emission tomography showed decreased uptake of indium-111 labelled neutrophils in the infarcted myocardium (as judged from technetium-99m pyrophosphate) in those who had received thrombolysis, suggesting a decreased inflammatory response. The results indicate increased neutrophil activation and free radical production after myocardial infarction; they also suggest that thrombolysis does not amplify the inflammatory response and may indeed suppress it. Images PMID:2317413

Elastase and matrix metalloproteinase activities are associated with pulmonary vascular disease in the nitrofen rat model of congenital diaphragmatic hernia.

PubMed

Wild, Benjamin; St-Pierre, Marie-Eve; Langlois, Stéphanie; Cowan, Kyle N

2017-05-01

Pulmonary vascular disease (PVD) is a leading cause of congenital diaphragmatic hernia (CDH) mortality. Progression of PVD involves extracellular matrix remodeling by elastases and matrix metalloproteinases (MMP), concomitant with proliferation of smooth muscle cells in a growth factor-enriched environment. Blockade of this pathway reversed primary pulmonary hypertension and improved survival. This study was designed to determine whether a similar pathway is induced in PVD secondary to CDH. Fetal rats exposed to nitrofen at gestational day 9 developed left-sided CDH and were compared at term to their non-CDH littermates by assessing histologic and biochemical features of PVD. Rats with CDH displayed right ventricle hypertrophy, increased pulmonary artery medial wall thickness and muscularization, and decreased lumen size. As revealed by in situ zymography and immunohistochemistry, this was associated with an induction of elastolytic and MMP activities as well as an elevation of epidermal growth factor and osteopontin levels in the diseased lung vasculature. CDH-associated PVD involves an induction of elastase and MMP activities and increased osteopontin deposition in an epidermal growth factor-rich environment. Inhibition of this pathway may thus represent a novel therapeutic approach for the treatment of CDH-associated PVD. Level I (Basic Science Study). Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of preoperative nutritional status on clinical outcomes after pancreatoduodenectomy.

PubMed

Kim, Eunjung; Kang, Jae Seung; Han, Youngmin; Kim, Hongbeom; Kwon, Wooil; Kim, Jae Ri; Kim, Sun-Whe; Jang, Jin-Young

2018-06-07

This study investigated the clinical outcomes according to the preoperative nutritional status and to identify factors influencing long-term unrecovered nutritional status. Data were prospectively collected from 355 patients who underwent PD between 2008 and 2014. Nutritional status was evaluated by Mini Nutrition Assessment (MNA) and patients were classified into group A (malnourished), group B (risk-of-malnutrition), or group C (well-nourished). MNA score, complications, body mass index (BMI), stool elastase level, biochemical parameters, and quality-of-life (QOL) were collected serially for 1 year. Preoperatively, 60 patients were categorized into group A, 224 into group B, and 71 into group C. Overall complication and pancreatic fistula were higher in groups A and B compared with group C (P = 0.003 vs P = 0.004). QOL, biochemical parameters, BMI and stool elastase level were lowest in group A preoperatively. BMI and stool elastase level remained low after surgery in all groups. Advanced age, low BMI, pre-existing diabetes mellitus, jaundice, exocrine insufficiency and adjuvant therapy were factors influencing long-term unrecovered nutritional status. Preoperative malnourished patients suffer from poor clinical outcomes. Therefore, those with risk factors of malnutrition should be monitored and vigorous efforts are needed to improve their nutrition. Copyright © 2018 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

PubMed Central

Gomis-Rüth, F X; Gómez, M; Bode, W; Huber, R; Avilés, F X

1995-01-01

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far. Images PMID:7556081

Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat.

PubMed

Fujita, M; Hong, K; Ito, Y; Fujii, R; Kariya, K; Nishimuro, S

1995-10-01

Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase (UK; 50000 IU/kg, i.v.) or tissue plasminogen activator (tPA; 13300 IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of nattokinase using this model and compared it with fibrino(geno)lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with nattokinase, plasmin and elastase were 62.0 +/- 5.3%, 15.8 +/- 0.7% and 0%, respectively. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo.

A preliminary study of laser tissue soldering as arterial wall reinforcement in an acute experimental aneurysm model.

PubMed

Oskoui, Philip; Stadler, Istvan; Lanzafame, Raymond J

2003-01-01

Aneurysm formation results from destruction of structural arterial wall connective tissue, leading to wall weakening and rupture. The purpose of this study was to demonstrate that reinforcement of the arterial wall using laser tissue soldering contributes to arterial wall stabilization and rupture prevention in an acute experimental model. Elastase (10 U/mg protein, Sigma-Aldrich Co., St. Louis, MO) was applied with a fine paint brush on femoral artery segments to cause fusiform aneurysm formation. After aneurysms formed (approximately 45 minutes after treatment), elastase was rinsed out and indocyanine green (ICG) and albumin soldering mixture (2.5 mg/ml ICG in 50% albumin) was delivered to the arterial segment, followed by laser irradiation at 830 nm, (15mW output for 20 minutes). In situ pressure burst measurements were then performed. In situ burst pressures were > 503 mmHg for normal arteries and 181 +/- 26.0 mmHg, for Elastase treated segments. (P < 0.0001) Treatment of experimental aneurysms laser tissue soldering returned burst strengths to > 503 mmHg. These results indicate laser tissue soldering reinforcement of weak arterial walls, is possible and may reduce the likelihood of acute rupture. Further development of this technique for aneurysm management is warranted. Copyright 2003 Wiley-Liss, Inc.

Skin whitening and anti-corrugation activities of glycoprotein fractions from liquid extracts of boiled sea cucumber.

PubMed

Kim, So Jung; Park, So Yun; Hong, Sun-Mee; Kwon, Eun-Hye; Lee, Taek-Kyun

2016-10-01

To determine skin whitening and wrinkle improvement efficacy, glycoprotein fractions were extracted from liquid extracts of boiled sea cucumber and their effects on tyrosine and elastase inhibitory activities were assayed. Fractions above and below 50 kDa (>50 kDa and <50 kDa) were extracted via a series of steps involving: boiling, filtering, desalting and freeze drying. Cytotoxicity, skin whitening and wrinkle-removing effects of boiled liquid were determined. Our MTT data showed that neither glycoprotein fraction of boiled liquid induces cellular cytotoxicity up to a concentration of 10 mg/mL treatment of the mouse melanoma cell line, B16F10, with 10 mg/mL >50 kDa enhanced tyrosinase and elastase inhibitory activities by 50.84% and 28.78%, respectively. Correlations of the >50 kDa concentration with tyrosinase inhibitory (R2 = 0.968) and elastase inhibitory (R2 = 0.983) efficacy were significant. >50 kDa glycoprotein fraction isolated from liquid extracts of boiled sea cucumber, which can serve as a functional cosmetic ingredient for whitening and wrinkle improvement of skin. Copyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

Ozone-induced airway hyperresponsiveness in patients with asthma: role of neutrophil-derived serine proteinases.

PubMed

Hiltermann, T J; Peters, E A; Alberts, B; Kwikkers, K; Borggreven, P A; Hiemstra, P S; Dijkman, J H; van Bree, L A; Stolk, J

1998-04-01

Proteinase inhibitors may be of potential therapeutic value in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Our aim was to study the role of neutrophils, and neutrophil-derived serine proteinases in an acute model in patients with asthma. Exposure to ozone induces an acute neutrophilic inflammatory reaction accompanied by an increase in airway hyperresponsiveness. It is thought that these two effects of ozone are linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined the effect of recombinant antileukoprotease (rALP), one of the major serine proteinase inhibitors in the lung, on ozone-induced changes in airway hyperresponsiveness in this model. We observed that 16 h after exposure to ozone, airway hyperresponsiveness to methacholine was increased both following placebo and rALP treatment. There was no significant difference between placebo and rALP treatment (change in area under the dose-response curve to methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the immediate decrease in FEV1 after ozone exposure was not significantly different between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In addition, no significant differences were observed in plasma levels of fibrinogen degradation products generated by neutrophil serine proteinases before and after exposure to ozone. We conclude that neutrophil-derived serine proteinases are not important mediators for ozone-induced hyperresponsiveness.

Investigation of long-term retention of unchanged (-)-N-{2-[(R)-3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide, a novel "funny" If current channel inhibitor, and its metabolites in the eyeball and thoracic aorta of rats.

PubMed

Umehara, Ken-ichi; Nakada, Naoyuki; Noguchi, Kiyoshi; Iwatsubo, Takafumi; Usui, Takashi; Kamimura, Hidetaka

2009-11-01

(-)-N-{2-[(R)-3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a novel "funny" If current channel inhibitor, was being developed as a treatment for stable angina and atrial fibrillation. After a single oral administration of (14)C-YM758, extensive accumulation and long-term retention of radioactivity were observed in the eyeballs of nonalbino rats and in the thoracic aorta of albino/nonalbino rats. Radioluminograms of the eyeballs of nonalbino rats indicated that the radioactivity was localized to the uveal tract, which suggests that the radioactivity may be positively charged and bound mainly to the melanins. Treatment with a mixture of 2 mol/l hydrochloric acid and methanol (5:95, v/v) allowed for the recovery of the major portion of radioactivity from the eyeball, which suggests reversible binding. The radioactive constituents in eyeballs consisted of the unchanged drug (YM758) and three metabolites [mainly 6,7-dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124)]. Using the organic solvent mixture described above, almost all of the radioactivity was not collected from the thoracic aorta, and approximately 90% was recovered by treatment with elastase, which suggests that some metabolites covalently bind to the elastin fiber localized in the tunica media.

Effect of Elastase-induced Emphysema on the Force-generating Ability of the Diaphragm

PubMed Central

Supinski, Gerald S.; Kelsen, Steven G.

1982-01-01

The effect of emphysema on the ability of the diaphragm to generate force was examined in costal diaphragm muscle strips from 10 Golden hamsters killed 18 mo after intratracheal injection of pancreatic elastase in a dose producing hyperinflation (mean total lung capacity [TLC] = 163% of control) and generalized panacinar emphysema. 13 saline-injected normal animals served as controls. The time course of isometric tension and the effect of alterations in muscle fiber and sarcomere length on the isometric tension (T) generated in response to tetanizing electrical stimuli (length-tension [L-T] relationship) were examined. Elastase administration caused an increase in diaphragm muscle thickness and reduction in the length of costal diaphragm muscle fibers measured in situ. Emphysema significantly increased the maximum tetanic tension as a result of hypertrophy. Maximal tension corrected for increases in muscle cross-sectional area (T/cm2), however, was the same in emphysematous (E) and control (C) animals. Emphysema also shifted the muscle fiber L-T curve of the diaphragm but not of a control muscle, the soleus, toward shorter lengths. In contrast to the effects of E on the diaphragm muscle fiber L-T curve, the sarcomere L-T curve was the same in E and C. Since the length at which tension was maximal correlated closely with sarcomere number (r = 0.94; P < 0.001) reduction in the number of sarcomeres in series in muscles from emphysematous animals appeared to explain the shift in the muscle fiber L-T curve. We conclude that in elastase-induced emphysema adaptive changes both in diaphragm cross-sectional area and sarcomere number augment the force-generating ability of the diaphragm. We speculate that changes in sarcomere number compensate for alterations in muscle fiber length resulting from chronic hyperinflation of the thorax, while diaphragmatic muscle hypertrophy represents a response to changes in respiratory load and/or diaphragm configuration (LaPlace relationship). Images PMID:6922866

Development and proof-of-concept of three-dimensional lung histology volumes

NASA Astrophysics Data System (ADS)

Mathew, Lindsay; Alabousi, Mostafa; Wheatley, Andrew; Aladl, Usaf; Slipetz, Deborah; Hogg, James C.; Fenster, Aaron; Parraga, Grace

2012-03-01

Most medical imaging is inherently three-dimensional (3D) but for validation of pathological findings, histopathology is commonly used and typically histopathology images are acquired as twodimensional slices with quantitative analysis performed in a single dimension. Histopathology is invasive, labour-intensive, and the analysis cannot be performed in real time, yet it remains the gold standard for the pathological diagnosis and validation of clinical or radiological diagnoses of disease. A major goal worldwide is to improve medical imaging resolution, sensitivity and specificity to better guide therapy and biopsy and to one day delay or replace biopsy. A key limitation however is the lack of tools to directly compare 3D macroscopic imaging acquired in patients with histopathology findings, typically provided in a single dimension (1D) or in two dimensions (2D). To directly address this, we developed methods for 2D histology slice visualization/registration to generate 3D volumes and quantified tissue components in the 3D volume for direct comparison to volumetric micro-CT and clinical CT. We used the elastase-instilled mouse emphysema lung model to evaluate our methods with murine lungs sectioned (5 μm thickness/10 μm gap) and digitized with 2μm in-plane resolution. 3D volumes were generated for wildtype and elastase mouse lung sections after semi-automated registration of all tissue slices. The 1D mean linear intercept (Lm) for wildtype (WT) (47.1 μm +/- 9.8 μm) and elastase mouse lung (64.5 μm +/- 14.0 μm) was significantly different (p<.001). We also generated 3D measurements based on tissue and airspace morphometry from the 3D volumes and all of these were significantly different (p<.0001) when comparing elastase and WT mouse lung. The ratio of the airspace-to-lung volume for the entire lung volume was also significantly and strongly correlated with Lm.

Smoking affects diagnostic salivary periodontal disease biomarker levels in adolescents.

PubMed

Heikkinen, Anna Maria; Sorsa, Timo; Pitkäniemi, Janne; Tervahartiala, Taina; Kari, Kirsti; Broms, Ulla; Koskenvuo, Markku; Meurman, Jukka H

2010-09-01

The effects of smoking on periodontal biomarkers in adolescents are unknown. This study investigates matrix metalloproteinase (MMP)-8 and polymorphonuclear leukocyte elastase levels in saliva together with periodontal health indices accounting for body mass index and smoking in a birth cohort from Finland. The oral health of boys (n = 258) and girls (n = 243) aged 15 to 16 years was examined clinically. Health habits were assessed by questionnaire. Saliva samples were collected and analyzed by immunofluorometric and peptide assays for MMP-8 levels and polymorphonuclear leukocyte elastase activities, and investigated statistically with the background factors. Median MMP-8 values of male smokers were 112.03 microg/l compared to 176.89 microg/l of non-smokers (P = 0.05). For female smokers corresponding values were 170.88 microg/l versus 177.92 microg/l in non-smokers (not statistically significant). Elastase values in male smokers were 5.88 x 10(-3) Delta OD(405)/h versus 11.0 x 10(-3) Delta OD(405)/h in non-smokers (P = 0.02), and in female smokers 9.16 x 10(-3) Delta OD(405)/h versus 10.88 x 10(-3) Delta OD(405)/h in non-smokers (P = 0.72). The effect was strengthened by high pack-years of smoking (MMP-8, P = 0.04; elastase, P = 0.01). Both biomarkers increased with gingival bleeding. However, statistically significant associations were observed with bleeding on probing and MMP-8 (P = 0.04); MMP-8 was suggestively associated with probing depth (P = 0.09) in non-smoking boys. In smokers with calculus, MMP-8 increased after adjusting with body mass index (P = 0.03). No corresponding differences were seen in girls. Smoking significantly decreased both biomarkers studied. Compared to girls, boys seem to have enhanced susceptibility for periodontitis as reflected in salivary MMP-8 values.

Comparison of fecal elastase-1 and pancreatic function testing in children.

PubMed

Wali, Prateek D; Loveridge-Lenza, Beth; He, Zhaoping; Horvath, Karoly

2012-02-01

The fecal pancreatic elastase-1 (FE-1) test is considered a simple, noninvasive, indirect measure of pancreatic function. We aimed to evaluate the performance of the FE-1 test compared with the direct pancreatic function test (PFT) with secretin stimulation in children. Data of 70 children (6 months-17 years of age) who had both FE-1 test and PFT were analyzed. The average FE-1 concentration was 403 ± 142 μg/g. Eleven children had concentrations below 200  μg/g, 23 between 201 to 500 μg/g, and 36 were above 500 μg/g. The average pancreatic elastase activity measured on direct stimulation was 49.1 ± 38.6  μmol · min (-1)· ml(-1) and 11 children had activity below the established cutoff (10.5 μmol · min(-1) · ml(-1)). Among the 11 children with pathologic PFT, 7 had normal FE-1, 4 were in the intermediate range (201-500 μg/g), and none were in the low range (<200 μg/g). Among the 59 children with normal direct PFT 11 (19%) had pathologic (<200 μg/g) and 19 (32%) had intermediate FE-1 tests. Twenty-nine children had both normal FE-1 concentration and normal PFT, giving a negative predictive value of 80%. The correlation between pancreatic elastase activity and FE-1 concentration was poor (r = 0.190). The sensitivity of the FE-1 test was found to be 41.7%, whereas the specificity was 49.2%. The positive predictive value of the FE-1 test was only 14%. The FE-1 test is a simple, noninvasive, indirect method; however, ordering physicians should be aware of its limitations. It can give false-positive results and has low sensitivity in children with mild pancreatic insufficiency without cystic fibrosis and in those with isolated pancreatic enzyme deficiencies.

Association between injury pattern of patients with multiple injuries and circulating levels of soluble tumor necrosis factor receptors, interleukin-6 and interleukin-10, and polymorphonuclear neutrophil elastase.

PubMed

Hensler, Thorsten; Sauerland, Stefan; Bouillon, Bertil; Raum, Marcus; Rixen, Dieter; Helling, Hanns-J; Andermahr, Jonas; Neugebauer, Edmund A M

2002-05-01

Our knowledge about the bidirectional interactions between brain and whole organism after trauma is still limited. It was the purpose of this prospective clinical study to determine the influence of severe head trauma (SHT) as well as trauma in different anatomic injury regions on posttraumatic inflammatory mediator levels from patients with multiple injuries. Thirty-five healthy controls, 33 patients with an isolated SHT, 47 patients with multiple injuries without SHT, and 45 patients with both SHT and multiple injuries were studied. The posttraumatic plasma levels of soluble tumor necrosis factor receptors p55 and p75, interleukin (IL)-6, IL-10, and polymorphonuclear neutrophil (PMN) elastase were monitored using enzyme-linked immunosorbent assay technique. The influence of head injuries as well as thorax, abdomen, and extremity injuries on the mediator release from patients with multiple injuries was investigated by multivariate linear regression models. The soluble tumor necrosis factor receptor p55/p75 ratio was significantly elevated within 3 hours of trauma in all three injury groups and returned to reference ratios after 12 hours. The lowest increase was found in patients suffering from an isolated SHT. Lowest mediator levels in this patient population were also found for IL-6, IL-10, and PMN elastase during the first 36 hours after trauma. Additional injuries to the head, thorax, abdomen, and extremity modulated mediator levels to a different degree. No specific effect was found for SHT when compared with other injury groups. Thorax injuries caused the quickest rise in mediator levels, whereas abdominal injuries significantly increased PMN elastase levels 12 to 24 hours after trauma. Traumatic injuries cause the liberation of various mediators, without any specific association between anatomic injury pattern and the pattern of mediator release.

Inhibitory effect of burdock leaves on elastase and tyrosinase activity.

PubMed

Horng, Chi-Ting; Wu, Hsing-Chen; Chiang, Ni-Na; Lee, Chiu-Fang; Huang, Yu-Syuan; Wang, Hui-Yun; Yang, Jai-Sing; Chen, Fu-An

2017-10-01

Burdock ( Arctium lappa L.) leaves generate a considerable amount of waste following burdock root harvest in Taiwan. To increase the use of burdock leaves, the present study investigated the optimal methods for producing burdock leaf extract (BLE) with high antioxidant polyphenolic content, including drying methods and solvent extraction concentration. In addition, the elastase and tyrosinase inhibitory activity of BLE was examined. Burdock leaves were dried by four methods: Shadow drying, oven drying, sun drying and freeze-drying. The extract solution was then subjected to total polyphenol content analysis and the method that produced BLE with the highest amount of total antioxidant components was taken forward for further analysis. The 1,1-diphenyl-2-pycrylhydrazyl scavenging, antielastase and antityrosinase activity of the BLE were measured to enable the evaluation of the antioxidant and skin aging-associated enzyme inhibitory activities of BLE. The results indicated that the total polyphenolic content following extraction with ethanol (EtOH) was highest using the freeze-drying method, followed by the oven drying, shadow drying and sun drying methods. BLE yielded a higher polyphenol content and stronger antioxidant activity as the ratio of the aqueous content of the extraction solvent used increased. BLE possesses marked tyrosinase and elastase inhibitory activities, with its antielastase activity notably stronger compared with its antityrosinase activity. These results indicate that the concentration of the extraction solvent was associated with the antioxidant and skin aging-associated enzyme inhibitory activity of BLE. The reactive oxygen species scavenging theory of skin aging may explain the tyrosinase and elastase inhibitory activity of BLE. In conclusion, the optimal method for obtaining BLE with a high antioxidant polyphenolic content was freeze-drying followed by 30-50% EtOH extraction. In addition, the antielastase and antityrosinase activities of the BLE produced may be aid in the development of skincare products with antiwrinkle and skin-evening properties.

Undiagnosed pancreatic exocrine insufficiency and chronic pancreatitis in functional GI disorder patients with diarrhea or abdominal pain.

PubMed

Talley, Nicholas J; Holtmann, Gerald; Nguyen, Quoc Nam; Gibson, Peter; Bampton, Peter; Veysey, Martin; Wong, James; Philcox, Stephen; Koloski, Natasha; Bunby, Lisa; Jones, Michael

2017-11-01

A previous UK study showed that 6.1% of patients with diarrhea-predominant irritable bowel syndrome (IBS-D) had evidence of severe pancreatic exocrine insufficiency (PEI), but these findings need replication. We aimed to identify the prevalence of PEI based on fecal elastase stool testing in consecutive outpatients presenting with chronic unexplained abdominal pain and/or diarrhea and/or IBS-D. Patients aged over 40 years presenting to hospital outpatient clinics from six sites within Australia with unexplained abdominal pain and/or diarrhea for at least 3 months and/or IBS-D were studied. Patients completed validated questionnaires and donated a stool sample in which elastase concentration was measured by ELISA. A concentration of < 100 mcg/g stool represented severe and < 200 mcg/g mild to moderate PEI. Patients whose fecal elastase was < 200 mcg/g underwent testing for pancreatic pathology with an endoscopic ultrasound or abdominal CT. Two hundred eighteen patients (mean age of 60 years, 29.4% male) were studied. PEI was found in 4.6% (95% CI 2.2-8.3%) (n = 10), with five patients (2.3% (95% CI 0.8-5.3%) having severe PEI. Only male sex and heavy alcohol use were significantly associated with abnormal versus normal pancreatic functioning. Of seven patients who underwent endoscopic ultrasound or CT, two had features indicative of chronic pancreatitis. One in 50 patients with IBS-D or otherwise unexplained abdominal pain or diarrhea have an abnormal fecal elastase, but unexpected pancreatic insufficiency was detected in only a minority of these. This study failed to confirm the high prevalence of PEI among patients with unexplained GI symptoms previously reported. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Inhibitory effect of burdock leaves on elastase and tyrosinase activity

PubMed Central

Horng, Chi-Ting; Wu, Hsing-Chen; Chiang, Ni-Na; Lee, Chiu-Fang; Huang, Yu-Syuan; Wang, Hui-Yun; Yang, Jai-Sing; Chen, Fu-An

2017-01-01

Burdock (Arctium lappa L.) leaves generate a considerable amount of waste following burdock root harvest in Taiwan. To increase the use of burdock leaves, the present study investigated the optimal methods for producing burdock leaf extract (BLE) with high antioxidant polyphenolic content, including drying methods and solvent extraction concentration. In addition, the elastase and tyrosinase inhibitory activity of BLE was examined. Burdock leaves were dried by four methods: Shadow drying, oven drying, sun drying and freeze-drying. The extract solution was then subjected to total polyphenol content analysis and the method that produced BLE with the highest amount of total antioxidant components was taken forward for further analysis. The 1,1-diphenyl-2-pycrylhydrazyl scavenging, antielastase and antityrosinase activity of the BLE were measured to enable the evaluation of the antioxidant and skin aging-associated enzyme inhibitory activities of BLE. The results indicated that the total polyphenolic content following extraction with ethanol (EtOH) was highest using the freeze-drying method, followed by the oven drying, shadow drying and sun drying methods. BLE yielded a higher polyphenol content and stronger antioxidant activity as the ratio of the aqueous content of the extraction solvent used increased. BLE possesses marked tyrosinase and elastase inhibitory activities, with its antielastase activity notably stronger compared with its antityrosinase activity. These results indicate that the concentration of the extraction solvent was associated with the antioxidant and skin aging-associated enzyme inhibitory activity of BLE. The reactive oxygen species scavenging theory of skin aging may explain the tyrosinase and elastase inhibitory activity of BLE. In conclusion, the optimal method for obtaining BLE with a high antioxidant polyphenolic content was freeze-drying followed by 30–50% EtOH extraction. In addition, the antielastase and antityrosinase activities of the BLE produced may be aid in the development of skincare products with antiwrinkle and skin-evening properties. PMID:28912875

Quorum sensing inhibitors from marine bacteria Oceanobacillus sp. XC22919.

PubMed

Chen, Xiaochun; Chen, Jianwei; Yan, Yinchun; Chen, Su; Xu, Xuewei; Zhang, Huawei; Wang, Hong

2018-02-12

In this study, three active compounds isolated from Oceanobacillus sp. XC22919 were identified as 2-methyl-N-(2'-phenylethyl) butyramide (1), 3-methyl-N-(2'-phenylethyl)-butyramide (2) and benzyl benzoate (3), and were first reported to exhibit the apparent quorum sensing inhibitory activities against C. violaceum 026 and P. aeruginosa. Compounds 1-3 inhibited violacein production of C. violaceum 026 by 10.5-55.7, 11.2-55.7, and 27.2%-95.7%, respectively, and inhibited pyocyanin production of P. aeruginosa by 1.7-50.8, 39.1-90.7, and 57.2%-98.7%, respectively. The azocasein-degrading proteolytic rates of P. aeruginosa were observed by 13.4-31.5, 13.4-28.8, and 11.3%-21.1%, respectively. With respect to elastase, the range of inhibition of activity of compounds 1-3 was 2.1-30.3, 4.2-18.2, and 8.9%-15.7%, respectively. Compounds 1 and 3 also showed a concentration-dependent attenuation in biofilm formation, with the maximum of 50.6% inhibition, and 37.7% inhibition at 100 μg/mL, respectively.

Eunicellin-based diterpenoids from the Formosan soft coral Klyxum molle with inhibitory activity on superoxide generation and elastase release by neutrophils.

PubMed

Lin, Ming-Chang; Chen, Bo-Wei; Huang, Chiung-Yao; Dai, Chang-Feng; Hwang, Tsong-Long; Sheu, Jyh-Horng

2013-09-27

Eleven new eunicellin-based diterpenoids possessing a cladiellane skeleton with a C-2, C-9 ether bridge, klymollins I-S (1-11), have been isolated from the EtOAc extract of the soft coral Klyxum molle from Taiwan waters. The structures of compounds 1-11 were elucidated by extensive spectroscopic analysis, including 2D NMR spectroscopy (COSY, HSQC, HMBC, and NOESY). Compound 5 exhibited cytotoxicity toward several cancer cell lines. Compound 5 is the first eunicellin-based metabolite bearing a phenyl group and displays significant inhibition of both superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophils.

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine

DOE PAGES

Julier, Ziad; Martino, Mikaël M.; de Titta, Alexandre; ...

2015-02-24

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with itsmore » neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. We reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8 + T cell responses in two murine cancer models.« less

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

PubMed

Edwards, J Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle Nee; French, Alfred D; Condon, Brian D

2018-03-13

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity ( K m ) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased K m observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency ( k cat / K m ), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

PubMed

Nickerson, Nicholas N; Joag, Vineet; McGavin, Martin J

2008-09-01

The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.

Inhibition of elastase-pulmonary emphysema in dominant-negative MafB transgenic mice.

PubMed

Aida, Yasuko; Shibata, Yoko; Abe, Shuichi; Inoue, Sumito; Kimura, Tomomi; Igarashi, Akira; Yamauchi, Keiko; Nunomiya, Keiko; Kishi, Hiroyuki; Nemoto, Takako; Sato, Masamichi; Sato-Nishiwaki, Michiko; Nakano, Hiroshi; Sato, Kento; Kubota, Isao

2014-01-01

Alveolar macrophages (AMs) play important roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously demonstrated upregulation of the transcription factor MafB in AMs of mice exposed to cigarette smoke. The aim of this study was to elucidate the roles of MafB in the development of pulmonary emphysema. Porcine pancreatic elastase was administered to wild-type (WT) and dominant-negative (DN)-MafB transgenic (Tg) mice in which MafB activity was suppressed only in macrophages. We measured the mean linear intercept and conducted cell differential analysis of bronchoalveolar lavage (BAL) cells, surface marker analysis using flow cytometry, and immunohistochemical staining using antibodies to matrix metalloproteinase (MMP)-9 and MMP-12. Airspace enlargement of the lungs was suppressed significantly in elastase-treated DN-MafB Tg mice compared with treated WT mice. AMs with projected pseudopods were decreased in DN-MafB Tg mice. The number of cells intermediately positive for F4/80 and weakly or intermediately positive for CD11b, which are considered cell subsets of matured AMs, decreased in the BAL of DN-MafB Tg mice. Furthermore, MMP-9 and -12 were significantly downregulated in BAL cells of DN-MafB Tg mice. Because MMPs exacerbate emphysema, MafB may be involved in pulmonary emphysema development through altered maturation of macrophages and MMP expression.

Anti-wrinkle effects of a tuna heart H2O fraction on Hs27 human fibroblasts.

PubMed

Kim, Young-Min; Jung, Hee-Jin; Choi, Jae-Sue; Nam, Taek-Jeong

2016-01-01

With the increase in life expectancy, there is also growing interest in anti-aging treatments and technologies. The development of anti-aging functional drugs for the skin, and foods from natural sources, may offer solutions to this global matter. Aging involves structural, functional and biochemical changes that occur throughout cells and bodily tissues; the amount of hormones secreted from of all human organs, including the skin, decreases over time. Matrix metalloproteinase (MMP) genes (MMP-1 and -8) play an important role in the aging of skin fibroblasts. For example, an increased MMP expression causes accelerated aging and the degradation of skin elasticity-related genes. In the present study, we examined the anti-wrinkle effects of tuna heart extract which are mediated through the inhibition of MMPs in skin cells. Generally, tuna contains high concentrations of selenium and antioxidants, which serve to remove free radicals, and is known to delay skin and body aging. In addition, unsaturated fatty acids in tuna help to maintain the natural glossy look of skin, and increase skin elasticity, providing moisture for dry skin. A recent study confirmed the various bio-effects of boiled tuna extract and muscle. However, bioactivity studies using tuna heart are limited. Thus, in the present study, we obtained extracts and fractions of tuna heart, and examined their effects on Hs27 human fibroblast proliferation using an MTS assay. In addition, we measured procollagen type 1 levels and elastase activity, and performed β-galactosidase staining. We then measured the expression levels of phosphatidylinositol 3-kinase/Akt and MMP-related genes by western blot analysis and RT-PCR. Our results revealed that tuna heart extract decreased MMP expression by upregulating tissue inhibitors of metalloproteinase-1 (TIMP-1) and decreasing elastase activity, thus exerting anti-aging and anti-wrinkle effects by increasing collagen synthesis and promoting skin fibroblast proliferation. Thus, our data suggest that tuna heart (TH)-H2O fractions exert anti-wrinkle effects on Hs27 cells.

Effect of Alpinia zerumbet components on antioxidant and skin diseases-related enzymes

PubMed Central

2012-01-01

Background The skin is chronically exposed to endogenous and environmental pro-oxidant agents, leading to the harmful generation of reactive oxygen species. Antioxidant is vital substances which possess the ability to protect the body from damage cause by free radicals induce oxidative stress. Alpinia zerumbet, a traditionally important economic plant in Okinawa, contains several interesting bioactive constituents and possesses health promoting properties. In this regard, we carried out to test the inhibitory effect of crude extracts and isolated compounds from A. zerumbet on antioxidant and skin diseases-related enzymes. Methods The antioxidant activities were examined by DPPH, ABTS and PMS-NADH radical scavenging. Collagenase, elastase, hyaluronidase and tyrosinase were designed for enzymatic activities to investigate the inhibitory properties of test samples using a continuous spectrophotometric assay. The inhibitory capacity of test samples was presented at half maximal inhibitory concentration (IC50). Results The results showed that aqueous extract of the rhizome was found to have greater inhibitory effects than the others on both of antioxidant and skin diseases-related enzymes. Furthermore, 5,6-dehydrokawain (DK), dihydro-5,6-dehydrokawain (DDK) and 8(17),12-labdadiene-15,16-dial (labdadiene), isolated from rhizome, were tested for antioxidant and enzyme inhibitions. We found that DK showed higher inhibitory activities on DPPH, ABTS and PMS-NADH scavenging (IC50 = 122.14 ± 1.40, 110.08 ± 3.34 and 127.78 ± 4.75 μg/ml, respectively). It also had stronger inhibitory activities against collagenase, elastase, hyaluronidase and tyrosinase (IC50 = 24.93 ± 0.97, 19.41 ± 0.61, 19.48 ± 0.24 and 76.67 ± 0.50 μg/ml, respectively) than DDK and labdadiene. Conclusion Our results indicate that the rhizome aqueous extract proved to be the source of bioactive compounds against enzymes responsible for causing skin diseases. Moreover, DK could be used as a potent inhibitor and be further exploited to be used in anti-skin disease formulations. PMID:22827920

[In vitro and in vivo studies on the cardioprotective action of oligomeric procyanidins in a Crataegus extract of leaves and blooms].

PubMed

Chatterjee, S S; Koch, E; Jaggy, H; Krzeminski, T

1997-07-01

Cardioprotective effects of a standardized extract from leaves with flowers of Crataegus (WS-1442; content of oligomeric procyandins [OPC]: 18.75%) have recently been demonstrated in an ischemia-reperfusion model in rats. Further studies were now conducted to clarify the mechanism of action and to identify active constituents involved in these effects of WS-1442. Exhausting partitioning between ethyl acetate/water and successive ultrafiltration of the aqueous layer led to the quantitative recovery of three fractions, which were tested for their in vitro radical scavenging (RS) and human neutrophil elastase (HNE) inhibitory activity. The lipophilic ethylacetate-soluble fraction A, enriched in flavone derivatives and constituting 14.9% of WS-1442, was as active as WS-1442 in inhibiting HNE. However, its RS activity was only about half that of the primary extract. Although 67.9% of WS-1442 was recovered in a water-soluble low molecular weight fraction B, this fraction displayed only weak RS and HNE inhibiting activity. In contrast, the RS and HNE inhibiting potencies of an essentially flavone-free and OPC-rich fraction C (21.3% of WS-1442) were significantly higher (inhibition of lipid peroxidation: IC50 0.3 microgram/ml; inhibition of HNE: IC50 0.84 microgram/ml) as those of WS-1442. The RS and HNE inhibitory activities of the extract and those of its fractions correlated well with their OPC-content but not with their concentration of flavonols. These results demonstrate that OPCs of Crataegus extracts possess stronger radical scavenging activities than flavone derivatives or other constituents. In addition, the oligomeric components are potent inhibitors of HNE. Oral administration of 20 mg/kg/d of the OPC-rich fraction C to rats afforded similar protection against ischemia-reperfusion induced pathologies as treatment with WS-1442 at a dose of 100 mg/kg/d. These observations indicate that radical scavenging and elastase inhibitory activities could indeed be involved in the observed cardioprotective effects of WS-1442, and demonstrate that OPCs are major orally active constituents of WS-1442. Thus, Crataegus extracts used therapeutically for cardiovascular diseases should be analyzed and standardized for their OPC-content.

Honokiol Dimers and Magnolol Derivatives with New Carbon Skeletons from the Roots of Magnolia officinalis and Their Inhibitory Effects on Superoxide Anion Generation and Elastase Release

PubMed Central

Chen, Hung-Chung; Kuo, Ping-Chung; Lee, E-Jian; Lee, Kuo-Hsiung; Wu, Tian-Shung

2013-01-01

Two honokiol dimers, houpulins A and B (1 and 2), and two magnolol derivatives, houpulins C and D (3 and 4), were isolated and characterized from an ethanol extract obtained from the roots of Magnolia officinalis. The chemical structures were determined based on spectroscopic and physicochemical analyses, which included 1D and 2D NMR, as well as mass spectrometry data. These four oligomers possess new carbon skeletons postulated to be biosynthesized from the coupling of three or four C6-C3 subunits. In addition, the new oligomers were evaluated for inhibition of superoxide anion generation and elastase release, and houpulin B (2) was identified as a new anti-inflammatory lead compound. PMID:23667420

Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities.

PubMed

Chlapanidas, Theodora; Faragò, Silvio; Lucconi, Giulia; Perteghella, Sara; Galuzzi, Marta; Mantelli, Melissa; Avanzini, Maria Antonietta; Tosca, Marta Cecilia; Marazzi, Mario; Vigo, Daniele; Torre, Maria Luisa; Faustini, Massimo

2013-07-01

Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFNγ, while no effects were observed on TNFα and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use. Copyright © 2013 Elsevier B.V. All rights reserved.

Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells.

PubMed

Si-Tahar, M; Merlin, D; Sitaraman, S; Madara, J L

2000-06-01

Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.

A Pathogenic Nematode Targets Recognition Proteins to Avoid Insect Defenses

PubMed Central

Toubarro, Duarte; Avila, Mónica Martinez; Montiel, Rafael; Simões, Nelson

2013-01-01

Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions. PMID:24098715

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

PubMed Central

Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

2014-01-01

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity. PMID:24865846

Roflumilast attenuates allergen-induced inflammation in mild asthmatic subjects.

PubMed

Gauvreau, Gail M; Boulet, Louis-Philippe; Schmid-Wirlitsch, Christine; Côté, Johanne; Duong, Mylinh; Killian, Kieran J; Milot, Joanne; Deschesnes, Francine; Strinich, Tara; Watson, Richard M; Bredenbröker, Dirk; O'Byrne, Paul M

2011-10-26

Phosphodiesterase 4 (PDE4) inhibitors increase intracellular cyclic adenosine monophosphate (cAMP), leading to regulation of inflammatory cell functions. Roflumilast is a potent and targeted PDE4 inhibitor. The objective of this study was to evaluate the effects of roflumilast on bronchoconstriction, airway hyperresponsiveness (AHR), and airway inflammation in mild asthmatic patients undergoing allergen inhalation challenge. 25 subjects with mild allergic asthma were randomized to oral roflumilast 500 mcg or placebo, once daily for 14 days in a double-blind, placebo-controlled, crossover study. Allergen challenge was performed on Day 14, and FEV1 was measured until 7 h post challenge. Methacholine challenge was performed on Days 1 (pre-dose), 13 (24 h pre-allergen), and 15 (24 h post-allergen), and sputum induction was performed on Days 1, 13, 14 (7 h post-allergen), and 15. Roflumilast inhibited the allergen-induced late phase response compared to placebo; maximum % fall in FEV1 (p = 0.02) and the area under the curve (p = 0.01). Roflumilast had a more impressive effect inhibiting allergen-induced sputum eosinophils, neutrophils, and eosinophil cationic protein (ECP) at 7 h post-allergen (all p = 0.02), and sputum neutrophils (p = 0.04), ECP (p = 0.02), neutrophil elastase (p = 0.0001) and AHR (p = 0.004) at 24 h post-allergen. This study demonstrates a protective effect of roflumilast on allergen-induced airway inflammation. The observed attenuation of sputum eosinophils and neutrophils demonstrates the anti-inflammatory properties of PDE4 inhibition and supports the roles of both cell types in the development of late phase bronchoconstriction and AHR. ClinicalTrials.gov: NCT01365533.

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria

PubMed Central

White, Phillipa C.; Milward, Michael R.; Cooper, Paul R.

2017-01-01

ABSTRACT Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii, stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. PMID:28947649

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

PubMed

Hirschfeld, Josefine; White, Phillipa C; Milward, Michael R; Cooper, Paul R; Chapple, Iain L C

2017-12-01

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii , stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. Copyright © 2017 American Society for Microbiology.

Creatinine

MedlinePlus

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Microgravity

NASA Image and Video Library

1989-02-03

(PCG) Protein Crystal Growth Porcine Elastase. This enzyme is associated with the degradation of lung tissue in people suffering from emphysema. It is useful in studying causes of this disease. Principal Investigator on STS-26 was Charles Bugg.

Toxoplasmosis Testing

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Helicobacter pylori Test

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VMA Test

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Vitamin A Test

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B Vitamins Test

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Celiac Disease Tests

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Plasma Free Metanephrines

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Dengue Fever Testing

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Lactose Tolerance Tests

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Uric Acid Test

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Comprehensive Metabolic Panel

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5-HIAA Test

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Phosphorus Test

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Peritoneal Fluid Analysis

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Serotonin Test

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Throat Culture

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TB Screening Tests

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PTH Test

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Blood Typing

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AMA Test

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Progesterone Test

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Gram Stain

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IL-23 Is Essential for the Development of Elastase-Induced Pulmonary Inflammation and Emphysema.

PubMed

Fujii, Utako; Miyahara, Nobuaki; Taniguchi, Akihiko; Waseda, Koichi; Morichika, Daisuke; Kurimoto, Etsuko; Koga, Hikari; Kataoka, Mikio; Gelfand, Erwin W; Cua, Daniel J; Yoshimura, Akihiko; Tanimoto, Mitsune; Kanehiro, Arihiko

2016-11-01

We recently reported that IL-17A plays a critical role in the development of porcine pancreatic elastase (PPE)-induced emphysema. The proliferation of T-helper type 17 (Th17) cells was induced by IL-23. To determine the contribution of IL-23 to the development of pulmonary emphysema, a mouse model of PPE-induced emphysema was used in which responses of IL-23p19-deficient (IL-23 -/- ) and wild-type (WT) mice were compared. Intratracheal instillation of PPE induced emphysematous changes in the lungs and was associated with increased levels of IL-23 in lung homogenates. Compared with WT mice, IL-23 -/- mice developed significantly lower static compliance values and markedly reduced emphysematous changes on histological analyses after PPE instillation. These changes were associated with lower levels of IL-17A and fewer Th17 cells in the lung. The neutrophilia seen in bronchoalveolar lavage fluid of WT mice was attenuated in IL-23 -/- mice, and the reduction was associated with decreased levels of keratinocyte-derived cytokine and macrophage inflammatory protein-2 in bronchoalveolar lavage fluid. Treatment with anti-IL-23p40 monoclonal antibody significantly attenuated PPE-induced emphysematous changes in the lungs of WT mice. These data identify the important contributions of IL-23 to the development of elastase-induced pulmonary inflammation and emphysema, mediated through an IL-23/IL-17 pathway. Targeting IL-23 in emphysema is a potential therapeutic strategy for delaying disease progression.

Subclinical exocrine pancreatic dysfunction resulting from decreased cholecystokinin secretion in the presence of intestinal villous atrophy.

PubMed

Nousia-Arvanitakis, Sanda; Fotoulaki, Maria; Tendzidou, Kyriaki; Vassilaki, Constantina; Agguridaki, Christina; Karamouzis, Michael

2006-09-01

The aim of this study was to evaluate the concept that pancreatic dysfunction in patients having gluten sensitivity (celiac disease [CD]) or cow's milk protein enteropathy (CMPE) may result from the lack of pancreatic enzyme stimulation in the absence or decrease of cholecystokinin (CCK) secretion caused by villous atrophy. The following parameters were measured: plasma CCK in response to a fatty meal and human pancreatic fecal elastase in 24 patients with CD while on gluten-free diet and after gluten provocation and in 12 patients with CMPE at diagnosis and after a 6-month period of cow's milk-free diet. Intestinal mucosa morphology was examined by small bowel biopsy. Sixty-three controls having no organic gastrointestinal problems were investigated once at the time of diagnostic evaluation. Fasting CCK, obtained at a time when patients with CD or CMPE had normal intestinal mucosa, was significantly different from postprandial and comparable to that of the control group. Fasting CCK obtained from patients with villous atrophy was also statistically different, but not significantly, from the postprandial. Fasting and postprandial plasma CCK and fecal pancreatic elastase values from patients having normal intestinal mucosa were significantly higher than those obtained from patients with villous atrophy. Significant correlation of intestinal mucosa morphology and CCK with fecal elastase concentration was documented. Exocrine pancreatic dysfunction in individuals having villous atrophy may be the consequence of decreased CCK secretion. Cholecystokinin and pancreatic secretion is restored to normal, with intestinal mucosa regeneration.

Review of commonly used clinical pathology parameters for general gastrointestinal disease with emphasis on small animals.

PubMed

Steiner, Jörg M

2014-01-01

A wide variety of markers are available to assess the function and pathology of the gastrointestinal (GI) tract. This review describes some of these markers with special emphasis given to markers used in dogs and cats. Small intestinal disease can be confirmed and localized by the measurement of serum concentrations of folate and cobalamin. Fecal α1-proteinase inhibitor concentration can increase in individuals with excessive GI protein loss. A wide variety of inflammatory markers are available for a variety of species that can be used to assess the inflammatory activity of various types of inflammatory cells in the GI tract, although most of these markers assess neutrophilic inflammation, such as neutrophil elastase, calprotectin, or S100A12. N-methylhistamine can serve as a marker of mast cell infiltration. Markers for lymphocytic or eosinophilic inflammation are currently under investigation. Exocrine pancreatic function can be assessed by measurement of serum concentrations of pancreatic lipase immunoreactivity (PLI) and trypsin-like immunoreactivity (TLI). Serum PLI concentration is increased in individuals with pancreatitis and has been shown to be highly specific for exocrine pancreatic function and sensitive for pancreatitis. Serum TLI concentration is severely decreased in individuals with exocrine pancreatic insufficiency.

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill L; Hook, Vivian Y H

2005-02-18

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

von Willebrand Factor Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

ADH (Antidiuretic Hormone) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Anti-Müllerian Hormone

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

C-Reactive Protein (CRP) Test

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GGT (Gamma-Glutamyl Transferase) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Catecholamines, Plasma and Urine Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

PT and INR Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Pleural Fluid Analysis Test

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T3 (Triiodothyronine) Test

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Tips on Blood Testing

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

CEA (Carcinoembryonic Antigen) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Urine Albumin and Albumin/ Creatinine Ratio

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Total Protein and Albumin/Globulin Ratio Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Liberation of Desmosine and Isodesmosine as Amino Acids from Insoluble Elastin by Elastolytic Proteases

PubMed Central

Umeda, Hideyuki; Aikawa, Masanori; Libby, Peter

2011-01-01

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation. PMID:21726534

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

PubMed Central

Bossa, Francesco; Barra, Donatella; Carloni, Massimo; Fasella, Paolo; Riva, Francesca; Doonan, Shawn; Doonan, Hilary J.; Hanford, Robin; Vernon, Charles A.; Walker, John M.

1973-01-01

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. PMID:4748834

Superabsorbent polymer-containing wound dressings have a beneficial effect on wound healing by reducing PMN elastase concentration and inhibiting microbial growth.

PubMed

Wiegand, C; Abel, M; Ruth, P; Hipler, U C

2011-11-01

A comprehensive in vitro approach was used to assess the effects of superabsorbent polymer (SAP) containing wound dressings in treatment of non-healing wounds. A slight negative effect on HaCaT cells was noted in vitro which is most likely due to the Ca(2+) deprivation of the medium by binding to the SAP. It could be shown that SAP wound dressings are able to bind considerable amounts of elastase reducing enzyme activity significantly. Furthermore, SAP's inhibit the formation of free radicals. The SAP-containing wound dressings tested also exhibited a significant to strong antimicrobial activity effectively impeding the growth of gram-negative and gram-positive bacteria as well as yeasts. In conclusion, in vitro data confirm the positive effect of SAP wound dressings observed in vivo and suggest that they should be specifically useful for wound cleansing.

Nucleosomes and neutrophil activation in sickle cell disease painful crisis

PubMed Central

Schimmel, Marein; Nur, Erfan; Biemond, Bart J.; van Mierlo, Gerard J.; Solati, Shabnam; Brandjes, Dees P.; Otten, Hans-Martin; Schnog, John-John; Zeerleder, Sacha

2013-01-01

Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ0-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ+-thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome. PMID:23911704

From Bench to Bedside: Utility of the Rabbit Elastase Aneurysm Model in Pre-Clinical Studies of Intracranial Aneurysm Treatment

PubMed Central

Brinjikji, Waleed; Ding, Yong H; Kallmes, David F; Kadirvel, Ramanathan

2016-01-01

Summary Pre-clinical studies are important in helping practitioners and device developers improve techniques and tools for endovascular treatment of intracranial aneurysms. Thus, an understanding of the major animal models used in such studies is important. The New Zealand rabbit elastase induced arterial aneurysm of the common carotid artery is one of the most commonly used models in testing the safety and efficacy of new endovascular devices. In this review we discuss 1) various techniques used to create the aneurysm, 2) complications of aneurysm creation, 3) natural history of the arterial aneurysm, 4) histopathologic and hemodynamic features of the aneurysm 5) devices tested using this model and 6) weaknesses of the model. We demonstrate how pre-clinical studies using this model are applied in treatment of intracranial aneurysms in humans. The model has a similar hemodynamic, morphological and histologic characteristics to human aneurysms and demonstrates similar healing responses to coiling as human aneurysms. Despite these strengths however, the model does have many weaknesses including the fact that the model does not emulate the complex inflammatory processes affecting growing and ruptured aneurysms. Furthermore the model’s extracranial location affects its ability to be used in preclinical safety assessments of new devices. We conclude that the rabbit elastase model has characteristics that make it a simple and effective model for preclinical studies on the endovascular treatment of intracranial aneurysms however further work is needed to develop aneurysm models that simulate the histopathologic and morphologic characteristics of growing and ruptured aneurysms. PMID:25904642

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

PubMed

Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

2015-06-01

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. © FASEB.

Inhibition of early AAA formation by aortic intraluminal pentagalloyl glucose (PGG) infusion in a novel porcine AAA model.

PubMed

Kloster, Brian O; Lund, Lars; Lindholt, Jes S

2016-05-01

The vast majority of abdominal aortic aneurysms found in screening programs are small, and as no effective treatment exits, many will expand until surgery is indicated. Therefore, it remains intriguing to develop a safe and low cost treatment of these small aneurysms, that is able to prevent or delay their expansion. In this study, we investigated whether intraluminal delivered pentagalloyl glucose (PGG) can impair the early AAA development in a porcine model. The infrarenal aorta was exposed in thirty pigs. Twenty underwent an elastase based AAA inducing procedure and ten of these received an additional intraluminal PGG infusion. The final 10 were sham operated and served as controls. All pigs who only had an elastase infusion developed macroscopically expanding AAAs. In pigs treated with an additional PGG infusion the growth rate of the AP-diameter rapidly returned to physiological values as seen in the control group. In the elastase group, histology revealed more or less complete resolution of the elastic lamellae in the media while they were more abundant, coherent and structurally organized in the PGG group. The control group displayed normal physiological growth and histology. In our model, intraluminal delivered PGG is able to penetrate the aortic wall from the inside and impair the early AAA development by stabilizing the elastic lamellae and preserving their integrity. The principle holds a high clinical potential if it can be translated to human conditions, since it, if so, potentially could represent a new drug for stabilizing small abdominal aneurysms.

Superiority of PC-SOD to other anti-COPD drugs for elastase-induced emphysema and alteration in lung mechanics and respiratory function in mice.

PubMed

Tanaka, Ken-Ichiro; Sato, Keizo; Aoshiba, Kazutetsu; Azuma, Arata; Mizushima, Tohru

2012-06-15

Bronchodilators (such as ipratropium bromide), steroids (such as fluticasone propionate), and newly developed anti-inflammatory drugs (such as roflumilast) are used for patients with chronic obstructive pulmonary disease (COPD). We recently reported that lecithinized superoxide dismutase (PC-SOD) confers a protective effect in mouse models of COPD. We here examined the therapeutic effect of the combined administration of PC-SOD with ipratropium bromide on pulmonary emphysema and compared the effect of PC-SOD to other types of drugs. The severity of emphysema in mice was assessed by various criteria. Lung mechanics (elastance) and respiratory function (ratio of forced expiratory volume in the first 0.05 s to forced vital capacity) were assessed. Administration of PC-SOD by inhalation suppressed elastase-induced pulmonary emphysema, alteration of lung mechanics, and respiratory dysfunction. The concomitant intratracheal administration of ipratropium bromide did not alter the ameliorating effects of PC-SOD. Administration of ipratropium bromide, fluticasone propionate, or roflumilast alone did not suppress the elastase-induced increase in the pulmonary level of superoxide anion, pulmonary inflammatory response, pulmonary emphysema, alteration of lung mechanics, or respiratory dysfunction as effectively as did PC-SOD. PC-SOD, but not the other drugs, showed a therapeutic effect even when the drug was administered after the development of emphysema. PC-SOD also suppressed the cigarette smoke-induced pulmonary inflammatory response and increase in airway resistance. Based on these results, we consider that the inhalation of PC-SOD would be therapeutically beneficial for COPD.

In-vitro evaluation of antioxidant, anti-elastase, anti-collagenase, anti-hyaluronidase activities of safranal and determination of its sun protection factor in skin photoaging.

PubMed

Madan, Kumud; Nanda, Sanju

2018-04-01

Safranal, a monoterpene aldehyde, is present as one of the main volatile constituents of Crocus sativus Linn. (saffron flowers). This volatile constituent not only contributes to the aroma of saffron but has been reported to possess antidiabetic, antiulcer, antiasthamatic, anticonvulsant, antidepressant, cardioprotective, anticancer and UV protective properties. Most of these therapeutic actions are contributed by its potential to quench reactive oxygen species (ROS). Antioxidant properties of phytoconstituents are now being explored for developing photoprotective skin formulations. These bioactives have the potential to protect the epidermal and dermal layers of the skin which mainly comprises of elastin and collagen. When UV rays penetrate the dermal layers, there is an increased production of elastase, collagenase and hyaluronidase leading to degradation of collagen, elastin and hyaluronic acid respectively. These dermal components are responsible to provide strength, elasticity and moisture to the skin. Due to frequent exposure to sunlight, these conditions tend to augment leading to wrinkle formation and sagging of skin. Although antioxidant properties of safranal have been established on various cell lines but till date no studies have been reported regarding the dermal enzyme inhibition activities. In the current research work, a comprehensive in vitro evaluation of antioxidant, anti-elastase, anti-collagenase, anti-hyaluronidase activities of safranal along with determination of sun protection factor (SPF) was carried out. The in vitro antioxidant activity was carried out by diphenylpicrylhydrazyl (DPPH) method and its IC 50 value was found to be 22.7 μg/ml. The enzyme inhibition IC 50 values of safranal for anti elastase activity were found to be 43.6 μg/ml, 70 μg/ml for antihyaluronidase activity and 9.4 μg/ml for anticollagenase activity. Photoprotective activity of safranal was determined by UV absorbance method and SPF calculated by Mansur equation which was found to be 6.6. The significant inhibitory activity of safranal on matrix metalloproteinases (MMPs) responsible for aging and a higher SPF established that this bioorganic molecule is a strong photoprotective agent. Its established free radical scavenging capability along with above characteristics make it a valuable component to be incorporated into herbal antiaging formulations. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways.

PubMed

Reihill, James A; Walker, Brian; Hamilton, Robert A; Ferguson, Timothy E G; Elborn, J Stuart; Stutts, M Jackson; Harvey, Brian J; Saint-Criq, Vinciane; Hendrick, Siobhan M; Martin, S Lorraine

2016-09-15

In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Co-incubation of PMN and CaCo-2 cells modulates inflammatory potential.

PubMed

Schaefer, M B; Schaefer, C A; Hecker, M; Morty, R E; Witzenrath, M; Seeger, W; Mayer, K

2017-05-20

Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

Polymorphism of alpha-1-antitrypsin in hematological malignancies

PubMed Central

2009-01-01

Alpha-1-antitrypsin (AAT) or serine protease inhibitor A1 (SERPINA1) is an important serine protease inhibitor in humans. The main physiological role of AAT is to inhibit neutrophil elastase (NE) released from triggered neutrophils, with an additional lesser role in the defense against damage inflicted by other serine proteases, such as cathepsin G and proteinase 3. Although there is a reported association between AAT polymorphism and different types of cancer, this association with hematological malignancies (HM) is, as yet, unknown. We identified AAT phenotypes by isoelectric focusing (in the pH 4.2-4.9 range) in 151 serum samples from patients with HM (Hodgkins lymphomas, non-Hodgkins lymphomas and malignant monoclonal gammopathies). Healthy blood-donors constituted the control group (n = 272). The evaluated population of patients as well as the control group, were at Hardy-Weinberg equilibrium for the AAT gene (χ2 = 4.42, d.f.11, p = 0.96 and χ2 = 4.71, d.f.11, p = 0.97, respectively). There was no difference in the frequency of deficient AAT alleles (Pi Z and Pi S) between patients and control. However, we found a significantly higher frequency of PiM1M1 homozygote and PiM1 allele in HM patients than in control (for phenotype: f = 0.5166 and 0.4118 respectively, p = 0.037; for allele: f = 0.7020 and 0.6360 respectively, p = 0.05). In addition, PiM homozygotes in HM-patients were more numerous than in controls (59% and 48%, respectively, p = 0.044). PiM1 alleles and PiM1 homozygotes are both associated with hematological malignancies, although this is considered a functionally normal AAT variant. PMID:21637443

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins.

PubMed

Stapels, Daphne A C; Woehl, Jordan L; Milder, Fin J; Tromp, Angelino T; van Batenburg, Aernoud A; de Graaf, Wilco C; Broll, Samuel C; White, Natalie M; Rooijakkers, Suzan H M; Geisbrecht, Brian V

2018-02-01

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes. © 2017 The Protein Society.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

PubMed

Nustede, Rainer; Klimiankou, Maksim; Klimenkova, Olga; Kuznetsova, Inna; Zeidler, Cornelia; Welte, Karl; Skokowa, Julia

2016-01-01

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR. © 2015 John Wiley & Sons Ltd.

Proteomic analysis of rainbow trout (Oncorhynchus mykiss) intestinal epithelia: physiological acclimation to short-term starvation.

PubMed

Baumgarner, Bradley L; Bharadwaj, Anant S; Inerowicz, Dorota; Goodman, Angela S; Brown, Paul B

2013-03-01

The intestinal epithelia form the first line of defense against harmful agents in the gut lumen of most monogastric vertebrates, including teleost fishes. Previous investigations into the effect of starvation on the intestinal epithelia of teleost fishes have focused primarily on changes in morphological characteristics and targeted molecular analysis of specific enzymes. The goal of this study was to use a comprehensive approach to help reveal how the intestinal epithelia of carnivorous teleost fishes acclimate to short-term nutrient deprivation. We utilized two-dimensional gel electrophoresis (2-DE) to conduct the proteomic analysis of the mucosal and epithelial layer of the anterior gut intestinal tract (GIT) from satiation fed vs. 4 week starved rainbow trout (Oncorhynchus mykiss). A total of 40 proteins were determined to be differentially expressed and were subsequently picked for in-gel trypsin digestion. Peptide mass fingerprint analysis was conducted using matrix assisted laser desorption time-of-flight/time-of-flight. Nine of the 11 positively identified proteins were directly related to innate immunity. The expression of α-1 proteinase inhibitor decreased in starved vs. fed fish. Also, the concentration of one leukocyte elastase inhibitor (LEI) isomer decreased in starved fish, though the concentration of another LEI isomer increased in due to starvation. In addition, starvation promoted an increased concentration of the important xenobiotic-transporter p-glycoprotein. Finally, starvation resulted in a significant increase in type II keratin E2. Overall, our results indicate that starvation promoted a reduced capacity to inhibit enzymatic stress but increased xenobiotic resistance and paracellular permeability of epithelial cells in the anterior intestine of rainbow trout. Copyright © 2012 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Frederick J.; Reynolds, Lucy J.; Toward, Toby J.

This study investigated whether a correlation between leukocyte-derived elastolytic activity, alveolar epithelial type-1 cell damage, and leukocyte infiltration of the airways existed in guinea-pigs chronically exposed to inhaled lipopolysaccharide (LPS). The airway pathology of this model, notably the neutrophilia, resembles chronic obstructive pulmonary disease (COPD). The effect of the corticosteroid, dexamethasone, or the phosphodiesterase-4 (PDE4)-inhibitor, rolipram, on these features was studied. Conscious guinea-pigs were exposed for 1 h to single or repeated (nine) doses of LPS (30 {mu}g ml{sup -1}). Dexamethasone (20 mg kg{sup -1}, ip) or rolipram (1 mg kg{sup -1}, ip) was administered 24 and 0.5 h beforemore » the first exposure and daily thereafter. Bronchoalveolar lavage fluid (BALF) was removed and elastolytic activity determined as the elastase-like release of Congo Red from impregnated elastin. The presence of the specific epithelial cell type-1 protein (40-42 kDa) RT1{sub 40} in BALF was identified by Western blotting using a rat monoclonal antibody and semi-quantified by dot-blot analysis. The antibody was found to identify guinea-pig RT1{sub 40}. BALF inflammatory cells, particularly neutrophils and macrophages, and elastolytic activity were increased in chronic LPS-exposed guinea-pigs, the latter by 90%. Chronic LPS exposure also increased (10.5-fold) RT1{sub 40} levels, indicating significant alveolar epithelial type-1 cell damage. Dexamethasone or rolipram treatment reduced the influx of inflammatory cells, the elastolytic activity (by 40% and 38%, respectively), and RT1{sub 40} levels (by 50% and 57%, respectively). In conclusion, chronic LPS-exposed guinea-pigs, like COPD, exhibit elastolytic lung damage. This was prevented by a PDE4 inhibitor and supports their development for suppressing this leukocyte-mediated pathology.« less

LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

PubMed Central

Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

2012-01-01

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib. PMID:22479189

Peptide-cellulose conjugates for protease point of care diagnostics and treatment

USDA-ARS?s Scientific Manuscript database

Peptide-cellulose conjugates containing Human Neutrophil Elastase substrate sequences with both colorimetric and fluorometric signal molecules have been synthesized on a variety of cellulosic and nanocellulosic substrates including cotton and wood nanocrystals, wood nanocomposites, cotton-based aero...

Peptide-nanocellulose sensor for human neutrophil elastase detection

USDA-ARS?s Scientific Manuscript database

Advances in biosensor technology promise to revolutionize healthcare and diagnosis with noninvasive methods. This is especially the case in the area of sensors for wound healing, where approaches for biochemical and cellular markers are emerging. Nanomaterials with high surface area and biocompatibl...

Creating a model of diseased artery damage and failure from healthy porcine aorta.

PubMed

Noble, Christopher; Smulders, Nicole; Green, Nicola H; Lewis, Roger; Carré, Matt J; Franklin, Steve E; MacNeil, Sheila; Taylor, Zeike A

2016-07-01

Large quantities of diseased tissue are required in the research and development of new generations of medical devices, for example for use in physical testing. However, these are difficult to obtain. In contrast, porcine arteries are readily available as they are regarded as waste. Therefore, reliable means of creating from porcine tissue physical models of diseased human tissue that emulate well the associated mechanical changes would be valuable. To this end, we studied the effect on mechanical response of treating porcine thoracic aorta with collagenase, elastase and glutaraldehyde. The alterations in mechanical and failure properties were assessed via uniaxial tension testing. A constitutive model composed of the Gasser-Ogden-Holzapfel model, for elastic response, and a continuum damage model, for the failure, was also employed to provide a further basis for comparison (Calvo and Peña, 2006; Gasser et al., 2006). For the concentrations used here it was found that: collagenase treated samples showed decreased fracture stress in the axial direction only; elastase treated samples showed increased fracture stress in the circumferential direction only; and glutaraldehyde samples showed no change in either direction. With respect to the proposed constitutive model, both collagenase and elastase had a strong effect on the fibre-related terms. The model more closely captured the tissue response in the circumferential direction, due to the smoother and sharper transition from damage initiation to complete failure in this direction. Finally, comparison of the results with those of tensile tests on diseased tissues suggests that these treatments indeed provide a basis for creation of physical models of diseased arteries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activated human neutrophil response to perfluorocarbon nanobubbles: oxygen-dependent and -independent cytotoxic responses.

PubMed

Hwang, Tsong-Long; Fang, Chia-Lang; Al-Suwayeh, Saleh A; Yang, Li-Jia; Fang, Jia-You

2011-06-10

Nanobubbles, a type of nanoparticles with acoustically active properties, are being utilized as diagnostic and therapeutic nanoparticles to better understand, detect, and treat human diseases. The objective of this work was to prepare different nanobubble formulations and investigate their physicochemical characteristics and toxic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated human neutrophils. The nanobubbles were prepared using perfluoropentane and coconut oil as the respective core and shell, with soybean phosphatidylcholine (SPC) and/or cationic surfactants as the interfacial layers. The cytotoxic effect of the nanobubbles on neutrophils was determined by extracellular O₂(.)⁻ release, intracellular reactive oxygen species (ROS), lactate dehydrogenase (LDH), and elastase release. Particle sizes of the nanobubbles with different percentages of perfluorocarbon, oil, and surfactants in ranged 186-432 nm. The nanobubbles were demonstrated to inhibit the generation of superoxide and intracellular ROS. The cytotoxicity of nanobubbles may be mainly associated with membrane damage, as indicated by the high LDH leakage. Systems with Forestall (FE), a cationic surfactant, or higher SPC contents exhibited the greatest LDH release by 3-fold compared to the control. The further addition of an oil component reduced the cytotoxicity induced by the nanobubbles. Exposure to most of the nanobubble formulations upregulated elastase release by activated neutrophils. Contrary to this result, stearylamine (SA)-containing systems slightly but significantly suppressed elastase release. FE and SA in a free form caused stronger responses by neutrophils than when they were incorporated into nanobubbles. In summary, exposure to nanobubbles resulted in a formulation-dependent toxicity toward human neutrophils that was associated with both oxygen-dependent and -independent pathways. Clinicians should therefore exercise caution when using nanobubbles in patients for diagnostic and drug targeting aims. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Anti-skin-aging benefits of exopolymers from Aureobasidium pullulans SM2001.

PubMed

Kim, Kyung Hu; Park, Soo Jin; Lee, Ji Eun; Lee, Young Joon; Song, Chang Hyun; Choi, Seong Hun; Ku, Sae Kwang; Kang, Su Jin

2014-01-01

There have been many attempts to search for affordable and effective functional cosmetic ingredients, especially from natural sources. As research into developing a functional cosmetic ingredient, we investigated whether exopolymers from Aureobasidium pullulans SM2001 (E-AP-SM2001) exert antioxidant, antiwrinkle, whitening, and skin moisturizing effects. Antioxidant effects of E-AP-SM2001 were determined by measuring free radical scavenging capacity and superoxide dismutase (SOD)-like activity. Antiwrinkle effects were assessed through the inhibition of hyaluronidase, elastase, collagenase, and matrix metalloproteinase (MMP)-1. Whitening effects were measured by tyrosinase inhibition assay, and by melanin formation test in B16/F10 melanoma cells. Skin moisturizing effects were detected by mouse skin water content test. E-AP-SM2001 showed potent DPPH radical scavenging activity and SOD-like effects. Additionally, hyaluronidase, elastase, collagenase, and MMP-1 activities were significantly inhibited by E-AP-SM2001. We also observed that E-AP-SM2001 effectively reduced melanin production by B16/F10 melanoma cells and mushroom tyrosinase activities. Furthermore, significant increases in skin water content were detected in E-AP-SM2001- treated mouse skin, as compared with vehicle-treated control skin. Notably, a mask pack containing E-AP-SM2001 showed a >twofold more extensive moisturizing effect compared with one containing Saccharomycopsis ferment filtrate. Our results suggest that E-AP-SM2001 has adequate antiaging, antiwrinkle, and whitening benefits and skin moisturizing effect. These effects involve reducing hyaluronidase, elastase, collagenase, and MMP-1 activities, as well as inhibition of melanin production and tyrosinase activities. Therefore, the antioxidant E-AP-SM2001 may serve as a predictable functional ingredient.

Epidemic Population Structure of Pseudomonas aeruginosa: Evidence for a Clone That Is Pathogenic to the Eye and That Has a Distinct Combination of Virulence Factors

PubMed Central

Lomholt, Jeanet A.; Poulsen, Knud; Kilian, Mogens

2001-01-01

The genetic structure of a population of Pseudomonas aeruginosa, isolated from patients with keratitis, endophthalmitis, and contact lens-associated red eye, contact lens storage cases, urine, ear, blood, lungs, wounds, feces, and the environment was determined by multilocus enzyme electrophoresis. The presence and characteristics of virulence factors were determined by restriction fragment length polymorphism analysis with DNA probes for lasA, lasB, aprA, exoS, exoT, exoU, and ctx and by zymography of staphylolysin, elastase, and alkaline protease. These analyses revealed an epidemic population structure of P. aeruginosa, characterized by frequent recombination in which a particular successful clone may increase, predominate for a time, and then disasappear as a result of recombination. Epidemic clones were found among isolates from patients with keratitis. They were characterized by high activity of a hitherto-unrecognized size variant of elastase, high alkaline protease activity, and possession of the exoU gene encoding the cytotoxic exoenzyme U. These virulence determinants are not exclusive traits in strains causing keratitis, as strains with other properties may cause keratitis in the presence of predisposing conditions. There were no uniform patterns of characteristics of isolates from other types of infection; however, all strains from urinary tract infections possessed the exoS gene, all strains from environment and feces and the major part of keratitis and wound isolates exhibited high elastase and alkaline protease activity, and all strains from feces showed high staphylolysin activity, indicating that these virulence factors may be important in the pathogenesis of these infectious diseases. PMID:11553572

Experimental progressive emphysema in BALB/cJ mice as a model for chronic alveolar destruction in humans

PubMed Central

Limjunyawong, Nathachit; Craig, John M.; Lagassé, H. A. Daniel; Scott, Alan L.

2015-01-01

Emphysema, one of the major components of chronic obstructive pulmonary disease (COPD), is characterized by the progressive and irreversible loss of alveolar lung tissue. Even though >80% of COPD cases are associated with cigarette smoking, only a relatively small proportion of smokers develop emphysema, suggesting a potential role for genetic factors in determining individual susceptibility to emphysema. Although strain-dependent effects have been shown in animal models of emphysema, the molecular basis underlying this intrinsic susceptibility is not fully understood. In this present study, we investigated emphysema development using the elastase-induced experimental emphysema model in two commonly used mouse strains, C57BL/6J and BALB/cJ. The results demonstrate that mice with different genetic backgrounds show disparate susceptibility to the development of emphysema. BALB/cJ mice were found to be much more sensitive than C57BL/6J to elastase injury in both a dose-dependent and time-dependent manner, as measured by significantly higher mortality, greater body weight loss, greater decline in lung function, and a greater loss of alveolar tissue. The more susceptible BALB/cJ strain also showed the persistence of inflammatory cells in the lung, especially macrophages and lymphocytes. A comparative gene expression analysis following elastase-induced injury showed BALB/cJ mice had elevated levels of il17A mRNA and a number of classically (M1) and alternatively (M2) activated macrophage genes, whereas the C57BL/6J mice demonstrated augmented levels of interferon-γ. These findings suggest a possible role for these cellular and molecular mediators in modulating the severity of emphysema and highlight the possibility that they might contribute to the heterogeneity observed in clinical emphysema outcomes. PMID:26232300

Experimental progressive emphysema in BALB/cJ mice as a model for chronic alveolar destruction in humans.

PubMed

Limjunyawong, Nathachit; Craig, John M; Lagassé, H A Daniel; Scott, Alan L; Mitzner, Wayne

2015-10-01

Emphysema, one of the major components of chronic obstructive pulmonary disease (COPD), is characterized by the progressive and irreversible loss of alveolar lung tissue. Even though >80% of COPD cases are associated with cigarette smoking, only a relatively small proportion of smokers develop emphysema, suggesting a potential role for genetic factors in determining individual susceptibility to emphysema. Although strain-dependent effects have been shown in animal models of emphysema, the molecular basis underlying this intrinsic susceptibility is not fully understood. In this present study, we investigated emphysema development using the elastase-induced experimental emphysema model in two commonly used mouse strains, C57BL/6J and BALB/cJ. The results demonstrate that mice with different genetic backgrounds show disparate susceptibility to the development of emphysema. BALB/cJ mice were found to be much more sensitive than C57BL/6J to elastase injury in both a dose-dependent and time-dependent manner, as measured by significantly higher mortality, greater body weight loss, greater decline in lung function, and a greater loss of alveolar tissue. The more susceptible BALB/cJ strain also showed the persistence of inflammatory cells in the lung, especially macrophages and lymphocytes. A comparative gene expression analysis following elastase-induced injury showed BALB/cJ mice had elevated levels of il17A mRNA and a number of classically (M1) and alternatively (M2) activated macrophage genes, whereas the C57BL/6J mice demonstrated augmented levels of interferon-γ. These findings suggest a possible role for these cellular and molecular mediators in modulating the severity of emphysema and highlight the possibility that they might contribute to the heterogeneity observed in clinical emphysema outcomes. Copyright © 2015 the American Physiological Society.

Emphysema induced by elastase alters the mRNA relative levels from DNA repair genes in acute lung injury in response to sepsis induced by lipopolysaccharide administration in Wistar rats.

PubMed

Sergio, Luiz Philippe S; Lucinda, Leda M F; Reboredo, Maycon M; de Paoli, Flavia; Fonseca, Lídia M C; Pinheiro, Bruno V; Mencalha, Andre L; Fonseca, Adenilson S

2018-03-01

Purpose/Aim of the study: Patients suffering from chronic obstructive pulmonary disease (COPD) in association with acute respiratory distress syndrome (ARDS) present oxidative stress in lung cells, with production of free radicals and DNA lesions in pulmonary and adjacent cells. Once the DNA molecule is damaged, a set of enzymatic mechanisms are trigged to preserve genetic code integrity and cellular homeostasis. These enzymatic mechanisms include the base and the nucleotide excision repair pathways, as well as telomere regulation. Thus, the aim of this work was to evaluate the mRNA levels from APEX1, ERCC2, TP53, and TRF2 genes in lung tissue from Wistar rats affected by acute lung injury in response to sepsis and emphysema. Adult male Wistar rats were randomized into 4 groups (n = 6, for each group): control, emphysema, sepsis, and emphysema with sepsis. Pulmonary emphysema was induced by intratracheal instillation of elastase (12 IU/animal) and sepsis induced by intraperitoneal Escherichia coli lipopolysaccharide (LPS) injection (10 mg/kg). Lungs were removed, and samples were withdrawn for histological analysis and total RNA extraction, cDNA synthesis, and mRNA level evaluation by real time quantitative polymerase chain reaction. Data show acute lung injury by LPS and emphysema by elastase and that APEX1, ERCC2, TP53, and TRF2 mRNA levels are increased significantly (p < 0.01) in emphysema with sepsis group. Our results suggest that alteration in mRNA levels from DNA repair and genomic stability could be part of cell response to acute lung injury in response to emphysema and sepsis.

Should we Investigate Gastroenterology Patients for Pancreatic Exocrine Insufficiency? A Dual Centre UK Study.

PubMed

Campbell, Jennifer A; Sanders, David S; Francis, Katherine A; Kurien, Matthew; Lee, Sai; Taha, Hatim; Ramadas, Arvind; Joy, Diamond; Hopper, Andrew D

2016-09-01

Pancreatic exocrine insufficiency may be under recognised in gastroenterological practice. We aimed to identify the prevalence of pancreatic insufficiency in secondary care gastroenterology clinics and determine if co-morbidity or presenting symptoms could predict diagnosis. A secondary aim was to assess response to treatment. A dual centre retrospective analysis was conducted in secondary care gastroenterology clinics. Patients tested for pancreatic exocrine insufficiency with faecal elastase-1 (FEL-1) between 2009 and 2013 were identified in two centres. Demographics, indication and co-morbidities were recorded in addition to dose and response to pancreatic enzyme replacement therapy. Binary logistic regression was used to assess if symptoms or co-morbidities could predict pancreatic insufficiency. 1821 patients were tested, 13.1% had low FEL-1 (<200µg/g). This prevalence was sub-analysed with 5.4% having FEL-1 100-200µg/g (mild insufficiency) and 7.6% having faecal elastase readings <100µg/g. Low FEL-1 was most significantly associated with weight loss or steatorrhoea. Co-morbidity analysis showed that low levels were significantly associated with excess alcohol intake, diabetes mellitus or human immunodeficiency virus; 80.0% treated with enzyme supplements reported symptomatic benefit with no difference in response between high and low dose supplementation (p=0.761). Targeting the use of FEL-1 in individuals with specific symptoms and associated conditions can lead to improved recognition of pancreatic exocrine insufficiency in a significant proportion of secondary care patients. Intervening with lifestyle advice such as smoking cessation and minimising alcohol intake could improve outcomes. In addition, up to 80% of patients with low faecal elastase respond to supplementation.

Immunohistochemical analysis of the gingiva with periodontitis of type I plasminogen deficiency compared to gingiva with gingivitis and periodontitis and healthy gingiva.

PubMed

Kurtulus Waschulewski, Idil; Gökbuget, Aslan Y; Christiansen, Nina M; Ziegler, Maike; Schuster, Volker; Wahl, Gerhard; Götz, Werner

2016-12-01

Type I plasminogen deficiency (Plgdef) is an uncommon chronic inflammation of mucous membranes. Gingival enlargements usually proceed with progressive periodontal destruction and tooth-loss. Plasmin(ogen)-independent enzymatic mechanisms for fibrin clearance have already been discussed in the literature. Our primary objective was to verify, immunohistochemically, the occurrence of different enzymatic factors involved in tissue breakdown of inflamed compared to healthy gingiva. Secondly, we tried to find out, if these patients have a similar microbiological profile to the patients with known gingivitis and periodontitis. Immunohistochemical analysis of enzymes elastase, plasminogen (plg), cathepsin G, matrix-metalloproteinase (MMP)-3 and MMP-7 and of glycoprotein fibrinogen were performed with gingival tissues from 3 healthy controls, 8 patients with Plgdef and 3 patients with gingivitis and periodontitis. Furthermore, plaque from 5 patients with plasminogen deficiency were also obtained to determine the microbiological profile. Significantly high numbers of elastase positive leukocytes were detected in all samples. Staining for MMP-3 and MMP-7 was seen in samples with gingivitis and periodontitis with a stronger staining in samples with periodontitis by Plgdef. Fibrinogen was detectable in all samples. Staining for plg was stronger in samples with periodontitis than in other samples. Staining for cathepsin G was weak in gingivitis and periodontitis. Subgingival microbial flora showed elevated colony forming units of Prevotella intermedia/nigrescens, Fusobacterium spp., Eikenella corrodens, Porphyromonas gingivalis and viridans streptococci. Strong staining of elastase, MMP-3 and MMP-7 and weak staining of plg in Plgdef samples supports the plasmin(ogen) - independent fibrin clearance. Similar subgingival microbiological flora was observed in periodontitis with Plgdef as in other periodontal diseases. Further investigations should determine the exact pathomechanism and focus on effective treatment methods of this entity. Copyright © 2016. Published by Elsevier Ltd.

Poly herbal formulation with anti-elastase and anti-oxidant properties for skin anti-aging.

PubMed

Kalyana Sundaram, Induja; Sarangi, Deepika Deeptirekha; Sundararajan, Vignesh; George, Shinomol; Sheik Mohideen, Sahabudeen

2018-01-29

Skin forms an important part of human innate immune system. Wrinkles, thinning and roughening of skin are some of the symptoms that affect the skin as it ages. Reactive oxygen species induced oxidative stress plays a major role in skin aging by modulating the elastase enzyme level in the skin. Extrinsic factors that affect skin aging such as UV radiation can also cause malignant melanoma. Here we selected four medicinal plant materials, namely, leaves of Nyctanthes arbor-tristis, unripe and ripe Aegle marmelos fruit pulp and the terminal meristem of Musa paradisiaca flower and investigated their anti-aging properties and cytotoxicity in vitro individually as well as in a poly herbal formulation containing the four plant extracts in different ratios. The phytochemical contents of the plant extracts were investigated for radical scavenging activity and total reducing power. Based upon its anti-oxidant properties, a poly herbal formulation containing leaves of Nyctanthes arbor-tristis, unripe and ripe fruit pulp of Aegle marmelos, and the terminal meristem of Musa paradisiaca flower in the ratio 6:2:1:1 (Poly Herbal Formulation 1) and 1:1:1:1 (Poly Herbal Formulation 2), respectively were formulated. It has been observed that the Poly Herbal Formulation 1 was more potent than Poly Herbal Formulation 2 due to better anti-oxidant and anti-elastase activities in NIH3T3 fibroblast cells. In addition Poly Herbal formulation 1 also had better anti-cancer activity in human malignant melanoma cells. Based on these results these beneficial plant extracts were identified for its potential application as an anti-aging agent in skin creams as well as an anti-proliferation compound against cancer cells.

Elastase-2, an angiotensin II-generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction.

PubMed

Becari, Christiane; Silva, Marcondes A B; Durand, Marina T; Prado, Cibele M; Oliveira, Eduardo B; Ribeiro, Mauricio S; Salgado, Helio C; Salgado, Maria Cristina O; Tostes, Rita C

2017-05-01

Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI). MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries. These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS. © 2017 The British Pharmacological Society.

Tranexamic Acid Attenuates The Loss of Lung Barrier Function in a Rat Model of Polytrauma And Hemorrhage With Resuscitation.

PubMed

Wu, Xiaowu; Dubick, Michael A; Schwacha, Martin G; Cap, Andrew P; Darlington, Daniel N

2017-04-01

Severe trauma, hemorrhage, and resuscitation can lead to a trauma-related acute lung injury that involves rapid infiltration of immune cells and platelets. This infiltration involves exymatic degradation of matrix proteins, including plasmin, and causes loss of barrier function. Since tranexamic acid (TXA) inhibits plasminogen/ plasmin binding to target substrates, it may attenuate loss of barrier function after severe trauma, hemorrhage, and resuscitation. Sprague-Dawley rats were subjected to polytrauma (laparotomy, and trauma to intestines, liver, right leg skeletal muscle, and right femur fracture), then bled 40% of their blood volume. One hour after completion of polytrauma and hemorrhage, resuscitation was begun with fresh whole blood (FWB) or FWB with prior bolus administration of TXA (10 mg/kg in 0.2 mL). Polytrauma, hemorrhage, and resuscitation with FWB led to an elevation in lung water content that was significantly reduced with TXA administration. Polytrauma and hemorrhage led to rise in the number of neutrophils/monocytes and platelets in the lungs, and a rise in myeloperoxidase (MPO), neutrophil elastase and complement C5a content. While resuscitation with FWB significantly reduced the cellular infiltrate and MPO, FWB/TXA further reduced the levels of neutrophil/monocytes, neutrophil elastase, and complement C5a. Polytrauma and hemorrhage led to rise in lung plasmin activity that was significantly reduced with either FWB or FWB/TXA resuscitation. Severe trauma and hemorrhage leads to increases in lung water content, and immune cell, platelets, MPO, elastase, and C5a content in lung tissue, all markers of inflammation and acute lung injury. The addition of TXA to FWB resuscitation markedly attenuated the rise in these parameters suggesting its utility in treating acute lung injury.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

PubMed

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Miller, Derek; Unkel, Ronald; Shaman, Majid; Jacques, Suzanne M; Panaitescu, Bogdan; Garcia-Flores, Valeria; Hassan, Sonia S

2017-08-01

Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep

PubMed Central

McPherson, Andrew S.; Dhungyel, Om P.

2017-01-01

ABSTRACT Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%). PMID:28202796

In vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion.

PubMed

Llewellyn-Jones, C G; Johnson, M M; Mitchell, J L; Pye, A; Okafor, V C; Hill, S L; Stockley, R A

1995-09-01

Bronchiectasis is associated with sputum containing high levels of the proteolytic enzyme elastase, which is thought to be involved in the pathogenesis of the disease. Agents which inhibit neutrophil function and interfere with neutrophil elastase release may have a beneficial effect on the development and progression of such diseases. We have studied the effects of the nonsteroidal anti-inflammatory agent indomethacin on neutrophil function in nine patients with clinically stable bronchiectasis. All patients remained clinically stable during the study. We observed a significant reduction in peripheral neutrophil chemotaxis to 10 nmol.L-1 N-formyl-methionyl-leucyl-phenylalanine (FMLP) from a mean of 19.86 (SEM 1.35) to 8.46 (0.68) cells.field-1 after 4 weeks of therapy. There was also a significant reduction in fibronectin degradation both by resting and FMLP-stimulated neutrophils, from a mean of 1.90 (0.19) micrograms x 3 x 10(5) cells at the start of therapy to 0.87 (0.08) micrograms after 4 weeks, and from 3.17 (0.35) micrograms to 1.48 (0.05) micrograms, respectively. There was no effect on spontaneous or stimulated superoxide anion generation by neutrophils. Despite the marked changes in peripheral neutrophil function, no adverse effect was observed on viable bacterial load in the bronchial secretions. In addition, there was no difference in sputum albumin, elastase or myeloperoxidase levels, and only minor changes in the chemotactic activity of the sputum. These results suggest that nonsteroidal anti-inflammatory agents have a major effect on peripheral neutrophil function but do not appear to have an adverse effect on bacterial colonization of the airways.

Effect of fluticasone propionate on sputum of patients with chronic bronchitis and emphysema.

PubMed

Llewellyn-Jones, C G; Harris, T A; Stockley, R A

1996-02-01

The effects of fluticasone propionate (FP) on sputum chemotactic activity, elastase inhibitory potential, albumin concentrations, and peripheral neutrophil function were studied in a group of patients with clinically stable, smoking-related chronic bronchitis and emphysema. Seventeen patients (50 to 75 yr of age) were entered into a double-blind, placebo-controlled study of 1.5 mg inhaled FP/d for 8 wk. Following treatment with FP the chemotactic activity of the sputum sol phase was lower than the corresponding values for the placebo group (p < 0.01). Values fell from a mean of 21.75 (+/- 1.58) during the run-in period to 18.37 (+/- 1.46; p < 0.01) after 4 wk and 17.63 (+/- 1.86; p < 0.05) after 8 wk treatment returning to 22.08 (+/- 1.26) cell/field after the washout period. The neutrophil elastase inhibitory capacity of the sputum sol phase increased (p < 0.025) with treatment from a mean of 0.177 microM elastase inhibited/L (+/- 0.05) pretreatment to 0.413 microM (+/- 0.054) after 4 wk and 0.415 microM (+/- 0.054) after 8 wk returning to 0.270 microM (+/- 0.07) after the washout period. Treatment with FP did not result in a change in the peripheral neutrophil functions studied or sputum albumin and myeloperoxidase concentrations. The results suggest that FP may play a protective role in these patients through a reduction in the chemotactic activity of lung secretions and potentially a reduction in the recruitment of neutrophils to the lung, and also by directly affecting the proteinase/antiproteinase balance, in favor of antiproteinases, within lung secretions.

A New Flow-Diverter (the FloWise): In-Vivo Evaluation in an Elastase-Induced Rabbit Aneurysm Model.

PubMed

Kim, Byung Moon; Kim, Dong Joon; Kim, Dong Ik

2016-01-01

We aimed to evaluate the efficacy and safety of a newly developed, partially retrievable flow-diverter (the FloWise) in an elastase-induced rabbit aneurysm model. We developed a partially retrievable flow diverter composed of 48 strands of Nitinol and platinum wire. The FloWise is compatible with any microcatheter of 0.027-inch inner diameter, and is retrievable up to 70% deployment. The efficacy and safety of the FloWise were evaluated in the elastase-induced rabbit aneurysm model. The rate of technical success (full coverage of aneurysm neck) and assessment of aneurysm occlusion and stent patency was conducted by angiograms and histologic examinations at the 1-month, 3-month, and 6-month follow-up. The patency of small arterial branches (intercostal or lumbar arteries) covered by the FloWise were also assessed in the 5 subjects. We attempted FloWise insertion in a total of 32 aneurysm models. FloWise placement was successful in 31 subjects (96.9%). Two stents (6.2%) were occluded at the 3-month follow-up, but there was no evidence of in-stent stenosis in other subjects. All stented aneurysms showed progressive occlusion: grade I (complete aneurysm occlusion) in 44.4% and grade II (aneurysm occlusion > 90%) in 55.6% at 1 month; grade I in 90% and II in 10% at 3 months; and grade I in 90% and II in 10% at 6 months. All small arterial branches covered by the FloWise remained patent. A newly developed, partially retrievable flow-diverter seems to be a safe and effective tool of aneurysm occlusion, as evaluated in the rabbit aneurysm model.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep.

PubMed

McPherson, Andrew S; Dhungyel, Om P; Whittington, Richard J

2017-05-01

Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2 , and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%). Copyright © 2017 McPherson et al.

ELANE Mutations in Cyclic and Severe Congenital Neutropenia—Genetics and Pathophysiology

PubMed Central

Horwitz, Marshall S.; Corey, Seth J.; Grimes, H. Leighton; Tidwell, Timothy

2012-01-01

There are two main forms of hereditary neutropenia: cyclic and severe congenital neutropenia (SCN). Cyclic neutropenia is an autosomal dominant disorder in which neutrophil counts fluctuate between nearly normal levels and close to zero with 21-day periodicity. In contrast, SCN, also known as Kostmann syndrome, consists of chronic and profound neutropenia, with a characteristic promyelocytic maturation arrest in the bone marrow. Unlike cyclic neutropenia, SCN displays frequent acquisition of somatic mutations in the gene, CSF3R, encoding the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR), and a strong predisposition to developing myelodysplasia (MDS) and/or acute myeloid leukemia (AML). Cyclic neutropenia is caused by heterozygous mutations in the gene, ELANE (formerly known as ELA2), encoding the neutrophil granule serine protease, neutrophil elastase. SCN is genetically heterogeneous, but it is most frequently associated with ELANE mutations. While some of the different missense mutations in ELANE exhibit phenotype-genotype correlation, the same mutations are sometimes found in patients with either form of inherited neutropenia. The mutations lead to production of a mutant polypeptide, but no common biochemical abnormality, including effects on proteolysis, has been identified. Two non-mutually exclusive theories have been advanced to explain how the mutations might produce neutropenia. The mislocalization hypothesis states that mutations within neutrophil elastase or involving other proteins responsible for its intracellular trafficking cause neutrophil elastase to accumulate in inappropriate subcellular compartments. The misfolding hypothesis proposes that mutations prevent the protein from properly folding, thereby inducing the stress response pathway within the endoplasmic reticulum (ER). We discuss how the mutations themselves provide clues into pathogenesis, describe supporting and contradictory observations for both theories, and highlight outstanding questions relating to pathophysiology of neutropenia. PMID:23351986

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

PubMed

Guentsch, A; Puklo, M; Preshaw, P M; Glockmann, E; Pfister, W; Potempa, J; Eick, S

2009-06-01

This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient's own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol-dependent and isoluminol-dependent chemiluminescence. Polymorphonuclear neutrophils from patients with chronic (62.16 +/- 19.39%) and aggressive (43.26 +/- 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 +/- 19.87%) at the 30-min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.

Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs.

PubMed

Masic, Aleksandar; Lu, Xinya; Li, Junwei; Mutwiri, George K; Babiuk, Lorne A; Brown, Earl G; Zhou, Yan

2010-10-08

Influenza A virus is an important respiratory pathogen of swine that causes significant morbidity and economic impact on the swine industry. Vaccination is the first choice for prevention and control of influenza infections. Live attenuated influenza vaccines (LAIV) are approved for use in humans and horses and their application provides broad protective immunity, however no LAIV against swine influenza virus (SIV) exists in the market. Previously we reported that an elastase-dependent mutant SIV A/Sw/Sk-R345V (R345V) derived from A/Sw/Saskatchewan/18789/02 (H1N1) (SIV/Sk02) is highly attenuated in pigs. Two intratracheal administrations of R345V induced strong cell-mediated and humoral immune responses and provided a high degree of protection to antigenically different SIV infection in pigs. Here we evaluated the immunogenicity and the protective efficacy of R345V against SIV infection by intranasal administration, the more practical route for vaccination of pigs in the field. Our data showed that intranasally administered R345V live vaccine is capable of inducing strong antigen-specific IFN-γ response from local tracheo-bronchial lymphocytes and antibody responses in serum and respiratory mucosa after two applications. Intranasal vaccination of R345V provided pigs with complete protection not only from parental wild type virus infection, but also from homologous antigenic variant A/Sw/Indiana/1726/88 (H1N1) infection. Moreover, intranasal administration of R345V conferred partial protection from heterologous subtypic H3N2 SIV infection in pigs. Thus, R345V elastase-dependent mutant SIV can serve as a live vaccine against antigenically different swine influenza viruses in pigs. Copyright © 2010 Elsevier Ltd. All rights reserved.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

PubMed

Ortiz, Gustavo; Salica, Juan P; Chuluyan, Eduardo H; Gallo, Juan E

2014-11-18

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus.

PubMed

Boguś, M I; Włóka, E; Wrońska, A; Kaczmarek, A; Kazek, M; Zalewska, K; Ligęza-Żuber, M; Gołębiowski, M

2017-03-01

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions. © 2016 The Royal Entomological Society.

The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheffield, William P., E-mail: sheffiel@mcmaster.ca; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario; Bhakta, Varsha

The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assaysmore » of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.« less

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

PubMed

Nishizaki, Tomoyuki

2018-01-01

In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis. © 2018 The Author(s). Published by S. Karger AG, Basel.

SPONTANEOUSLY HYPERTENSIVE RATS ARE SUSCEPTIBLE TO AIRWAY DISEASE INDUCED BY SULFUR DIOXIDE

EPA Science Inventory

Rodent models of chronic pulmonary diseases induced by sulfur dioxide (SO2), elastase or tobacco smoke have limited utility because of their lack of chronicity of inflammation, and they demonstrate limited sensitivity to a given experimental manipulation. We hypothesized that dis...

Paraformaldehyde fixation of neutrophils for immunolabeling of granule antigens in cryoultrasections.

PubMed

Elliott, E; Dennison, C; Fortgens, P H; Travis, J

1995-10-01

Paraformaldehyde (PFA) fixation was optimized to facilitate the immobilization and labeling of multiple granule antigens, using short fixation regimens and cryoultramicrotomy of unembedded neutrophils (PMNs). In the optimal protocol, extraction of azurophil granule antigens (especially of the abundant elastase) was obviated by manipulating the polymeric state of PFA, and hence its rate of cross-linking, by altering its concentration and pH in a multistep process. Primary fixation conditions used (4% PFA, pH 8.0, 5 min) favor fixative penetration and rapid cross-linking. Stable cross-linking of the antigen was achieved in a secondary fixation step using conditions that favor larger, more cross-linking polymeric forms of PFA (8% PFA, pH 7.2, 15 min). Immobilization of granule antigens was enhanced by flotation of cut sections on fixative (8% PFA, pH 8.0) before labeling and by using post-labeling fixation with 1% glutaraldehyde. The optimized protocol facilitated immobilization and immunolabeling of elastase, myeloperoxidase, lactoferrin, and cathepsin D in highly hydrated, unembedded PMNs.

Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen.

PubMed

Nidialkova, N A; Varbanets, L D; Chernyshenko, V O

2016-01-01

Peptidase of Bacillus thuringiensis var. israelensis IМV В-7465 was isolated from culture supernatant using consecutive fractionations by an ammonium sulphate (60% saturation), ion-exchange chromatography and gel-filtration on the TSK-gels Toyoperl HW-55 and DEAE 650(M). Specific elastase (442 U∙mg of protein-1) and collagenase (212.7 U∙mg of protein-1) activities of the purified enzyme preparation were 8.0- and 6.1-fold, respectively higher than ones of the culture supernatant. Peptidase yields were 33.5% for elastase activity and 30.1% for collagenase activity. It was established that the enzyme is serine metal-dependent alkaline peptidase with Mr about 37 kDa. Maximal hydrolysis of elastin and collagen occurs at the optimum pH 8.0 and t° – 40 and 50 °С, respectively. The purified preparation has high stability at pH in the range of 7.0 to 10.0 and 40-50 °С.

Mechanisms implicated in the effects of boron on wound healing.

PubMed

Nzietchueng, Rosine Mayap; Dousset, Brigitte; Franck, Patricia; Benderdour, Mohamed; Nabet, Pierre; Hess, Ketsia

2002-01-01

Recently, we demonstrated that boron modulates the turnover of the extracellular matrix and increases TNFalpha release. In the present study, we used an in vitro test to investigate the direct effect of boron on specific enzymes (elastase, trypsin-like enzymes, collagenase and alkaline phosphatase) implicated in extracellular matrix turnover. Boron decreased the elastase and alkaline phosphatase activity, but had no effect on trypsin and collagenase activities. The effect of boron on the enzyme activities was also tested in fibroblasts considered as an in vivo test. In contrast to the results obtained in vitro, boron enhanced the trypsin-like, collagenase, and cathepsin D activities in fibroblasts. Boron did not modify the generation of free radicals compared to the control and did not seem to act on the intracellular alkaline phosphatase activity, However, as it did enhance phosphorylation, it can be hypothesized that boron may affect living cells via a mediator, which could be TNFalpha whose transduction signal involves a cascade of phosphorylations.

A study of elastase peptides from bovine white matter proteolipid.

PubMed

Lees, M B; Macklin, W B; Chao, B H

1981-10-01

Bovine white matter proteolipid has been digested with elastase in the presence of deoxycholate. After acidification, the digest was separated into an acid-soluble and an acid-insoluble fraction. The acid-insoluble fraction was enriched in nonpolar amino acids and, by a combination of solvent fractionation and chromatography, a fraction was obtained which consisted of a mixture of two peptides with a molecular weight of approximately 4000 daltons. The acid-soluble peptides were separated by molecular sieve, ion exchange and high performance liquid chromatography (HPLC) in the reverse phase mode. The purified peptides were smaller than expected on the basis of their elution position from a molecular sieve column, suggesting they were in an aggregated state during the initial chromatography. Reverse phase HPLC was shown to be useful for fingerprinting these peptide mixtures. The data demonstrate the difficulties associated with the study of this proteolipid and emphasize the tendency of both the protein and the peptides derived from it to aggregate.

The effect of mangiferin on skin: Penetration, permeation and inhibition of ECM enzymes.

PubMed

Ochocka, Renata; Hering, Anna; Stefanowicz-Hajduk, Justyna; Cal, Krzysztof; Barańska, Helena

2017-01-01

Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a polyphenol with strong antioxidant properties. Mangiferin is obtained from the mango tree (Mangifera indica L., Anacardiaceae). It has been proven that mangiferin exhibits many pharmacological activities. The aim of this study was to analyze the penetration of mangiferin into the human skin and through the skin. According to our knowledge, skin penetration and permeation studies of mangiferin have not been analyzed so far. Additionally, the influence of mangiferin on two Extracellular Matrix Enzymes (ECM): collagenase and elastase, was evaluated for the first time. It has been indicated that mangiferin is able to permeate the stratum corneum and penetrate into the epidermis and dermis in comparable amounts. For confirmation of the obtained results, fluorescence microscopy was successfully utilized. The analysis revealed the capability of mangiferin to reversibly inhibit elastase and collagenase activity. The mechanism of mangiferin interaction with both enzymes was estimated as a noncompetitive inhibition.

The effect of mangiferin on skin: Penetration, permeation and inhibition of ECM enzymes

PubMed Central

Hering, Anna; Stefanowicz–Hajduk, Justyna; Cal, Krzysztof; Barańska, Helena

2017-01-01

Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a polyphenol with strong antioxidant properties. Mangiferin is obtained from the mango tree (Mangifera indica L., Anacardiaceae). It has been proven that mangiferin exhibits many pharmacological activities. The aim of this study was to analyze the penetration of mangiferin into the human skin and through the skin. According to our knowledge, skin penetration and permeation studies of mangiferin have not been analyzed so far. Additionally, the influence of mangiferin on two Extracellular Matrix Enzymes (ECM): collagenase and elastase, was evaluated for the first time. It has been indicated that mangiferin is able to permeate the stratum corneum and penetrate into the epidermis and dermis in comparable amounts. For confirmation of the obtained results, fluorescence microscopy was successfully utilized. The analysis revealed the capability of mangiferin to reversibly inhibit elastase and collagenase activity. The mechanism of mangiferin interaction with both enzymes was estimated as a noncompetitive inhibition. PMID:28750062

Cotton Study: Albumin Binding and its Effect on Elastase Activity in the Chronic Non-healing Wound

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro, Nathan J.; Goheen, Steven C.

A comparative examination of two methods, the classical- and chromatographic, commonly used to study adsorption isotherms is presented. Both methods were used to study the solid/liquid interface of two different derivatives of cotton fiber and bovine serum albumin (BSA).

Importance of Flagella and Enterotoxins for Aeromonas Virulence in a Mouse Model

EPA Science Inventory

A genetic characterization of eight virulence factor genes, elastase, lipase, polar flagella (flaA/flaB, flaG), lateral flagella (lafA), and the enterotoxins alt, act, and ast, was performed using polymerase chain reaction with 55 drinking water and nine clinical isolates. When 1...

In vitro hemostatic, hydrogen peroxide production and elastase sequestration properties of nonwoven ultra clean greige cotton dressing

USDA-ARS?s Scientific Manuscript database

Nonwoven UltraCleanTM Cotton (highly cleaned and hydroentangled, greige cotton) retains the native wax and pectin content (~2%) of the cotton fiber traditionally removed from scoured and bleached cotton gauze, yet potentially affording wound healing properties. In vitro thromboelastography, hydrog...

A dietary supplement improves facial photoaging and skin sebum, hydration and tonicity modulating serum fibronectin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins.

PubMed

Di Cerbo, Alessandro; Laurino, Carmen; Palmieri, Beniamino; Iannitti, Tommaso

2015-03-01

Excessive exposure to the sun can cause severe photoaging as early as the second decade of life resulting in a loss of physiological elastic fiber functions. We designed a first study to assess differences in facial skin pH, sebum, elasticity, hydration and tonicity and serum levels of fibronectin, elastin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins between patients affected by facial photoaging and healthy controls. In a second study we tested the hypothesis that a dietary supplement would improve facial photoaging, also promoting changes in the above mentioned skin and serum parameters. In the first study we enrolled 30 women [age: 47.5 ± 1.6 years (mean ± standard error of the mean)] affected by moderate facial photoaging (4 cm ≤ Visual Analogue Scale (VAS)<7 cm) and 30 healthy women [age: 45.9 ± 1.6 years (mean ± standard error of the mean)]. In the second study we enrolled a cohort of 30 women [age: 43.6 ± 1.2 years (mean ± standard error of the mean)], affected by moderate (n = 22) and severe (VAS ≥ 7 cm; n = 8) facial photoaging, who were randomized to receive a pharmaceutical formulation (VISCODERM Pearls; IBSA FARMACEUTICI ITALIA Srl, Lodi, Italy) containing Pycnogenol, collagen, coenzyme Q10, low-molecular-weight hyaluronic acid, chondroitin sulfate and glucosamine sulfate (n = 15) or placebo (n = 15). Dietary supplement and placebo were administered 2 times a day for 4 weeks. Facial photoaging was assessed by VAS in the first cohort of patients affected by facial photoaging and healthy controls and, at baseline and 2 weeks after the end of treatment, in the second cohort of patients who underwent treatment with VISCODERM Pearls and placebo. Skin Tester was used to analyze differences in facial skin parameters between patients affected by facial photoaging and healthy controls. Skin Tester was also used to assess the effect of VISCODERM Pearls on facial skin parameters and compared with placebo 2 weeks after the end of treatment. Serum levels of fibronectin, elastin, neutrophil elastase 2, hyaluronic acid and carbonylated proteins were measured by enzyme-linked immunosorbent assay in the first cohort of patients affected by facial photoaging and healthy controls and, at baseline and 2 weeks after the end of treatment, in the second cohort of patients who underwent treatment with VISCODERM Pearls and placebo. VAS photoaging score was higher in patients affected by photoaging, if compared with healthy controls (p < 0.0001). pH and sebum were increased in patients affected by photoaging, if compared with healthy controls (both p < 0.0001), while elasticity, hydration and tonicity were decreased in patients affected by photoaging, if compared with healthy controls (all p < 0.0001). Serum fibronectin and hyaluronic acid concentrations were lower in patients affected by photoaging, if compared with healthy controls (both p < 0.0001). Serum neutrophil elastase 2, elastin and carbonylated protein concentrations were higher in patients affected by photoaging, if compared with healthy controls (p < 0.01, p < 0.01 and p < 0.0001, respectively). Dietary supplement administration resulted in an improvement in VAS photoaging score, if compared with placebo (p < 0.0001), as observed 2 weeks after the end of treatment. Facial sebum, hydration and tonicity were increased in the active treatment group vs. placebo (p < 0.0001, p < 0.0001 and p < 0.05, respectively) 2 weeks after the end of treatment. Serum fibronectin and hyaluronic acid concentrations were increased in the dietary supplement group, if compared with placebo (p < 0.01 and p < 0.001) 2 weeks after the end of treatment, while no statistical difference in serum elastin concentration was observed between the two groups. Serum neutrophil elastase 2 and carbonylated protein concentrations were decreased in the dietary supplement group 2 weeks after the end of treatment, if compared with placebo (p < 0.001 and p < 0.0001). We found significantly increased serum levels of neutrophil elastase 2, elastin and carbonylated proteins and decreased levels of hyaluronic acid and fibronectin in patients affected by facial photoaging, if compared with healthy controls. These findings coupled with a significant decrease in skin hydration, tonicity and elasticity and increased skin pH and sebum. Treatment with the dietary supplement VISCODERM Pearls significantly improved VAS photoaging score and skin hydration, sebum and tonicity 2 weeks after the end of a 4-week treatment period in patients affected by moderate to severe facial photoaging. These findings coupled with a significant increase in serum fibronectin and hyaluronic acid and a decrease in serum carbonylated proteins and neutrophil elastase 2 in the active treatment group, if compared with placebo. Our findings suggest that VISCODERM Pearls is effective for treatment of facial photoaging but further studies in larger cohorts of patients are required. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The alpha-tocopherol form of vitamin E boosts elastase activity of human PMNs and their ability to kill Streptococcus pneumoniae

USDA-ARS?s Scientific Manuscript database

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pul...

Biocompatibility differences with respect to the dialyzer sterilization method.

PubMed

Müller, T F; Seitz, M; Eckle, I; Lange, H; Kolb, G

1998-01-01

The impact of the method of sterilization (steam vs. ethylene oxide, ETO) on indices of biocompatibility is investigated using polysulfone membranes. Eight patients were treated with a random choice of the high-flux membranes F60S (steam) and F60 (ETO) and the low-flux membrane F6 (ETO). Blood samples were taken prior to and 5, 15, 30, 60, and 180 min after the start of hemodialysis. White blood cell count, platelet count, and plasma concentrations of polymorphonuclear neutrophil elastase, complements C3a and C5a, and beta2-microglobulin were determined. The dialysis procedure was associated with a significant decrease in white blood cell count and beta2-microglobulin level and a significant increase in polymorphonuclear neutrophil elastase and complement C3a and C5a levels. However, the steam-sterilized F60S membrane had a significantly lower impact on the biocompatibility indices than the ETO-sterilized F60 and F6 membranes (p < 0.05 or p < 0.001 for the individual markers). We conclude that using steam instead of ETO for sterilization may improve the biocompatibility of membranes.

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

PubMed Central

Pandeeti, Emmanuel Vijay Paul; Pitchika, Gopi Krishna; Jotshi, Jyotsna; Nilegaonkar, Smita S.; Kanekar, Pradnya P.; Siddavattam, Dayananda

2011-01-01

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme. PMID:21347249

Anti-Inflammatory and Neuroprotective Constituents from the Peels of Citrus grandis.

PubMed

Kuo, Ping-Chung; Liao, Yu-Ren; Hung, Hsin-Yi; Chuang, Chia-Wei; Hwang, Tsong-Long; Huang, Shiow-Chyn; Shiao, Young-Ji; Kuo, Daih-Huang; Wu, Tian-Shung

2017-06-09

A series of chromatographic separations performed on the ethanol extracts of the peels of Citrus grandis has led to the characterization of forty compounds, including seventeen coumarins, eight flavonoids, two triterpenoids, four benzenoids, two steroids, one lignan, one amide, and five other compounds, respectively. The chemical structures of the purified constituents were identified on the basis of spectroscopic elucidation, including 1D- and 2D-NMR, UV, IR, and mass spectrometric analysis. Most of the isolated compounds were examined for their inhibition of superoxide anion generation and elastase release by human neutrophils. Among the isolates, isomeranzin ( 3 ), 17,18-dihydroxybergamottin ( 12 ), epoxybergamottin ( 13 ), rhoifolin ( 19 ), vitexicarpin ( 22 ) and 4-hydroxybenzaldehyde ( 29 ) displayed the most significant inhibition of superoxide anion generation and elastase release with IC 50 values ranged from 0.54 to 7.57 μM, and 0.43 to 4.33 μM, respectively. In addition, 7-hydroxy-8-(2'-hydroxy-3'-methylbut-3'-enyl)coumarin ( 8 ) and 17,18-dihydroxybergamottin ( 12 ) also exhibited the protection of neurons against A-mediated neurotoxicity at 50 μM.

Transferrin-derived synthetic peptide induces highly conserved pro-inflammatory responses of macrophages.

PubMed

Haddad, George; Belosevic, Miodrag

2009-02-01

We examined the induction of macrophage pro-inflammatory responses by transferrin-derived synthetic peptide originally identified following digestion of transferrin from different species (murine, bovine, human N-lobe and goldfish) using elastase. The mass spectrometry analysis of elastase-digested murine transferrin identified a 31 amino acid peptide located in the N2 sub-domain of the transferrin N-lobe, that we named TMAP. TMAP was synthetically produced and shown to induce a number of pro-inflammatory genes by quantitative PCR. TMAP induced chemotaxis, a potent nitric oxide response, and TNF-alpha secretion in different macrophage populations; P338D1 macrophage-like cells, mouse peritoneal macrophages, mouse bone marrow-derived macrophages (BMDM) and goldfish macrophages. The treatment of BMDM cultures with TMAP stimulated the production of nine cytokines and chemokines (IL-6, MCP-5, MIP-1 alpha, MIP-1 gamma, MIP-2, GCSF, KC, VEGF, and RANTES) that was measured using cytokine antibody array and confirmed by Western blot. Our results indicate that transferrin-derived peptide, TMAP, is an immunomodulating molecule capable of inducing pro-inflammatory responses in lower and higher vertebrates.

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

PubMed

Sato, Nori; Taniguchi, Takako; Goda, Yuichiro; Kosaka, Hirofumi; Higashino, Kosaku; Sakai, Toshinori; Katoh, Shinsuke; Yasui, Natsuo; Sairyo, Koichi; Taniguchi, Hisaaki

2016-12-02

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B4, interleukin-8, elastase, and myeloperoxidase in human neutrophils.

PubMed

Kiss, Anna K; Filipek, Agnieszka; Czerwińska, Monika; Naruszewicz, Marek

2010-09-22

In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-D-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-D-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2- and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-D-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-D-glucose of 17±1, 15±1, 6.5±2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-D-glucose on reactive oxygen species and especially on O2- production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.

Elastase-2, a Tissue Alternative Pathway for Angiotensin II Generation, Plays a Role in Circulatory Sympathovagal Balance in Mice

PubMed Central

Becari, Christiane; Durand, Marina T.; Guimaraes, Alessander O.; Lataro, Renata M.; Prado, Cibele M.; de Oliveira, Mauro; Candido, Sarai C. O.; Pais, Paloma; Ribeiro, Mauricio S.; Bader, Michael; Pesquero, Joao B.; Salgado, Maria C. O.; Salgado, Helio C.

2017-01-01

In vitro and ex vivo experiments indicate that elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, is an alternative pathway for angiotensin II (Ang II) generation. However, the role played by ELA-2 in vivo is unclear. We examined ELA-2 knockout (ELA-2KO) mice compared to wild-type (WT) mice and determined whether ELA-2 played a role in hemodynamics [arterial pressure (AP) and heart rate (HR)], cardiocirculatory sympathovagal balance and baroreflex sensitivity. The variability of systolic arterial pressure (SAP) and pulse interval (PI) for evaluating autonomic modulation was examined for time and frequency domains (spectral analysis), whereas a symbolic analysis was also used to evaluate PI variability. In addition, baroreflex sensitivity was examined using the sequence method. Cardiac function was evaluated echocardiographically under anesthesia. The AP was normal whereas the HR was reduced in ELA-2KO mice (425 ± 17 vs. 512 ± 13 bpm from WT). SAP variability and baroreflex sensitivity were similar in both strains. The LF power from the PI spectrum (33.6 ± 5 vs. 51.8 ± 4.8 nu from WT) and the LF/HF ratio (0.60 ± 0.1 vs. 1.45 ± 0.3 from WT) were reduced, whereas the HF power was increased (66.4 ± 5 vs. 48.2 ± 4.8 nu from WT) in ELA-2KO mice, indicating a shift toward parasympathetic modulation of HR. Echocardiographic examination showed normal fractional shortening and an ejection fraction in ELA-2KO mice; however, the cardiac output, stroke volume, and ventricular size were reduced. These findings provide the first evidence that ELA-2 acts on the sympathovagal balance of the heart, as expressed by the reduced sympathetic modulation of HR in ELA-2KO mice. PMID:28386233

Synthesized zinc peroxide nanoparticles (ZnO2-NPs): a novel antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory approach toward polymicrobial burn wounds.

PubMed

Ali, Sameh Samir; Morsy, Reda; El-Zawawy, Nessma Ahmed; Fareed, Mervat F; Bedaiwy, Mohamed Yaser

2017-01-01

Increasing of multidrug resistance (MDR) remains an intractable challenge for burn patients. Innovative nanomaterials are also in high demand for the development of new antimicrobial biomaterials that inevitably have opened new therapeutic horizons in medical approaches and lead to many efforts for synthesizing new metal oxide nanoparticles (NPs) for better control of the MDR associated with the polymicrobial burn wounds. Recently, it seems that metal oxides can truly be considered as highly efficient inorganic agents with antimicrobial properties. In this study, zinc peroxide NPs (ZnO 2 -NPs) were synthesized using the co-precipitation method. Synthesized ZnO 2 -NPs were characterized by X-ray diffraction, Fourier transformed infrared, transmission electron microscopy, thermogravimetric analysis, differential scanning calorimetry, and ultraviolet-visible spectroscopy. The characterization techniques revealed synthesis of the pure phase of non-agglomerated ZnO 2 -NPs having sizes in the range of 15-25 nm with a transition temperature of 211°C. Antimicrobial activity of ZnO 2 -NPs was determined against MDR Pseudomonas aeruginosa (PA) and Aspergillus niger (AN) strains isolated from burn wound infections. Both strains, PA6 and AN4, were found to be more susceptible strains to ZnO 2 -NPs. In addition, a significant decrease in elastase and keratinase activities was recorded with increased concentrations of ZnO 2 -NPs until 200 µg/mL. ZnO 2 -NPs revealed a significant anti-inflammatory activity against PA6 and AN4 strains as demonstrated by membrane stabilization, albumin denaturation, and proteinase inhibition. Moreover, the results of in vivo histopathology assessment confirmed the potential role of ZnO 2 -NPs in the improvement of skin wound healing in the experimental animal models. Clearly, the synthesized ZnO 2 -NPs have demonstrated a competitive capability as antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory candidates, suggesting that the ZnO 2 -NPs are promising metal oxides that are potentially valued for biomedical applications.

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

PubMed Central

Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

2015-01-01

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

Elastase-2, a Tissue Alternative Pathway for Angiotensin II Generation, Plays a Role in Circulatory Sympathovagal Balance in Mice.

PubMed

Becari, Christiane; Durand, Marina T; Guimaraes, Alessander O; Lataro, Renata M; Prado, Cibele M; de Oliveira, Mauro; Candido, Sarai C O; Pais, Paloma; Ribeiro, Mauricio S; Bader, Michael; Pesquero, Joao B; Salgado, Maria C O; Salgado, Helio C

2017-01-01

In vitro and ex vivo experiments indicate that elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, is an alternative pathway for angiotensin II (Ang II) generation. However, the role played by ELA-2 in vivo is unclear. We examined ELA-2 knockout (ELA-2KO) mice compared to wild-type (WT) mice and determined whether ELA-2 played a role in hemodynamics [arterial pressure (AP) and heart rate (HR)], cardiocirculatory sympathovagal balance and baroreflex sensitivity. The variability of systolic arterial pressure (SAP) and pulse interval (PI) for evaluating autonomic modulation was examined for time and frequency domains (spectral analysis), whereas a symbolic analysis was also used to evaluate PI variability. In addition, baroreflex sensitivity was examined using the sequence method. Cardiac function was evaluated echocardiographically under anesthesia. The AP was normal whereas the HR was reduced in ELA-2KO mice (425 ± 17 vs. 512 ± 13 bpm from WT). SAP variability and baroreflex sensitivity were similar in both strains. The LF power from the PI spectrum (33.6 ± 5 vs. 51.8 ± 4.8 nu from WT) and the LF/HF ratio (0.60 ± 0.1 vs. 1.45 ± 0.3 from WT) were reduced, whereas the HF power was increased (66.4 ± 5 vs. 48.2 ± 4.8 nu from WT) in ELA-2KO mice, indicating a shift toward parasympathetic modulation of HR. Echocardiographic examination showed normal fractional shortening and an ejection fraction in ELA-2KO mice; however, the cardiac output, stroke volume, and ventricular size were reduced. These findings provide the first evidence that ELA-2 acts on the sympathovagal balance of the heart, as expressed by the reduced sympathetic modulation of HR in ELA-2KO mice.

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

PubMed Central

Malmsten, Martin; Kasetty, Gopinath; Pasupuleti, Mukesh; Alenfall, Jan; Schmidtchen, Artur

2011-01-01

Background Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. Methodology and Principal Findings Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various “superbugs” including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. Conclusions/Significance Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise “tuning” of toxicity and proteolytic stability may be achieved by changing tag-length and adding W- or F-amino acid tags. PMID:21298015

Risk factors for bronchiectasis in children with cystic fibrosis.

PubMed

Sly, Peter D; Gangell, Catherine L; Chen, Linping; Ware, Robert S; Ranganathan, Sarath; Mott, Lauren S; Murray, Conor P; Stick, Stephen M

2013-05-23

Bronchiectasis develops early in the course of cystic fibrosis, being detectable in infants as young as 10 weeks of age, and is persistent and progressive. We sought to determine risk factors for the onset of bronchiectasis, using data collected by the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) intensive surveillance program. We examined data from 127 consecutive infants who received a diagnosis of cystic fibrosis after newborn screening. Chest computed tomography (CT) and bronchoalveolar lavage (BAL) were performed, while the children were in stable clinical condition, at 3 months and 1, 2, and 3 years of age. Longitudinal data were used to determine risk factors associated with the detection of bronchiectasis from 3 months to 3 years of age. The point prevalence of bronchiectasis at each visit increased from 29.3% at 3 months of age to 61.5% at 3 years of age. In multivariate analyses, risk factors for bronchiectasis were presentation with meconium ileus (odds ratio, 3.17; 95% confidence interval [CI], 1.51 to 6.66; P=0.002), respiratory symptoms at the time of CT and BAL (odds ratio, 2.27; 95% CI, 1.24 to 4.14; P=0.008), free neutrophil elastase activity in BAL fluid (odds ratio, 3.02; 95% CI, 1.70 to 5.35; P<0.001), and gas trapping on expiratory CT (odds ratio, 2.05; 95% CI, 1.17 to 3.59; P=0.01). Free neutrophil elastase activity in BAL fluid at 3 months of age was associated with persistent bronchiectasis (present on two or more sequential scans), with the odds seven times as high at 12 months of age and four times as high at 3 years of age. Neutrophil elastase activity in BAL fluid in early life was associated with early bronchiectasis in children with cystic fibrosis. (Funded by the National Health and Medical Research Council of Australia and Cystic Fibrosis Foundation Therapeutics.)

Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

PubMed

Gnat, Sebastian; Łagowski, Dominik; Nowakiewicz, Aneta; Zięba, Przemysław

2018-05-20

The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on construction of the profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase, and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. Profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by T. mentagrophytes, T. verrucosum, or M. canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains infecting humans. Haemolysis seems to be dependent on host factors and is more common in the case of human dermatophyte isolates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Angiogenin: a novel inhibitor of neutrophil lactoferrin release during extracorporeal circulation.

PubMed

Schmaldienst, Sabine; Oberpichler, André; Tschesche, Harald; Hörl, Walter H

2003-01-01

Degranulation of polymorphonuclear leukocytes (PMNL) occurs during extracorporeal circulation. A degranulation-inhibiting protein identical to angiogenin was recently isolated from high-flux dialyzer ultrafiltrate. This protein inhibits the release of lactoferrin and metalloproteinases from PMNL in vitro. In the present study, we investigated end-stage renal disease patients undergoing regular hemodialysis treatment with either high-flux dialyzers (n = 51) or low-flux dialyzers (n = 44), and chronically uremic patients undergoing hemodiafiltration (n = 30). Hemodialysis therapy with low-flux polysulfone or cellulose triacetate membranes caused no or only minimal reduction (

Tea polyphenols as an antivirulence compound Disrupt Quorum-Sensing Regulated Pathogenicity of Pseudomonas aeruginosa

PubMed Central

Yin, Honging; Deng, Yifeng; Wang, Huafu; Liu, Wugao; Zhuang, Xiyi; Chu, Weihua

2015-01-01

Green tea, a water extract of non-fermented leaves of Camellia sinensis L., is one of the nonalcoholic beverages in China. It is becoming increasingly popular worldwide, because of its refreshing, mild stimulant and medicinal properties. Here we examined the quorum sensing inhibitory potentials of tea polyphenols (TP) as antivirulence compounds both in vitro and in vivo. Biosensor assay data suggested minimum inhibitory concentrations (MICs) of TP against selected pathogens were 6.25 ~ 12.5 mg/mL. At sub-MIC, TP can specifically inhibit the production of violacein in Chromobacterium violaceum 12472 with almost 98% reduction at 3.125 mg/mL without affecting its growth rate. Moreover, TP exhibited inhibitory effects on virulence phenotypes regulated by QS in Pseudomonas aeruginosa. The total proteolytic activity, elastase, swarming motility and biofilm formation were reduced in a concentration-dependent manner. In vivo, TP treatment resulted in the reduction of P. aeruginosa pathogenicity in Caenorhabditis elegans. When its concentration was 3.125 mg/mL, the survival rate reached 63.3%. In the excision wound infection model, the wound contraction percentage in treatment groups was relatively increased and the colony-forming units (CFU) in the wound area were significantly decreased. These results suggested that TP could be developed as a novel non-antibiotic QS inhibitor without killing the bacteria but as an antivirulence compound to control bacterial infection. PMID:26548447

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

Proteome Analysis of Human Sebaceous Follicle Infundibula Extracted from Healthy and Acne-Affected Skin

PubMed Central

Bek-Thomsen, Malene; Lomholt, Hans B.; Scavenius, Carsten; Enghild, Jan J.; Brüggemann, Holger

2014-01-01

Acne vulgaris is a very common disease of the pilosebaceous unit of the human skin. The pathological processes of acne are not fully understood. To gain further insight sebaceous follicular casts were extracted from 18 healthy and 20 acne-affected individuals by cyanoacrylate-gel biopsies and further processed for mass spectrometry analysis, aiming at a proteomic analysis of the sebaceous follicular casts. Human as well as bacterial proteins were identified. Human proteins enriched in acne and normal samples were detected, respectively. Normal follicular casts are enriched in proteins such as prohibitins and peroxiredoxins which are involved in the protection from various stresses, including reactive oxygen species. By contrast, follicular casts extracted from acne-affected skin contained proteins involved in inflammation, wound healing and tissue remodeling. Among the most distinguishing proteins were myeloperoxidase, lactotransferrin, neutrophil elastase inhibitor and surprisingly, vimentin. The most significant biological process among all acne-enriched proteins was ‘response to a bacterium’. Identified bacterial proteins were exclusively from Propionibacterium acnes. The most abundant P. acnes proteins were surface-exposed dermatan sulphate adhesins, CAMP factors, and a so far uncharacterized lipase in follicular casts extracted from normal as well as acne-affected skin. This is a first proteomic study that identified human proteins together with proteins of the skin microbiota in sebaceous follicular casts. PMID:25238151

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

New markers of urinary tract infection.

PubMed

Masajtis-Zagajewska, Anna; Nowicki, Michal

2017-08-01

Urinary tract infection (UTI) is the most common bacterial infection independent of age. It is also one of the most common causes of hospitalizations for infections among elderly people and the most common indication for antibiotic prescriptions in primary care. Both diagnostics and management of lower and upper urinary tract infections provide challenges in clinical practice due to their high prevalence and recurrence, and worldwide increase of antibiotic resistance. The clinical symptoms of UTI are often uncharacteristic or asymptomatic. The accurate diagnosis and early treatment are crucial due to risk of septicaemia and long-term consequences. Currently the diagnosis of urinary tract infection is based on the presence of clinical symptoms in combination with the results of nitrite strip test indicating the presence of bacteria in urine and semi-quantitative measurement of white blood cells count in urine. Although urine culture is the gold standard in UTI diagnostics it is both time-consuming and costly. Searching for novel biomarkers of UTI has attracted much attention in recent years. The article reviews several promising serum and urine biomarkers of UTI such as leukocyte esterase, C-reactive protein, procalcitonin, interleukins, elastase alpha (1)-proteinase inhibitor, lactofferin, secretory immunoglobulin A, heparin-binding protein, xanthine oxidase, myeloperoxidase, soluble triggering receptor expressed on myeloid cells-1, α-1 microglobulin (α1Mg) and tetrazolium nitroblue test (TNB). Copyright © 2017 Elsevier B.V. All rights reserved.

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis

PubMed Central

Qu, Yanyan; Olonisakin, Tolani; Bain, William; Zupetic, Jill; Brown, Rebecca; Hulver, Mei; Xiong, Zeyu; Shanks, Robert M.Q.; Bomberger, Jennifer M.; Cooper, Vaughn S.; Zegans, Michael E.; Han, Jongyoon; Pilewski, Joseph; Ray, Anuradha; Ray, Prabir; Lee, Janet S.

2018-01-01

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1–deficient (Thbs1–/–) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1–/– mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger. PMID:29415890

Reporters to monitor cellular MMP12 activity

NASA Astrophysics Data System (ADS)

Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

2010-02-01

Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (1). Single-dose intravenous toxicity studies in rats and dogs].

PubMed

Yanagi, H; Yamaguchi, K; Shimizu, K; Shichino, Y; Nishiyama, K; Mori, H; Shinomiya, K; Ueda, H; Suzuki, Y; Yonezawa, H; Fujita, T

1998-07-01

Single-dose toxicity studies of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, were conducted in Sprague-Dawley (SD) rats and beagle dogs. The rats of both sexes were administered ONO-5046.Na intravenously at a single dose of 150, 300 or 450 mg/kg. The male dogs were also given ONO-5046.Na at a single dose of 75 or 150 mg/kg. In the rat study, hypoactivity, bradypnea and paleness of limbs and pinna were observed at doses of 300 mg/kg and above. In particular, one of six female rats in the 450 mg/kg group showed clonic convulsion and died. In surviving animals, those signs disappeared within 3 hr after administration. No effect on body weight gain was seen in either group. Necropsy findings showed a slight foamy fluid in the bronchus, hemorrhage at the right knee joint muscle, tendon and lung in a dead animal. In the dog study, no effects on clinical signs, body weight, food consumption and blood biochemistry were seen in any animals of the 75 and 150 mg/kg groups. It is concluded that the approximate lethal doses are 450 mg/kg in rats and 150 mg/kg and above in dogs.

The Role of Neprilysin in Regulating the Hair Cycle

PubMed Central

Morisaki, Naoko; Ohuchi, Atsushi; Moriwaki, Shigeru

2013-01-01

In most mammals, each hair follicle undergoes a cyclic process of growing, regressing and resting phases (anagen, catagen, telogen, respectively) called the hair cycle. Various biological factors have been reported to regulate or to synchronize with the hair cycle. Some factors involved in the extracellular matrix, which is a major component of skin tissue, are also thought to regulate the hair cycle. We have focused on an enzyme that degrades elastin, which is associated with skin elasticity. Since our previous study identified skin fibroblast elastase as neprilysin (NEP), we examined the fluctuation of NEP enzyme activity and its expression during the synchronized hair cycle of rats. NEP activity in the skin was elevated at early anagen, and decreased during catagen to telogen. The expression of NEP mRNA and protein levels was modulated similarly. Immunostaining showed changes in NEP localization throughout the hair cycle, from the follicular epithelium during early anagen to the dermal papilla during catagen. To determine whether NEP plays an important role in regulating the hair cycle, we used a specific inhibitor of NEP (NPLT). NPLT was applied topically daily to the dorsal skin of C3H mice, which had been depilated in advance. Mice treated with NPLT had significantly suppressed hair growth. These data suggest that NEP plays an important role in regulating the hair cycle by its increased expression and activity in the follicular epithelium during early anagen. PMID:23418484

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.; Aggeler, J.

1978-04-01

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continuedmore » presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.« less

IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin.

PubMed

Takagi, Naoyuki; Kawakami, Kazuyoshi; Kanno, Emi; Tanno, Hiromasa; Takeda, Atsushi; Ishii, Keiko; Imai, Yoshimichi; Iwakura, Yoichiro; Tachi, Masahiro

2017-02-01

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil-derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin-17A (IL-17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL-17A production affected the wound healing in skin. IL-17A-knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild-type (WT) mice. In contrast, the administration of recombinant IL-17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL-17A-administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL-17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL-17A may be associated with impaired wound healing in skin. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nanocellulose-based biosensors: design, preparation, and activity of peptide-linked cotton cellulose nanocrystals having fluorimetric and colorimetric elastase detection sensitivity

USDA-ARS?s Scientific Manuscript database

Nanocrystalline cellulose is an amphiphilic, high surface area material that can be easily functionalized and is biocom-patible and eco-friendly. It has been used singularly and in combination with other nanomaterials to optimize biosensor design. The attachment of peptides and proteins to nanocryst...

Onion Peel Ethylacetate Fraction and Its Derived Constituent Quercetin 4'-O-β-D Glucopyranoside Attenuates Quorum Sensing Regulated Virulence and Biofilm Formation.

PubMed

Al-Yousef, Hanan M; Ahmed, Atallah F; Al-Shabib, Nasser A; Laeeq, Sameen; Khan, Rais A; Rehman, Md T; Alsalme, Ali; Al-Ajmi, Mohamed F; Khan, Mohammad S; Husain, Fohad M

2017-01-01

The resistance and pathogenesis of bacteria could be related to their ability to sense and respond to population density, termed quorum sensing (QS). Inhibition of the QS system is considered as a novel strategy for the development of antipathogenic agents, especially for combating drug-resistant bacterial infections. In the present study, the anti-QS activity of Onion peel ethylacetate fraction (ONE) was tested against Chromobacterium violaceum CV12472 and Pseudomonas aeruginosa PAO1. ONE inhibit the QS-mediated virulence factors production such as violacein in C. violaceum and elastase, pyocyanin in P. aeruginosa . Further, the treatment with sub-MICs of ONE significantly inhibited the QS-mediated biofilm formation, EPS (Extracellular polymeric substances) production and swarming motility. Further, quercetin 4'- O -β-D glucopyranoside (QGP) was isolated from ONE and its anti-QS potential was confirmed after observing significant inhibition of QS-controlled virulence factors such as violacein, elastase, pyocyanin and biofilm formation in test pathogens. Molecular docking analysis predicted that QGP should be able to bind at the active sites of Vfr and LasR, and if so blocks the entry of active sites in Vfr and LasR.

Degradation of tropoelastin and skin elastin by neprilysin.

PubMed

Mora Huertas, Angela C; Schmelzer, Christian E H; Luise, Chiara; Sippl, Wolfgang; Pietzsch, Markus; Hoehenwarter, Wolfgang; Heinz, Andrea

2018-03-01

Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1' upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

IL-17 Plays a Role in Respiratory Syncytial Virus-induced Lung Inflammation and Emphysema in Elastase and LPS-injured Mice.

PubMed

Mebratu, Yohannes A; Tesfaigzi, Yohannes

2018-06-01

Respiratory syncytial virus (RSV) is associated with enhanced progression of chronic obstructive pulmonary disease (COPD) and COPD exacerbations. However, little is known about the role of IL-17 in RSV-induced lung injury. We first investigated the role of RSV infection in enhancing mucous cell hyperplasia (MCH) and airspace enlargement in the lungs of mice injured with elastase and LPS (E/LPS). Mice injured with E/LPS had an enhanced and prolonged neutrophilic response to RSV that was associated with decreased levels of type I IFN and increased levels of IL-17, IL-23, CXCL-1, granulocyte colony stimulating factor (GCSF), CXCL-5, and matrix metalloproteinase (MMP)-9. In addition, extent of MCH and mean weighted alveolar space were increased significantly in the lungs of E/LPS-injured mice infected with RSV compared with E/LPS-only or RSV-only controls. Interestingly, immunodepletion of IL-17 before viral infection diminished the RSV-driven MCH and airspace enlargement in the E/LPS-injured animals, suggesting that IL-17 may be a therapeutic target for MCH and airspace enlargement when enhanced by RSV infection.

Neutrophil elastase in cyclic and severe congenital neutropenia

PubMed Central

Duan, Zhijun; Korkmaz, Brice; Lee, Hu-Hui; Mealiffe, Matthew E.; Salipante, Stephen J.

2007-01-01

Mutations in ELA2 encoding the neutrophil granule protease, neutrophil elastase (NE), are the major cause of the 2 main forms of hereditary neutropenia, cyclic neutropenia and severe congenital neutropenia (SCN). Genetic evaluation of other forms of neutropenia in humans and model organisms has helped to illuminate the role of NE. A canine form of cyclic neutropenia corresponds to human Hermansky-Pudlak syndrome type 2 (HPS2) and results from mutations in AP3B1 encoding a subunit of a complex involved in the subcellular trafficking of vesicular cargo proteins (among which NE appears to be one). Rare cases of SCN are attributable to mutations in the transcriptional repressor Gfi1 (among whose regulatory targets also include ELA2). The ultimate biochemical consequences of the mutations are not yet known, however. Gene targeting of ELA2 has thus far failed to recapitulate neutropenia in mice. The cycling phenomenon and origins of leukemic transformation in SCN remain puzzling. Nevertheless, mutations in all 3 genes are capable of causing the mislocalization of NE and may also induce the unfolded protein response, suggesting that there might a convergent pathogenic mechanism focusing on NE. PMID:17053055

Protective effect of Cornus walteri Wangerin leaf against UVB irradiation induced photoaging in human reconstituted skin.

PubMed

Park, Hyun-Chul; Jung, Taek Kyu; Kim, Mi Jin; Yoon, Kyung-Sup

2016-12-04

Cornus walteri Wangerin has been used in oriental traditional medicine for the treatment of antidiarrheal and inflammation. The efficacy of Cornus walteri Wangerin on skin anti-photoaging was investigated. Hydrolyzed Cornus walteri Wangerin leaf was tested for the anti-photoaging effects against ultraviolet B (UVB)-induced matrix metalloproteinase (MMP)-1, pro-inflammatory cytokines using human reconstituted skin (KeraSkin™-FT) and also tested for elastase activity in vitro. The MMP-1 and pro-inflammatory cytokine levels of the extract were evaluated by enzyme-linked immunosorbent assay (ELISA). The extract of hydrolyzed Cornus walteri Wangerin leaf (CWE) had the elastase inhibitory activity (IC 50 : 0.457mg/mL). CWE inhibited MMP-1 expression up to 61% in comparison with the control group which was not treated using CWE, but exposed to UVB. CWE also showed an inhibitory effect on releasing pro-inflammatory cytokines (IL-6 and IL-8) in KeraSkin™-FT (30% and 57% inhibition at dose of 50μg/mL, respectively). CWE is a promising anti-photoaging agent for the treatment of UVB-induced skin. Copyright © 2016. Published by Elsevier Ireland Ltd.

Nanoparticles Effectively Target Rapamycin Delivery to Sites of Experimental Aortic Aneurysm in Rats.

PubMed

Shirasu, Takuro; Koyama, Hiroyuki; Miura, Yutaka; Hoshina, Katsuyuki; Kataoka, Kazunori; Watanabe, Toshiaki

2016-01-01

Several drugs targeting the pathogenesis of aortic aneurysm have shown efficacy in model systems but not in clinical trials, potentially owing to the lack of targeted drug delivery. Here, we designed a novel drug delivery system using nanoparticles to target the disrupted aortic aneurysm micro-structure. We generated poly(ethylene glycol)-shelled nanoparticles incorporating rapamycin that exhibited uniform diameter and long-term stability. When injected intravenously into a rat model in which abdominal aortic aneurysm (AAA) had been induced by infusing elastase, labeled rapamycin nanoparticles specifically accumulated in the AAA. Microscopic analysis revealed that rapamycin nanoparticles were mainly distributed in the media and adventitia where the wall structures were damaged. Co-localization of rapamycin nanoparticles with macrophages was also noted. Rapamycin nanoparticles injected during the process of AAA formation evinced significant suppression of AAA formation and mural inflammation at 7 days after elastase infusion, as compared with rapamycin treatment alone. Correspondingly, the activities of matrix metalloproteinases and the expression of inflammatory cytokines were significantly suppressed by rapamycin nanoparticle treatment. Our findings suggest that the nanoparticle-based delivery system achieves specific delivery of rapamycin to the rat AAA and might contribute to establishing a drug therapy approach targeting aortic aneurysm.

Nanoparticles Effectively Target Rapamycin Delivery to Sites of Experimental Aortic Aneurysm in Rats

PubMed Central

Shirasu, Takuro; Koyama, Hiroyuki; Miura, Yutaka; Hoshina, Katsuyuki; Kataoka, Kazunori; Watanabe, Toshiaki

2016-01-01

Several drugs targeting the pathogenesis of aortic aneurysm have shown efficacy in model systems but not in clinical trials, potentially owing to the lack of targeted drug delivery. Here, we designed a novel drug delivery system using nanoparticles to target the disrupted aortic aneurysm micro-structure. We generated poly(ethylene glycol)-shelled nanoparticles incorporating rapamycin that exhibited uniform diameter and long-term stability. When injected intravenously into a rat model in which abdominal aortic aneurysm (AAA) had been induced by infusing elastase, labeled rapamycin nanoparticles specifically accumulated in the AAA. Microscopic analysis revealed that rapamycin nanoparticles were mainly distributed in the media and adventitia where the wall structures were damaged. Co-localization of rapamycin nanoparticles with macrophages was also noted. Rapamycin nanoparticles injected during the process of AAA formation evinced significant suppression of AAA formation and mural inflammation at 7 days after elastase infusion, as compared with rapamycin treatment alone. Correspondingly, the activities of matrix metalloproteinases and the expression of inflammatory cytokines were significantly suppressed by rapamycin nanoparticle treatment. Our findings suggest that the nanoparticle-based delivery system achieves specific delivery of rapamycin to the rat AAA and might contribute to establishing a drug therapy approach targeting aortic aneurysm. PMID:27336852

Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa.

PubMed

Xu, G; Xiong, W; Hu, Q; Zuo, P; Shao, B; Lan, F; Lu, X; Xu, Y; Xiong, S

2010-10-01

To investigate the bactericidal activity of lactoferrin-derived peptides and a new LF-derived peptides chimera (LFchimera) against P. aeruginosa and the influence on virulence factors of P. aeruginosa. Lactoferricin (LFcin) and lactoferrampin (LFampin) are highly bioactive peptides isolated from the N-terminal region of lactoferrin (LF) by pepsin digestion. In this study, we designed LFchimera containing LFcin amino acids 17-30 and LFampin amino acids 268-284. Pseudomonas aeruginosa cells were incubated in medium with peptides at different concentrations, and then the assays of viability, pyocyanin, elastase activity and biofilm formation of P. aeruginosa were performed. We found that the concentration-dependent antibactericidal activity and down-regulating pyocyanin, elastase and biofilm formation of LFchimera were significantly stronger than those of LF, LFcin, LFampin or LFcin plus LFampin. Our results indicated that LF, LFcin, LFampin and LFchimera were potential candidates to combat P. aeruginosa, and LFchimera was the most effective in them. The new LFchimera has better activity against P. aeruginosa than LF, LFcin and LFampin and may be a promising new compound for treatment of P. aeruginosa infection. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2015-05-01

HCLS1 associated protein X-1 (HAX1), cause hereditary forms of neutropenia . Previously, competing hypotheses have posited that mutant forms of...derived induced pluripotent stem cell (iPSC) model of ELANE-associated neutropenia . During the second year of this project, in order to facilitate...pathology. 3 2. KEY WORDS neutropenia bone marrow failure neutrophil elastase ELANE HAX1 alternate translation induced pluripotent stem cells (iPSC

Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count.

PubMed

Albenzio, Marzia; Santillo, Antonella; Caroprese, Mariangela; Schena, Laura; Russo, Donatella Esterina; Sevi, Agostino

2011-11-01

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

PubMed

Bou Ghanem, Elsa N; Lee, James N; Joma, Basma H; Meydani, Simin N; Leong, John M; Panda, Alexander

2017-01-01

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae

PubMed Central

Bou Ghanem, Elsa N.; Lee, James N.; Joma, Basma H.; Meydani, Simin N.; Leong, John M.; Panda, Alexander

2017-01-01

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22–35 years) or elderly (65–69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection. PMID:28516066

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

PubMed

Silva, Lívia V; Galdino, Anna Clara M; Nunes, Ana Paula F; dos Santos, Kátia R N; Moreira, Beatriz M; Cacci, Luciana C; Sodré, Cátia L; Ziccardi, Mariangela; Branquinha, Marta H; Santos, André L S

2014-11-01

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-β-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 μg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa. Copyright © 2014 Elsevier GmbH. All rights reserved.

Pelvic Organ Support in Animals with Partial Loss of Fibulin-5 in the Vaginal Wall

PubMed Central

Shi, Haolin; Balgobin, Sunil; Montoya, T. Ignacio; Yanagisawa, Hiromi; Word, R. Ann

2016-01-01

Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5f/f/SMA++-rtTA/Cre+, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5f/f/SMA++-rtTA/Cre-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6–8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse. PMID:27124299

Synthesized zinc peroxide nanoparticles (ZnO2-NPs): a novel antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory approach toward polymicrobial burn wounds

PubMed Central

El-Zawawy, Nessma Ahmed; Fareed, Mervat F; Bedaiwy, Mohamed Yaser

2017-01-01

Increasing of multidrug resistance (MDR) remains an intractable challenge for burn patients. Innovative nanomaterials are also in high demand for the development of new antimicrobial biomaterials that inevitably have opened new therapeutic horizons in medical approaches and lead to many efforts for synthesizing new metal oxide nanoparticles (NPs) for better control of the MDR associated with the polymicrobial burn wounds. Recently, it seems that metal oxides can truly be considered as highly efficient inorganic agents with antimicrobial properties. In this study, zinc peroxide NPs (ZnO2-NPs) were synthesized using the co-precipitation method. Synthesized ZnO2-NPs were characterized by X-ray diffraction, Fourier transformed infrared, transmission electron microscopy, thermogravimetric analysis, differential scanning calorimetry, and ultraviolet-visible spectroscopy. The characterization techniques revealed synthesis of the pure phase of non-agglomerated ZnO2-NPs having sizes in the range of 15–25 nm with a transition temperature of 211°C. Antimicrobial activity of ZnO2-NPs was determined against MDR Pseudomonas aeruginosa (PA) and Aspergillus niger (AN) strains isolated from burn wound infections. Both strains, PA6 and AN4, were found to be more susceptible strains to ZnO2-NPs. In addition, a significant decrease in elastase and keratinase activities was recorded with increased concentrations of ZnO2-NPs until 200 µg/mL. ZnO2-NPs revealed a significant anti-inflammatory activity against PA6 and AN4 strains as demonstrated by membrane stabilization, albumin denaturation, and proteinase inhibition. Moreover, the results of in vivo histopathology assessment confirmed the potential role of ZnO2-NPs in the improvement of skin wound healing in the experimental animal models. Clearly, the synthesized ZnO2-NPs have demonstrated a competitive capability as antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory candidates, suggesting that the ZnO2-NPs are promising metal oxides that are potentially valued for biomedical applications. PMID:28860766

Pelvic Organ Support in Animals with Partial Loss of Fibulin-5 in the Vaginal Wall.

PubMed

Chin, Kathleen; Wieslander, Cecilia; Shi, Haolin; Balgobin, Sunil; Montoya, T Ignacio; Yanagisawa, Hiromi; Word, R Ann

2016-01-01

Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5f/f/SMA++-rtTA/Cre+, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5f/f/SMA++-rtTA/Cre-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6-8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse.

Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences

PubMed Central

Gardill, Bernd R.; Vogl, Michael R.; Lin, Hai-Yan; Hammond, Geoffrey L.; Muller, Yves A.

2012-01-01

Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of β-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species. PMID:23300763

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

A diketopiperazine factor from Rheinheimera aquimaris QSI02 exhibits anti-quorum sensing activity.

PubMed

Sun, Shiwei; Dai, Xiaoyun; Sun, Jiao; Bu, Xiangguo; Weng, Caihong; Li, Hui; Zhu, Hu

2016-12-21

An ethyl acetate (EtOAc) extract isolated from the marine bacterium, Rheinheimera aquimaris QSI02, was found to exhibit anti-quorum sensing (anti-QS) activity. A subsequent bioassay-guided isolation protocol led to the detection of an active diketopiperazine factor, cyclo(Trp-Ser). Biosensor assay data showed that the minimum inhibitory concentration (MIC) of cyclo(Trp-Ser) ranged from 3.2 mg/ml to 6.4 mg/m for several microorganisms, including Escherichia coli, Chromobacterium violaceum CV026, Pseudomonas aeruginosa PA01, Staphylococcus aureus, and Candida albicans. Additionally, sub-MICs of cyclo(Trp-Ser) decreased the QS-regulated violacein production in C. violaceum CV026 by 67%. Furthermore, cyclo(Trp-Ser) can decrease QS-regulated pyocyanin production, elastase activity and biofilm formation in P. aeruginosa PA01 by 65%, 40% and 59.9%, respectively. Molecular docking results revealed that cyclo(Trp-Ser) binds to CviR receptor more rigidly than C 6 HSL with lower docking energy -8.68 kcal/mol, while with higher binding energy of -8.40 kcal/mol than 3-oxo-C 12 HSL in LasR receptor. Molecular dynamics simulation suggested that cyclo(Trp-Ser) is more easy to bind to CviR receptor than natural signaling molecule, but opposite in LasR receptor. These results suggest that cyclo(Trp-Ser) can be used as a potential inhibitor to control QS systems of C. violaceum and P. aeruginosa and provide increased the understanding of molecular mechanism that influences QS-regulated behaviors.

[Alpha-1 antitrypsin deficiency. The experience of Pulido Valente Hospital with augmentation therapy].

PubMed

Alves Costa, Carla; Santos, Cristina

2009-01-01

Alpha-1 antitrypsin (AAT) is synthesised in the liver and has half-life of 4-5 days. AAT has antiprotease activity, with particular affinity for neutrophil elastase. Its deficiency leads to a lack of effective lung protection against activated neutrophil enzymes. Deficiency of AAT is a genetic disorder that occurs as a result of the inheritance of two protease inhibitor deficient alleles. Of the deficient alleles, Pi*Z is the most common, and the homozygous form Pi*ZZ results in the lowest serum levels, usually below 50 mg/ dl. The "protective threshold" is 80 mg/dl. Smoking increases the risk of emphysema. The current goal of augmentation therapy is to raise the plasma levels, above protective threshold and slow disease progression. The authors present the experience of the Day Care Hospital of the Pulido Valente Hospital with five male patients presenting emphysema due to AAT deficiency, receiving weekly intravenous treatment with Prolastin. We performed a clinical, respiratory functional and radiological evaluation between 2003 and 2007. The results point to a slower progression of the disease, with clinical and radiological stability and a reduced rate of FEV1 decline. Augmentation therapy is an expensive treatment and its use is lacking supportive evidence of efficacy by randomized controlled clinical trials. Evidence that it confers benefits is based on observational studies. Our experience is positive, showing clinical, radiological and functional benefits. The literature available points to a decrease in mortality, but we could not affirm so in our small population.

Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells.

PubMed

Kim, You-Sun; Kokturk, Nurdan; Kim, Ji-Young; Lee, Sei Won; Lim, Jaeyun; Choi, Soo Jin; Oh, Wonil; Oh, Yeon-Mok

2016-10-01

Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

PubMed Central

Stankovic, Marija; Divac-Rankov, Aleksandra; Petrovic-Stanojevic, Natasa; Mitic-Milikic, Marija; Nagorni-Obradovic, Ljudmila; Radojkovic, Dragica

2012-01-01

Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n=348), asthma (n=71), and bronchiectasis (n=35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1:5519, 1:38, and 1:5519). Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population. PMID:22971141

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Clinical utility of alpha-1 proteinase inhibitor in the management of adult patients with severe alpha-1 antitrypsin deficiency: a review of the current literature

PubMed Central

Parr, David G; Lara, Beatriz

2017-01-01

Alpha-1 antitrypsin (AAT) functions primarily to inhibit neutrophil elastase, and its deficiency predisposes individuals to the development of chronic obstructive pulmonary disease (COPD). The putative protective serum concentration is generally considered to be above a threshold of 11 μM/L, and therapeutic augmentation of AAT above this value is believed to retard the progression of emphysema. Several AAT preparations, all derived from human donor plasma, have been commercialized since approval by the US Food and Drug Administration (FDA) in 1987. Biochemical efficacy has been demonstrated by augmentation of pulmonary antiprotease activity, but demonstration of clinical efficacy in randomized, placebo-controlled trials has been hampered by the practical difficulties of performing conventional studies in a rare disease with a relatively long natural history. Computed tomography has been applied to measure lung density as a more specific and sensitive surrogate outcome measure of emphysema than physiologic indices, such as forced expiratory volume in 1 second, and studies consistently show a therapeutic reduction in the rate of lung density decline. However, convincing evidence of benefit using traditional clinical measures remains elusive. Intravenous administration of AAT at a dose of 60 mg/kg/week is the commonest regime in use and has well-documented safety and tolerability. International and national guidelines on the management of AAT deficiency recommend intravenous augmentation therapy to supplement optimized usual COPD treatment in patients with severe deficiency and evidence of lung function impairment. PMID:28769553

Detection of anti-neutrophil cytoplasmic antibodies after acute Plasmodium falciparum malaria.

PubMed Central

Wenisch, C; Wenisch, H; Bankl, H C; Exner, M; Graninger, W; Looareesuwan, S; Rumpold, H

1996-01-01

Four of 30 patients with Plasmodium falciparum infection in Bangkok, Thailand, were positive for anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence 1 month after antimalarial therapy. No myeloperoxidase, proteinase 3, lactoferrin, or elastase reactivity was found. Since no evidence of vasculitis was seen in these patients, anti-neutrophil cytoplasmic antibody production in malaria-infected susceptible patients probably represents a secondary response, indicating neutrophil activation. PMID:8770517

IMPACT OF PSEUDOMONAS AND STAPHYLOCOCCAL INFECTION ON INFLAMMATION AND CLINICAL STATUS IN YOUNG CHILDREN WITH CYSTIC FIBROSIS

PubMed Central

Sagel, Scott D.; Gibson, Ronald L.; Emerson, Julia; McNamara, Sharon; Burns, Jane L.; Wagener, Jeffrey S.; Ramsey, Bonnie W.

2009-01-01

Objectives To assess the effects of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) infection on lower airway inflammation and clinical status in young children with cystic fibrosis (CF). Study design We studied 111 children < 6 years of age who had two Pa positive oropharyngeal cultures within 12 months. We examined bronchoalveolar lavage fluid (BALF) inflammatory markers (cell count, differential, IL-8, IL-6, neutrophil elastase), CF-related bacterial pathogens, exotoxin A serology, and clinical indicators of disease severity. Results Young children with CF with both upper and lower airway Pa infection had higher neutrophil counts, IL-8 and free neutrophil elastase levels, increased likelihood of positive exotoxin A titers, and lower Shwachman scores compared with those with positive upper airway cultures only. Sa was associated with increased lower airway inflammation and the presence of both Pa and Sa had an additive effect on concentrations of lower airway inflammatory markers. BALF markers of inflammation increased with numbers of different bacterial pathogens detected. Conclusions Young children with CF with upper and lower airway Pa infection have heightened endobronchial inflammation and poorer clinical status compared with children with only upper airway Pa infection. The independent and additive effects of Sa on inflammation support the importance of polymicrobial infection in early CF lung disease. PMID:18822427

Pancreatic fibrosis correlates with exocrine pancreatic insufficiency after pancreatoduodenectomy.

PubMed

Tran, T C K; van 't Hof, G; Kazemier, G; Hop, W C; Pek, C; van Toorenenbergen, A W; van Dekken, H; van Eijck, C H J

2008-01-01

Obstruction of the pancreatic duct can lead to pancreatic fibrosis. We investigated the correlation between the extent of pancreatic fibrosis and the postoperative exocrine and endocrine pancreatic function. Fifty-five patients who were treated for pancreatic and periampullary carcinoma and 19 patients with chronic pancreatitis were evaluated. Exocrine pancreatic function was evaluated by fecal elastase-1 test, while endocrine pancreatic function was assessed by plasma glucose level. The extent of fibrosis, duct dilation and endocrine tissue loss was examined histopathologically. A strong correlation was found between pancreatic fibrosis and elastase-1 level less than 100 microg/g (p < 0.0001), reflecting severe exocrine pancreatic insufficiency. A strong correlation was found between pancreatic fibrosis and endocrine tissue loss (p < 0.0001). Neither pancreatic fibrosis nor endocrine tissue loss were correlated with the development of postoperative diabetes mellitus. Duct dilation alone was neither correlated with exocrine nor with endocrine function loss. The majority of patients develop severe exocrine pancreatic insufficiency after pancreatoduodenectomy. The extent of exocrine pancreatic insufficiency is strongly correlated with preoperative fibrosis. The loss of endocrine tissue does not correlate with postoperative diabetes mellitus. Preoperative dilation of the pancreatic duct per se does not predict exocrine or endocrine pancreatic insufficiency postoperatively. Copyright 2008 S. Karger AG, Basel.

Multiscale mechanical integrity of human supraspinatus tendon in shear after elastin depletion.

PubMed

Fang, Fei; Lake, Spencer P

2016-10-01

Human supraspinatus tendon (SST) exhibits region-specific nonlinear mechanical properties under tension, which have been attributed to its complex multiaxial physiological loading environment. However, the mechanical response and underlying multiscale mechanism regulating SST behavior under other loading scenarios are poorly understood. Furthermore, little is known about the contribution of elastin to tendon mechanics. We hypothesized that (1) SST exhibits region-specific shear mechanical properties, (2) fiber sliding is the predominant mode of local matrix deformation in SST in shear, and (3) elastin helps maintain SST mechanical integrity by facilitating force transfer among collagen fibers. Through the use of biomechanical testing and multiphoton microscopy, we measured the multiscale mechanical behavior of human SST in shear before and after elastase treatment. Three distinct SST regions showed similar stresses and microscale deformation. Collagen fiber reorganization and sliding were physical mechanisms observed as the SST response to shear loading. Measures of microscale deformation were highly variable, likely due to a high degree of extracellular matrix heterogeneity. After elastase treatment, tendon exhibited significantly decreased stresses under shear loading, particularly at low strains. These results show that elastin contributes to tendon mechanics in shear, further complementing our understanding of multiscale tendon structure-function relationships. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

PubMed

Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

2009-07-20

Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms

PubMed Central

Tidwell, Timothy; Wechsler, Jeremy; Nayak, Ramesh C.; Trump, Lisa; Salipante, Stephen J.; Cheng, Jerry C.; Donadieu, Jean; Glaubach, Taly; Corey, Seth J.; Grimes, H. Leighton; Lutzko, Carolyn; Cancelas, Jose A.

2014-01-01

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein. PMID:24184683

A prospective randomized study of systemic inflammation and immune response after laparoscopic nissen fundoplication performed with standard and low-pressure pneumoperitoneum.

PubMed

Schietroma, Mario; Carlei, Francesco; Cecilia, Emanuela M; Piccione, Federica; Sista, Federico; De Vita, Fabiola; Amicucci, Gianfranco

2013-04-01

The aim of this study was to compare changes in the systemic inflammation and immune response in the early postoperative (p.o.) period after laparoscopic Nissen fundoplication (LNF) was performed with standard-pressure and low-pressure carbon dioxide pneumoperitoneum. We studied 68 patients with documented gastroesophageal reflux disease and who underwent a LNF: 35 using standard-pressure (12 to 14 mmHg) and 33 low-pressure (6 to 8 mmHg) pneumoperitoneum. White blood cells, peripheral lymphocytes subpopulation, human leukocyte antigen-DR, neutrophil elastase, interleukin (IL)-6 and IL-1, and C-reactive protein were investigated. A significantly higher concentration of neutrophil elastase, IL-6 and IL-1, and C-reactive protein was detected postoperatively in the standard-pressure group of patients in comparison with the low-pressure group (P<0.05). A statistically significant change in human leukocyte antigen-DR expression was recorded p.o. at 24 hours, as a reduction of this antigen expressed on monocyte surface in patients from standard group; no changes were noted in low-pressure group patients (P<0.05). This study demonstrated that reducing the pressure of the pneumoperitoneum to 6 to 8 mm Hg during LNF is reduced p.o. inflammatory response and avoided p.o. immunosuppression.

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph.

PubMed

Yang, Fan; Wang, Yang; Sumathipala, Niranji; Cao, Xiaolong; Kanost, Michael R; Jiang, Haobo

2018-08-01

Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu 429 ) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu 429 . Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction. Copyright © 2018 Elsevier Ltd. All rights reserved.

A new hydroxychavicol dimer from the roots of Piper betle.

PubMed

Lin, Chwan-Fwu; Hwang, Tsong-Long; Chien, Chun-Chien; Tu, Huei-Yu; Lay, Horng-Liang

2013-02-26

A new hydroxychavicol dimer, 2-(g'-hydroxychavicol)-hydroxychavicol (1), was isolated from the roots of Piper betle Linn. along with five known compounds, hydroxychavicol (2), aristololactam A II (3), aristololactam B II (4), piperolactam A (5) and cepharadione A (6). The structures of these isolated compounds were elucidated by spectroscopic methods. Compounds 1 and 2 exhibited inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils.

Gateways to clinical trials.

PubMed

Tomillero, A; Moral, M A

2009-03-01

ABT-869, Acadesine, Acetylsalicylic acid/omeprazole, Adefovir, Adefovir dipivoxil, AEG-35156, Agatolimod sodium, Albiglutide, Alemtuzumab, Alipogene tiparvovec, Alogliptin benzoate, AMG-386, Amrubicin hydrochloride, Apremilast, Aripiprazole, Asoprisnil, Atorvastatin/fenofibrate, AVN-944, Axitinib; Belinostat, Bevacizumab, BHT-3021, BI-2536, Biapenem, Bilastine, Biphasic insulin aspart, Blinatumomab, Bortezomib, Bosentan; Catumaxomab, CD-NP, Cediranib, Certolizumab pegol, Cetuximab, Choline fenofibrate, Ciclesonide, CK-1827452,Clevudine, Clofarabine, CSL-360, CYT-997; Dapagliflozin, Darinaparsin, Denosumab, Densiron 68, Desloratadine, Dulanermin; Edoxaban tosilate, Emtricitabine, Entecavir, Erlotinib hydrochloride, Everolimus, Exenatide, Ezetimibe, Ezetimibe/simvastatin; Fidaxomicintiacumiv, Fulvestrant; G-207, GCR-8015, Gefitinib, Ghrelin (human), Glufosfamide; HPV16L1E7CVLP; Ibutamoren mesilate, Imatinib mesylate, Insulin detemir, Insulin glargine, Iodine (I131) tositumomab, Istaroxime, ITMN-191, Ixabepilone; JZP-4, Lenalidomide; Levetiracetam, Linaclotide acetate, Liposomal cytarabine/daunorubicin, Liposomal doxorubicin, Liraglutide, LY-518674; Milatuzumab, MMR-V, Motesanib diphosphate, Mycophenolic acid sodium salt; Niacin/simvastatin; Obatoclax mesylate, Odanacatib; Paclitaxel nanoparticles, Paclitaxel-eluting stent, Pazufloxacin, PBT-2, Pegfilgrastim, Peginterferon alfa-2a, Peginterferon alfa-2b, Peginterferon alfa-2b/ribavirin, Pemetrexed disodium, Perampanel, PfCP2.9, Pitavastatin calcium, Poly I:CLC, Pomalidomide, Pralatrexate, Pramlintide acetate, Prucalopride; rhGAD65, Roflumilast; RTS,S/AS02D; SCH-530348, Semagacestat, Sirolimus-eluting coronary stent, Sirolimus-Eluting Stent, SIR-Spheres, Sivelestat sodium hydrate, Sorafenib, Sunitinib malate; Tadalafil, Tafluprost, Tanespimycin, Teduglutide, Telaprevir, Telbivudine, Tenofovir disoproxil fumarate, Tiotropium bromide, TMC-435350, Tositumomab/iodine (I131) tositumomab, Travoprost/timolol, Triciribine phosphate; Vandetanib, VIA-2291, Vinflunine, Vorinostat; XL-019; Yttrium 90 (90Y) ibritumomab tiuxetan. Copyright 2009 Prous Science, S.A.U. or its licensors. All rights reserved.

Increased β-glucuronidase activity in bronchoalveolar lavage fluid of children with bacterial lung infection: A case-control study.

PubMed

Panagiotopoulou, Evgenia C; Fouzas, Sotirios; Douros, Konstantinos; Triantaphyllidou, Irene-Eva; Malavaki, Christina; Priftis, Kostas N; Karamanos, Nikos K; Anthracopoulos, Michael B

2015-11-01

β-Glucuronidase is a lysosomal enzyme released into the extracellular fluid during inflammation. Increased β-glucuronidase activity in the cerebrospinal and peritoneal fluid has been shown to be a useful marker of bacterial inflammation. We explored the role of β-glucuronidase in the detection of bacterial infection in bronchoalveolar lavage fluid (BALF) of paediatric patients. In this case-control study, % polymorphonuclear cell count (PMN%), β-glucuronidase activity, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and elastase were measured in culture-positive (≥10(4) cfu/mL, C+) and -negative (C-) BALF samples obtained from children. A total of 92 BALF samples were analysed. The median β-glucuronidase activity (measured in nanomoles of 4-methylumbelliferone (4-MU)/mL BALF/h) was 246.4 in C+ (interquartile range: 71.2-751) and 21.9 in C- (4.0-40.8) (P < 0.001). The levels of TNF-α and IL-8 were increased in C+ as compared with C- (5.4 (1.7-12.6) vs 0.7 (0.2-6.2) pg/mL, P < 0.001 and 288 (76-4300) vs 287 (89-1566) pg/mL, P = 0.042, respectively). Elastase level and PMN% did not differ significantly (50 (21-149) vs 26 (15-59) ng/mL, P = 0.051 and 20 (9-40) vs 18 (9-34) %, P = 0.674, respectively). The area under the curve of β-glucuronidase activity (0.856, 95% confidence interval (CI): 0.767-0.920) was higher than that of TNF-α (0.718; 95% CI: 0.614-0.806; P = 0.040), IL-8 (0.623; 95% CI: 0.516-0.722; P = 0.001), elastase (0.645; 95% CI: 0.514-0.761; P = 0.008) and PMN% (0.526; 95 % CI: 0.418-0.632; P < 0.001). This study demonstrates a significant increase of β-glucuronidase activity in BALF of children with culture-positive bacterial inflammation. In our population β-glucuronidase activity showed superior predictive ability for bacterial lung infection than other markers of inflammation. © 2015 Asian Pacific Society of Respirology.

Contact system activation and high thrombin generation in hyperthyroidism.

PubMed

Kim, Namhee; Gu, Ja-Yoon; Yoo, Hyun Ju; Han, Se Eun; Kim, Young Il; Nam-Goong, Il Sung; Kim, Eun Sook; Kim, Hyun Kyung

2017-05-01

Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized that the contact system is activated along with active NET formation in hyperthyroidism and that their markers correlate with disease severity. In 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors (fibrinogen, and factor VII, VIII, IX, XI and XII), D-dimer, thrombin generation assay (TGA) markers, NET formation markers (histone-DNA complex, double-stranded DNA and neutrophil elastase) and contact system markers (activated factor XII (XIIa), high-molecular-weight kininogen (HMWK), prekallikrein and bradykinin) were measured. Patients with hyperthyroidism showed higher levels of fibrinogen (median (interquartile range), 315 (280-344) vs 262 (223-300), P  = 0.001), D-dimer (103.8 (64.8-151.5) vs 50.7 (37.4-76.0), P  < 0.001), peak thrombin (131.9 (102.2-159.4) vs 31.6 (14.8-83.7), P  < 0.001) and endogenous thrombin potential (649 (538-736) vs 367 (197-1147), P  = 0.021) in TGA with 1 pM tissue factor, neutrophil elastase (1.10 (0.39-2.18) vs 0.23 (0.20-0.35), P  < 0.001), factor XIIa (66.9 (52.8-87.0) vs 73.0 (57.1-86.6), P  < 0.001), HMWK (6.11 (4.95-7.98) vs 3.83 (2.60-5.68), P  < 0.001), prekallikrein (2.15 (1.00-6.36) vs 1.41 (0.63-2.22), P  = 0.026) and bradykinin (152.4 (137.6-180.4) vs 118.3 (97.1-137.9), P  < 0.001) than did normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX and XIIa, D-dimer, peak thrombin, neutrophil elastase, HMWK and bradykinin showed significant odds ratios representing hyperthyroidism's contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX, D-dimer, double-stranded DNA and bradykinin. This study demonstrated that contact system activation and abundant NET formation occurred in the high thrombin generation state in hyperthyroidism and were correlated with free T4 level. © 2017 European Society of Endocrinology.

Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

PubMed Central

Halder, Luke D.; Abdelfatah, Mahmoud A.; Jo, Emeraldo A. H.; Jacobsen, Ilse D.; Westermann, Martin; Beyersdorf, Niklas; Lorkowski, Stefan; Zipfel, Peter F.; Skerka, Christine

2017-01-01

Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16−/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation. PMID:28133459

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation.

PubMed

Di Francesco, Andrea; Di Germanio, Clara; Panda, Amaresh C; Huynh, Phu; Peaden, Robert; Navas-Enamorado, Ignacio; Bastian, Paul; Lehrmann, Elin; Diaz-Ruiz, Alberto; Ross, David; Siegel, David; Martindale, Jennifer L; Bernier, Michel; Gorospe, Myriam; Abdelmohsen, Kotb; de Cabo, Rafael

2016-10-01

NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3' untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3'UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as an RNA-binding protein may help to explain its pleiotropic biological effects. Published by Elsevier Inc.

Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease?

PubMed

Bendiske, Jennifer; Bahr, Ben A

2003-05-01

Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

A randomized clinical trial of alpha(1)-antitrypsin augmentation therapy.

PubMed

Dirksen, A; Dijkman, J H; Madsen, F; Stoel, B; Hutchison, D C; Ulrik, C S; Skovgaard, L T; Kok-Jensen, A; Rudolphus, A; Seersholm, N; Vrooman, H A; Reiber, J H; Hansen, N C; Heckscher, T; Viskum, K; Stolk, J

1999-11-01

We have investigated whether restoration of the balance between neutrophil elastase and its inhibitor, alpha(1)-antitrypsin, can prevent the progression of pulmonary emphysema in patients with alpha(1)-antitrypsin deficiency. Twenty-six Danish and 30 Dutch ex-smokers with alpha(1)-antitrypsin deficiency of PI*ZZ phenotype and moderate emphysema (FEV(1) between 30% and 80% of predicted) participated in a double-blind trial of alpha(1)-antitrypsin augmentation therapy. The patients were randomized to either alpha(1)-antitrypsin (250 mg/kg) or albumin (625 mg/kg) infusions at 4-wk intervals for at least 3 yr. Self-administered spirometry performed every morning and evening at home showed no significant difference in decline of FEV(1) between treatment and placebo. Each year, the degree of emphysema was quantified by the 15th percentile point of the lung density histogram derived from computed tomography (CT). The loss of lung tissue measured by CT (mean +/- SEM) was 2.6 +/- 0.41 g/L/yr for placebo as compared with 1.5 +/- 0.41 g/L/yr for alpha(1)-antitrypsin infusion (p = 0.07). Power analysis showed that this protective effect would be significant in a similar trial with 130 patients. This is in contrast to calculations based on annual decline of FEV(1) showing that 550 patients would be needed to show a 50% reduction of annual decline. We conclude that lung density measurements by CT may facilitate future randomized clinical trials of investigational drugs for a disease in which little progress in therapy has been made in the past 30 yr.

A diketopiperazine factor from Rheinheimera aquimaris QSI02 exhibits anti-quorum sensing activity

PubMed Central

Sun, Shiwei; Dai, Xiaoyun; Sun, Jiao; Bu, Xiangguo; Weng, Caihong; Li, Hui; Zhu, Hu

2016-01-01

An ethyl acetate (EtOAc) extract isolated from the marine bacterium, Rheinheimera aquimaris QSI02, was found to exhibit anti-quorum sensing (anti-QS) activity. A subsequent bioassay-guided isolation protocol led to the detection of an active diketopiperazine factor, cyclo(Trp-Ser). Biosensor assay data showed that the minimum inhibitory concentration (MIC) of cyclo(Trp-Ser) ranged from 3.2 mg/ml to 6.4 mg/m for several microorganisms, including Escherichia coli, Chromobacterium violaceum CV026, Pseudomonas aeruginosa PA01, Staphylococcus aureus, and Candida albicans. Additionally, sub-MICs of cyclo(Trp-Ser) decreased the QS-regulated violacein production in C. violaceum CV026 by 67%. Furthermore, cyclo(Trp-Ser) can decrease QS-regulated pyocyanin production, elastase activity and biofilm formation in P. aeruginosa PA01 by 65%, 40% and 59.9%, respectively. Molecular docking results revealed that cyclo(Trp-Ser) binds to CviR receptor more rigidly than C6HSL with lower docking energy −8.68 kcal/mol, while with higher binding energy of −8.40 kcal/mol than 3-oxo-C12HSL in LasR receptor. Molecular dynamics simulation suggested that cyclo(Trp-Ser) is more easy to bind to CviR receptor than natural signaling molecule, but opposite in LasR receptor. These results suggest that cyclo(Trp-Ser) can be used as a potential inhibitor to control QS systems of C. violaceum and P. aeruginosa and provide increased the understanding of molecular mechanism that influences QS-regulated behaviors. PMID:28000767

Elephantiasis, elastin, and chronic wound healing: 19th century and contemporary viewpoints relevant to hypotheses concerning lymphedema, leprosy, erysipelas, and psoriasis--review and reflections.

PubMed

Ryan, T J

2009-03-01

Both wound healing and lymphedema have fibrosis of the skin in common. They also share destruction of elastin by elastases from neutrophils as a significant feature. These are not new observations, and the writings of Unna and Kaposi are recalled. The contemporary observations on elastin by Gerli and his team are discussed in the light of these much earlier opinions.

Zinc status in chronic pancreatitis and its relationship with exocrine and endocrine insufficiency.

PubMed

Girish, Banavara Narasimhamurthy; Rajesh, Gopalakrishna; Vaidyanathan, Kannan; Balakrishnan, Vallath

2009-11-05

A major role of the pancreas in zinc homeostasis has been suggested. To assess erythrocyte zinc status in chronic pancreatitis and to correlate it with pancreatic exocrine and endocrine insufficiency. One hundred and one patients with chronic pancreatitis (34 alcoholic chronic pancreatitis, 67 tropical chronic pancreatitis) were prospectively studied. Disease characteristics and imaging features were recorded. Erythrocyte zinc was estimated by flame atomic absorption spectrophotometry. Exocrine insufficiency was assessed using polyclonal antibody ELISA for pancreatic stool elastase1. Endocrine insufficiency was assessed by serum glucose levels and insulin requirement. Erythrocyte zinc was significantly lower in chronic pancreatitis patients than in the controls (26.5+/-9.5 microg/g Hb vs. 38.0+/-6.6 microg/g Hb; P<0.001), and in tropical chronic pancreatitis than in alcoholic chronic pancreatitis (25.0+/-10.4 microg/g Hb vs. 29.6+/-6.5 microg/g Hb, P=0.001). In chronic pancreatitis patients who had exocrine insufficiency, erythrocyte zinc positively correlated with stool elastase1 (r=0.587, P<0.001). Erythrocyte zinc levels were significantly lower in diabetic patients as compared to non-diabetics (P=0.036). This study demonstrates zinc deficiency in chronic pancreatitis patients, and that zinc deficiency correlates with exocrine and endocrine insufficiency. Further studies may clarify the possible benefits of zinc supplementation in chronic pancreatitis.

Anti-Inflammatory Activity and Changes in Antioxidant Properties of Leaf and Stem Extracts from Vitex mollis Kunth during In Vitro Digestion

PubMed Central

Morales-Del-Rio, Juan Alfredo; Gutiérrez-Lomelí, Melesio; Robles-García, Miguel Angel; Aguilar, Jose Antonio; Lugo-Cervantes, Eugenia; Guerrero-Medina, Pedro Javier; Ruiz-Cruz, Saul; Cinco-Moroyoqui, Francisco J.; Wong-Corral, Francisco J.; Del-Toro-Sánchez, Carmen Lizette

2015-01-01

Vitex mollis is used in traditional Mexican medicine for the treatment of some ailments. However, there are no studies on what happens to the anti-inflammatory activity or antioxidant properties and total phenolic content of leaves and stem extracts of Vitex mollis during the digestion process; hence, this is the aim of this work. Methanolic, acetonic, and hexanic extracts were obtained from both parts of the plant. Extract yields and anti-inflammatory activity (elastase inhibition) were measured. Additionally, changes in antioxidant activity (DPPH and ABTS) and total phenols content of plant extracts before and after in vitro digestion were determined. The highest elastase inhibition to prevent inflammation was presented by hexanic extracts (leaf = 94.63% and stem = 98.30%). On the other hand, the major extract yield (16.14%), antioxidant properties (ABTS = 98.51% and DPPH = 94.47% of inhibition), and total phenols (33.70 mg GAE/g of dried sample) were showed by leaf methanolic extract. Finally, leaf and stem methanolic extracts presented an antioxidant activity increase of 35.25% and 27.22%, respectively, in comparison to their initial values after in vitro digestion process. All samples showed a decrease in total phenols at the end of the digestion. These results could be the basis to search for new therapeutic agents from Vitex mollis. PMID:26451153

A Metabolic Trade-Off Modulates Policing of Social Cheaters in Populations of Pseudomonas aeruginosa

PubMed Central

Yan, Huicong; Wang, Meizhen; Sun, Feng; Dandekar, Ajai A.; Shen, Dongsheng; Li, Na

2018-01-01

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of public goods such as the secreted protease elastase. P. aeruginosa requires the LasI–LasR QS circuit to induce elastase and enable growth on casein as the sole carbon and energy source. The LasI–LasR system also induces a second QS circuit, the RhlI–RhlR system. During growth on casein, LasR-mutant social cheaters emerge, and this can lead to a population collapse. In a minimal medium containing ammonium sulfate as a nitrogen source, populations do not collapse, and cheaters and cooperators reach a stable equilibrium; however, without ammonium sulfate, cheaters overtake the cooperators and populations collapse. We show that ammonium sulfate enhances the activity of the RhlI–RhlR system in casein medium and this leads to increased production of cyanide, which serves to control levels of cheaters. This enhancement of cyanide production occurs because of a trade-off in the metabolism of glycine: exogenous ammonium ion inhibits the transformation of glycine to 5,10-methylenetetrahydrofolate through a reduction in the expression of the glycine cleavage genes gcvP1 and gcvP2, thereby increasing the availability of glycine as a substrate for RhlR-regulated hydrogen cyanide synthesis. Thus, environmental ammonia enhances cyanide production and stabilizes QS in populations of P. aeruginosa. PMID:29535700

Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

PubMed Central

Sun, Xingshen; Olivier, Alicia K.; Yi, Yaling; Pope, Christopher E.; Hayden, Hillary S.; Liang, Bo; Sui, Hongshu; Zhou, Weihong; Hager, Kyle R.; Zhang, Yulong; Liu, Xiaoming; Yan, Ziying; Fisher, John T.; Keiser, Nicholas W.; Song, Yi; Tyler, Scott R.; Goeken, J. Adam; Kinyon, Joann M.; Radey, Matthew C.; Fligg, Danielle; Wang, Xiaoyan; Xie, Weiliang; Lynch, Thomas J.; Kaminsky, Paul M.; Brittnacher, Mitchell J.; Miller, Samuel I.; Parekh, Kalpaj; Meyerholz, David K.; Hoffman, Lucas R.; Frana, Timothy; Stewart, Zoe A.; Engelhardt, John F.

2015-01-01

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients. PMID:24637292

Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis.

PubMed

Scott, Ann E; Milward, Mike; Linden, Gerard J; Matthews, John B; Carlile, Monica J; Lundy, Fionnuala T; Naeeni, Mojgan A; Lorraine Martin, S; Walker, Brian; Kinane, Denis; Brock, Gareth R; Chapple, Iain L C

2012-02-01

To characterize and map temporal changes in the biological and clinical phenotype during a 21-day experimental gingivitis study. Experimental gingivitis was induced over 21 days in healthy human volunteers (n = 56), after which normal brushing was resumed (resolution phase). Gingival and plaque indices were assessed. Gingival crevicular fluid was collected from four paired test and contra-lateral control sites in each volunteer during induction (Days 0, 7, 14 and 21) and resolution (Days 28 and 42) of experimental gingivitis. Fluid volumes were measured and a single analyte was quantified from each site-specific, 30s sample. Data were evaluated by analysis of repeated measurements and paired sample tests. Clinical indices and gingival crevicular fluid volumes at test sites increased from Day 0, peaking at Day 21 (test/control differences all p < 0.0001) and decreased back to control levels by Day 28. Levels of four inflammatory markers showed similar patterns, with significant differences between test and control apparent at Day 7 (substance P, cathepsin G, interleukin-1β, elastase: all p < 0.03) and peaking at Day 21 (all p < 0.002). Levels of α-1-antitrypsin showed no pattern. Levels of substance P, cathepsin G, interleukin-1β and neutrophil elastase act as objective biomarkers of gingival inflammation induction and resolution that typically precede phenotypical changes. © 2011 John Wiley & Sons A/S.

Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells.

PubMed

Hiramoto, Takafumi; Ebihara, Yasuhiro; Mizoguchi, Yoko; Nakamura, Kazuhiro; Yamaguchi, Kiyoshi; Ueno, Kazuko; Nariai, Naoki; Mochizuki, Shinji; Yamamoto, Shohei; Nagasaki, Masao; Furukawa, Yoichi; Tani, Kenzaburo; Nakauchi, Hiromitsu; Kobayashi, Masao; Tsuji, Kohichiro

2013-02-19

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

Promotion of adipogenesis by an EP2 receptor agonist via stimulation of angiogenesis in pulmonary emphysema.

PubMed

Tsuji, Takao; Yamaguchi, Kazuhiro; Kikuchi, Ryota; Itoh, Masayuki; Nakamura, Hiroyuki; Nagai, Atsushi; Aoshiba, Kazutetsu

2014-08-01

Body weight loss is a common manifestation in patients with chronic obstructive pulmonary disease (COPD), particularly those with severe emphysema. Adipose angiogenesis is a key mediator of adipogenesis and use of pro-angiogenic agents may serve as a therapeutic option for lean COPD patients. Since angiogenesis is stimulated by PGE2, we examined whether ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, might promote adipose angiogenesis and adipogenesis in a murine model of elastase-induced pulmonary emphysema (EIE mice). Mice were intratracheally instilled with elastase or saline, followed after 4 weeks by intraperitoneal administration of ONO-AE1-259 for 4 weeks. The subcutaneous adipose tissue (SAT) weight decreased in the EIE mice, whereas in the EIE mice treated with ONO-AE1-259, the SAT weight was largely restored, which was associated with significant increases in SAT adipogenesis, angiogenesis, and VEGF protein production. In contrast, ONO-AE1-259 administration induced no alteration in the weight of the visceral adipose tissue. These results suggest that in EIE mice, ONO-AE1-259 stimulated adipose angiogenesis possibly via VEGF production, and thence, adipogenesis. Our data pave the way for the development of therapeutic interventions for weight loss in emphysema patients, e.g., use of pro-angiogenic agents targeting the adipose tissue vascular component. Copyright © 2014 Elsevier Inc. All rights reserved.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

PubMed

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-06-29

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice

PubMed Central

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-01-01

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema. PMID:26160987

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

PubMed Central

Zhang, Chao; van der Voort, Dustin; Shi, Hong; Qing, Yulan; Hiraoka, Shuichi; Takemoto, Minoru; Yokote, Koutaro; Moxon, Joseph V.; Norman, Paul; Rittié, Laure; Atkins, G. Brandon; Gerson, Stanton L.; Shi, Guo-Ping; Golledge, Jonathan; Dong, Nianguo; Perbal, Bernard; Prosdocimo, Domenick A.

2016-01-01

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II–induced AAA and elastase perfusion–stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II–induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease. PMID:26974158

The Association of Viral Hepatitis and Acute Pancreatitis

PubMed Central

Geokas, Michael C.; Olsen, Harvey; Swanson, Virginia; Rinderknecht, Heinrich

1972-01-01

The histological features of 24 pancreases obtained from patients who died of causes other than hepatitis, pancreatitis or pancreatic tumors, included a variable degree of autolysis, rare foci of inflammatory reaction but no hemorrhagic fat necrosis or destruction of elastic tissue in vessel walls (elastolysis). Assays of elastase in extracts of these pancreases showed no free enzyme, but varying amounts of proelastase. A review of autopsy findings in 33 patients with fatal liver necrosis attributed to halothane anesthesia, demonstrated changes of acute pancreatitis only in two. On the other hand, a review of 16 cases of fulminant viral hepatitis revealed changes characteristic of acute pancreatitis in seven – interstitial edema, hemorrhagic fat necrosis, inflammatory reaction and frequently elastolysis in vessel walls. Determination of elastase in extracts of one pancreas showed the bulk of the enzyme in free form. Furthermore, assays of urinary amylase in 44 patients with viral hepatitis showed increased levels of this enzyme (2583 ± 398 mean value ± standard error, Somogyi units per 100 ml in 13, or 29.5 percent). The evidence suggests that acute pancreatitis may at times complicate viral hepatitis. Although direct proof of viral pancreatic involvement is not feasible at present, a rational hypothesis is advanced which underlines similar mechanisms of tissue involvement in both liver and pancreas that may be brought about by the hepatitis viruses. PMID:5070694

Effects of Egg Shell Membrane Hydrolysates on Anti-Inflammatory, Anti-Wrinkle, Anti-Microbial Activity and Moisture-Protection.

PubMed

Yoo, Jinhee; Park, Kimoon; Yoo, Youngji; Kim, Jongkeun; Yang, Heejin; Shin, Youngjae

2014-01-01

This study was conducted to examine the effects of eggshell membrane hydrolysates (ESMH) on the anti-inflammatory, anti-wrinkle, anti-microbial activity, and moisture-protection for cosmetic use. Whole ESMH (before fractionation), and fraction I (>10 kDa), fraction II (3-10 kDa), and fraction III (<3 kDa) of the hydrolysates were assessed in this experiment. As lipopolysaccharide (LPS) and IFN-γ caused the inflammation on Raw264.7 cell, whole ESMH and fraction I showed to be effective in inhibiting the induction of cell inflammation depending on the concentration, and also showed outstanding effect to suppress the skin inflammation. Fraction I inhibited collagenase and elastase activities to a greater extent than the other fractions, while all fractions had antibiotic effects at concentrations of 10 mg/disc and 20 mg/disc. In addition, it showed the moisture protection effects of skin on the holding amount and losing amount of moisture in upper-inner arm of the human body with a relatively low loss rate in skin, which confirmed that the hydrolyzed fractions of ESM helps to form the superior protective layer of moisture. It was concluded that ESMH fractions with different molecular weights, especially the 10 kDa fraction, have anti-lipopolysaccharide, anti-IFN-γ-induced inflammation, anti- collagenase and elastase activities, and thus can be used as a cosmetic agent to protect skin.

Inhibition of human neutrophil elastase by α1-antitrypsin functionalized colloidal microcarriers.

PubMed

Reibetanz, Uta; Schönberg, Maria; Rathmann, Sophie; Strehlow, Vincent; Göse, Martin; Leßig, Jacqueline

2012-07-24

Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of α(1)-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intra- and extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra- and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.

International Shock Congress (1st) and Annual Society Meeting (10th) Held in Montreal, Canada on 7-11 June 1987

DTIC Science & Technology

1987-10-01

demonstrated that endotoxin shock is associated with a decrease in compliance of the superior and inferior vena cavae and probably £ Abstracts 17 central...site of a burn wound. METHODS: Anesthetized 350 gram male Long Evans rats were prepared by intrarenal inferior vena cava (IVC) ligation. The rats...elastase has been investigated in the superior vena caval and left atrium blood collected from 167 patients who underwent open heart surgery. The effect

Cladielloides A and B: New Eunicellin-Type Diterpenoids from an Indonesian Octocoral Cladiella sp

PubMed Central

Chen, Yung-Husan; Tai, Chia-Ying; Hwang, Tsong-Long; Weng, Ching-Feng; Li, Jan-Jung; Fang, Lee-Shing; Wang, Wei-Hsien; Wu, Yang-Chang; Sung, Ping-Jyun

2010-01-01

Two new eunicellin-type diterpenoids, cladielloides A (1) and B (2), which were found to possess a 2-hydroxybutyroxy group in their structures, were isolated from an Indonesian octocoral identified as Cladiella sp. The structures of eunicellins 1 and 2 were elucidated by spectroscopic methods. Cladielloide B (2) exhibited moderate cytotoxicity toward CCRF-CEM tumor cells and this compound displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils. PMID:21339957

MrkD sub 1P from Klebsiella pneumoniae IA565 Allows for Co-existence with Pseudomonas aeruginosa and Protection from Protease-mediated Biofilm Detachment

DTIC Science & Technology

2013-11-01

secreted effectors, such as phenazines , rhamnolipids, cis-2-decenoic acid, alkaline protease, exotoxins, and elastase, which are used by P. aeruginosa...demonstrated with various types of microorganisms, including Candida albi- cans, which is sensitive to phenazine (41), and Staphylococcus au- reus...2013. Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines . mBio 4(1):e00526 – 00512. doi:10 .1128/mBio

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection.

PubMed

Gupta, P; Gupta, R K; Harjai, K

2013-01-01

Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.

Pancreatic endocrine and exocrine function in children following near-total pancreatectomy for diffuse congenital hyperinsulinism.

PubMed

Arya, Ved Bhushan; Senniappan, Senthil; Demirbilek, Huseyin; Alam, Syeda; Flanagan, Sarah E; Ellard, Sian; Hussain, Khalid

2014-01-01

Congenital hyperinsulinism (CHI), the commonest cause of persistent hypoglycaemia, has two main histological subtypes: diffuse and focal. Diffuse CHI, if medically unresponsive, is managed with near-total pancreatectomy. Post-pancreatectomy, in addition to persistent hypoglycaemia, there is a very high risk of diabetes mellitus and pancreatic exocrine insufficiency. International referral centre for the management of CHI. Medically unresponsive diffuse CHI patients managed with near-total pancreatectomy between 1994 and 2012. Near-total pancreatectomy. Persistent hypoglycaemia post near-total pancreatectomy, insulin-dependent diabetes mellitus, clinical and biochemical (faecal elastase 1) pancreatic exocrine insufficiency. Of more than 300 patients with CHI managed during this time period, 45 children had medically unresponsive diffuse disease and were managed with near-total pancreatectomy. After near-total pancreatectomy, 60% of children had persistent hypoglycaemia requiring medical interventions. The incidence of insulin dependent diabetes mellitus was 96% at 11 years after surgery. Thirty-two patients (72%) had biochemical evidence of severe pancreatic exocrine insufficiency (Faecal elastase 1<100 µg/g). Clinical exocrine insufficiency was observed in 22 (49%) patients. No statistically significant difference in weight and height standard deviation score (SDS) was found between untreated subclinical pancreatic exocrine insufficiency patients and treated clinical pancreatic exocrine insufficiency patients. The outcome of diffuse CHI patients after near-total pancreatectomy is very unsatisfactory. The incidence of persistent hypoglycaemia and insulin-dependent diabetes mellitus is very high. The presence of clinical rather than biochemical pancreatic exocrine insufficiency should inform decisions about pancreatic enzyme supplementation.

CXCL8 hyper-signaling in the aortic abdominal aneurysm.

PubMed

Kokje, Vivianne B C; Gäbel, Gabor; Dalman, Ron L; Koole, Dave; Northoff, Bernd H; Holdt, Lesca M; Hamming, Jaap F; Lindeman, Jan H N

2018-08-01

There are indications for elevated CXCL8 levels in abdominal aortic aneurysm disease (AAA). CXCL8 is concurrently involved in neutrophil-mediated inflammation and angiogenesis, two prominent and distinctive characteristics of AAA. As such we considered an evaluation of a role for CXCL8 in AAA progression relevant. ELISA's, real time PCR and array analysis were used to explore CXCL8 signaling in AAA wall samples. A role for CXCL8 in AAA disease was tested through the oral CXCR1/2 antagonist DF2156A in the elastase model of AAA disease. There is an extreme disparity in aortic wall CXCL8 content between AAA and aortic atherosclerotic disease (median [IQR] aortic wall CXCL8 content: 425 [141-1261] (AAA) vs. 23 [2.8-89] (atherosclerotic aorta) µg/g protein (P < 1 · 10 -14 )), and abundant expression of the CXCR1 and 2 receptors in AAA. Array analysis followed by pathway analysis showed that CXCL8 hyper-expression in AAA is followed increased by IL-8 signaling (Z-score for AAA vs. atherosclerotic control: 2.97, p < 0.0001). Interference with CXCL8 signaling through DF2156A fully abrogated AAA formation and prevented matrix degradation in the murine elastase model of AAA disease (p < 0.001). CXCL8-signaling is a prominent and distinctive feature of AAA, interference with the pathway constitutes a promising target for medical stabilization of AAA. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Anti-Oxidant, Anti-Aging, and Anti-Melanogenic Properties of the Essential Oils from Two Varieties of Alpinia zerumbet.

PubMed

Tu, Pham Thi Be; Tawata, Shinkichi

2015-09-14

Here, we investigated the anti-oxidant and anti-aging effects of essential oils (EOs) from the leaves of Alpinia zerumbet (tairin and shima) in vitro and anti-melanogenic effects in B16F10 melanoma cells. The anti-oxidant activities were performed with 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); nitric oxide; singlet oxygen; hydroxyl radical scavenging; and xanthine oxidase. The inhibitory activities against collagenase, elastase, hyaluronidase, and tyrosinase were employed for anti-aging. The anti-melanogenic was assessed in B16F10 melanoma cells by melanin synthesis and intracellular tyrosinase inhibitory activity. The volatile chemical composition of the essential oil was analyzed with gas chromatography-mass spectrometry (GC/MS). The EO was a complex mixture mainly consisting of monoterpenes and sesquiterpenes. The results revealed that tairin and shima EOs showed strong anti-oxidant activities against DPPH and nitric oxide, hydroxyl radical scavenging activity, and xanthine oxidase inhibition. Compared to shima EO; tairin EO exhibited strong anti-aging activity by inhibiting collagenase, tyrosinase, hyaluronidase, and elastase (IC50 = 11 ± 0.1; 25 ± 1.2; 83 ± 1.6; and 213 ± 2 μg/mL, respectively). Both EOs inhibited intracellular tyrosinase activity; thus, reducing melanin synthesis. These results suggest that tairin EO has better anti-oxidant/anti-aging activity than shima EO, but both are equally anti-melanogenic.

Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging II: Over-Expression of Neprilysin Plays an Essential Role

PubMed Central

Imokawa, Genji; Nakajima, Hiroaki; Ishida, Koichi

2015-01-01

Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts. PMID:25856676

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

PubMed

Sun, Xingshen; Olivier, Alicia K; Yi, Yaling; Pope, Christopher E; Hayden, Hillary S; Liang, Bo; Sui, Hongshu; Zhou, Weihong; Hager, Kyle R; Zhang, Yulong; Liu, Xiaoming; Yan, Ziying; Fisher, John T; Keiser, Nicholas W; Song, Yi; Tyler, Scott R; Goeken, J Adam; Kinyon, Joann M; Radey, Matthew C; Fligg, Danielle; Wang, Xiaoyan; Xie, Weiliang; Lynch, Thomas J; Kaminsky, Paul M; Brittnacher, Mitchell J; Miller, Samuel I; Parekh, Kalpaj; Meyerholz, David K; Hoffman, Lucas R; Frana, Timothy; Stewart, Zoe A; Engelhardt, John F

2014-05-01

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

PubMed

Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M; Borel, Florie; Reeves, Emer P; Calcedo, Roberto; Rouhani, Farshid N; Yachnis, Anthony; Humphries, Margaret; Campbell-Thompson, Martha; Messina, Louis; Chulay, Jeffrey D; Trapnell, Bruce; Wilson, James M; McElvaney, Noel G; Flotte, Terence R

2017-06-07

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Retinoic acid-related orphan receptor-α is induced in the setting of DNA damage and promotes pulmonary emphysema.

PubMed

Shi, Ying; Cao, Jiaofei; Gao, Jane; Zheng, Liang; Goodwin, Andrew; An, Chang Hyoek; Patel, Avignat; Lee, Janet S; Duncan, Steven R; Kaminski, Naftali; Pandit, Kusum V; Rosas, Ivan O; Choi, Augustine M K; Morse, Danielle

2012-09-01

The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. To determine the role of Rora in smoke-induced emphysema. Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

PubMed

Song, Eunju; Chung, Haeyon; Shim, Eugene; Jeong, Jung-Ky; Han, Bok-Kyung; Choi, Hyuk-Joon; Hwang, Jinah

2016-11-01

Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

Upregulation of MMP12 and its activity by UVA1 in human skin: potential implications for photoaging.

PubMed

Tewari, Angela; Grys, Katarzyna; Kollet, Jutta; Sarkany, Robert; Young, Antony R

2014-10-01

UVA1 constitutes around 75% of the terrestrial UV radiation, and most of the output of artificial tanning sources. However, the molecular effects of UVA1 in human skin in vivo are surprisingly poorly understood. We have examined time-dependent whole-genome expression, along with mRNA and protein changes in the skin after one minimal erythema dose of spectrally pure UVA1 (50 J cm(-2)) and 300 nm UVB (30 mJ cm(-2)). After 24 hours, the genes induced to the greatest extent were those involved in extracellular matrix remodeling with both UVA1 (P=5.5e-7) and UVB (P=2.9e-22). UVA1 and UVB caused different effects on matrix metalloproteinase (MMP) expression: UVB induced MMP1, MMP3, and MMP10 mRNA at 24 hours to a much greater extent than UVA1. MMP12 induction by UVA1 at 6 hours is marked and much greater than that by UVB. We have found that MMP12 mRNA induction by UVA1 resulted in expression of MMP12 protein, which is functional as an elastase. This induction of elastase activity did not occur with UVB. We hypothesize that the UVA1 induction of MMP12 mediates some of its photoaging effects, particularly by contributing to elastin degeneration in late solar elastosis. MMP12 is a good marker of UVA1 exposure.

Effects of low and standard intra-abdominal pressure on systemic inflammation and immune response in laparoscopic adrenalectomy: A prospective randomised study.

PubMed

Schietroma, Mario; Pessia, Beatrice; Stifini, Derna; Lancione, Laura; Carlei, Francesco; Cecilia, Emanuela Marina; Amicucci, Gianfranco

2016-01-01

The advantages of laparoscopic adrenalectomy (LA) over open adrenalectomy are undeniable. Nevertheless, carbon dioxide (CO2) pneumoperitoneum may have an unfavourable effect on the local immune response. The aim of this study was to compare changes in the systemic inflammation and immune response in the early post-operative (p.o.) period after LA performed with standard and low-pressure CO2 pneumoperitoneum. We studied, in a prospective randomised study, 51 patients consecutively with documented adrenal lesion who had undergone a LA: 26 using standard-pressure (12-14 mmHg) and 25 using low-pressure (6-8 mmHg) pneumoperitoneum. White blood cells (WBC), peripheral lymphocyte subpopulation, human leucocyte antigen-DR (HLA-DR), neutrophil elastase, interleukin (IL)-6 and IL-1, and C-reactive protein (CRP) were investigated. Significantly higher concentrations of neutrophil elastase, IL-6 and IL-1 and CRP were detected p.o. in the standard-pressure group of patients in comparison with the low-pressure group (P < 0.05). A statistically significant change in HLA-DR expression was recorded p.o. at 24 h, as a reduction of this antigen expressed on the monocyte surface in patients from the standard group; no changes were noted in low-pressure group patients (P < 0.05). This study demonstrated that reducing the pressure of the pneumoperitoneum to 6-8 mmHg during LA reduced p.o. inflammatory response and averted p.o. immunosuppression.

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

PubMed

Rajamanickam, Anuradha; Munisankar, Saravanan; Bhootra, Yukthi; Dolla, Chandra Kumar; Nutman, Thomas B; Babu, Subash

2018-01-01

Infection with the helminth parasite Strongyloides stercoralis ( Ss ) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

Peritonitis from perforated peptic ulcer and immune response.

PubMed

Schietroma, Mario; Piccione, Federica; Carlei, Francesco; Sista, Federico; Cecilia, Emanuela Marina; Amicucci, Gianfranco

2013-10-01

Elevated intra-abdominal pressure during the laparoscopy may promote bacteremia, endotoxemia, and systemic inflammatory response. In patients with generalized peritonitis from perforated peptic ulcer (PPU), we sought to compare acute phase response, immunologic status, and bacterial translocation from laparoscopic and open approach. From July 2005 to September 2011, 115 consecutive patients underwent peptic ulcer repair for PPU: 58 cases laparoscopic peptic ulcer repair (LR) and 57 cases open peptic ulcer repair (OR). Bacteremia, endotoxemia, white blood cells population, human leukocyte antigen-DR (HLA-DR), neutrophil-elastase, interleukin-1 and 6 (IL-1 and IL-6), and C-reactive protein (CRP) were investigated. Patients characteristics and grade of peritoneal contamination were similar in the two groups. One hour after intervention, bacteremia was significantly higher in the "open" group than in the laparoscopic group (p < .001). A significantly higher concentration of systemic endotoxin was detected intraoperatively in the "open" group of patients in comparison to the laparoscopic group (p < .0001). Laparotomy caused a significant increase in neutrophil concentration, neutrophil-elastase, IL-1 and IL-6, CRP, and decrease of HLA-DR. We recorded six cases (10.3%) of intra-abdominal abscess in the "open" group and one (1.7%) in laparoscopic group (p < .001). OR, in case of peritonitis after PPU, increased the incidence of bacteremia, endotoxemia, and systemic inflammation compared with LR. Early enhanced postoperative systemic inflammation may cause lower transient immunologic defense after laparotomy (decrease of HLA-DR), leading to enhanced sepsis in these patients.

A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise.

PubMed

Zhao, Hui; Gulesserian, Sara; Malinao, Maria Christina; Ganesan, Sathish Kumar; Song, James; Chang, Mi Sook; Williams, Melissa M; Zeng, Zhilan; Mattie, Michael; Mendelsohn, Brian A; Stover, David R; Doñate, Fernando

2017-09-01

Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866-76. ©2017 AACR See related article by Zhao et al., p. 1877 . ©2017 American Association for Cancer Research.

Exhaled breath condensate MMP-9 levels in children with bronchiectasis.

PubMed

Karakoc, Gulbin Bingol; Inal, Ayfer; Yilmaz, Mustafa; Altintas, Derya Ufuk; Kendirli, Seval Guneser

2009-10-01

Bronchiectasis (BE) is still an important cause of chronic supurative respiratory diseases in developing countries. Neutrophil-derived proteases such as neutrophil elastase and matrix metalloproteases (MMPs) are implicated in causing airway damage in chronic pulmonary disease. In this study, we aimed to evaluate the MMP-9 and its natural tissue inhibitors of metalloproteinases (TIMP-1) levels utilizing the exhaled breath condensate (EBC) method and their relationship with radiological findings and pulmonary functions in children with BE.Thirty-eight children with BE and 12 healthy children were included: Group 1 (cystic fibrosis [CF] BE), Group 2 (non-CF BE), Group 3 (control group). High-resolution computerized tomography (HRCT) scores were calculated according to the anatomic extent of BE. Pulmonary function tests were performed, and MMP-9 and TIMP-1 levels in EBC were analyzed by ELISA.Exhaled breath condensate MMP-9 level was 48.9 +/- 26.8 ng/ml for Group 1, and for Group 2, 42.8 +/- 18.1 ng/ml; and for Group 3, 30 +/- 3.7 ng/ml. Although no statistically significant difference was found between the Groups 1 and 2, a significant difference was detected between these groups and controls. No statistically significant difference was found in TIMP-1 levels regarding all groups. EBC MMP-9 levels were inversely correlated with pulmonary functions test, and positively with HRCT scores and annual number of pulmonary infections.In conclusion, this study showed that EBC of children with both CF BE and non-CF BE contained higher levels of MMP-9 in comparison to controls. We suggest that EBC MMP-9 level may be a useful marker of airway injury in patients with BE however prospective studies are needed.

Complement Activation in Relation to Capillary Leakage in Children with Septic Shock and Purpura

PubMed Central

Hazelzet, Jan A.; de Groot, Ronald; van Mierlo, Gerard; Joosten, Koen F. M.; van der Voort, Edwin; Eerenberg, Anke; Suur, Marja H.; Hop, Wim C. J.; Hack, C. Erik

1998-01-01

To assess the relationship between capillary leakage and inflammatory mediators during sepsis, blood samples were taken on hospital admission, as well as 24 and 72 h later, from 52 children (median age, 3.3 years) with severe meningococcal sepsis, of whom 38 survived and 14 died. Parameters related to cytokines (interleukin 6 [IL-6] IL-8, plasma phospholipase A2, and C-reactive protein [CRP]), to neutrophil degranulation (elastase and lactoferrin), to complement activation (C3a, C3b/c, C4b/c, and C3- and C4-CRP complexes), and to complement regulation (functional and inactivated C1 inhibitor and C4BP) were determined. The degree of capillary leakage was derived from the amount of plasma infused and the severity of disease by assessing the pediatric risk of mortality (PRISM) score. Levels of IL-6, IL-8, C3b/c, C3-CRP complexes, and C4BP on admission, adjusted for the duration of skin lesions, were significantly different in survivors and nonsurvivors (C3b/c levels were on average 2.2 times higher in nonsurvivors, and C3-CRP levels were 1.9 times higher in survivors). Mortality was independently related to the levels of C3b/c and C3-CRP complexes. In agreement with this, levels of complement activation products correlated well with the PRISM score or capillary leakage. Thus, these data show that complement activation in patients with severe meningococcal sepsis is associated with a poor outcome and a more severe disease course. Further studies should reveal whether complement activation may be a target for therapeutical intervention in this disease. PMID:9784543

Prostanoid-dependent spontaneous pain and PAR2-dependent mechanical allodynia following oral mucosal trauma

PubMed Central

Ito, Misa; Hitomi, Suzuro; Nodai, Tomotaka; Sago, Teppei; Yamaguchi, Kiichiro; Harano, Nozomu; Gunjigake, Kaori; Hosokawa, Ryuji; Kawamoto, Tatsuo; Inenaga, Kiyotoshi

2017-01-01

During dental treatments, intraoral appliances frequently induce traumatic ulcers in the oral mucosa. Such mucosal injury-induced mucositis leads to severe pain, resulting in poor quality of life and decreased cooperation in the therapy. To elucidate mucosal pain mechanisms, we developed a new rat model of intraoral wire-induced mucositis and investigated pain mechanisms using our proprietary assay system for conscious rats. A thick metal wire was installed in the rats between the inferior incisors for one day. In the mucosa of the mandibular labial fornix region, which was touched with a free end of the wire, traumatic ulcer and submucosal abscess were induced on day 1. The ulcer was quickly cured until next day and abscess formation was gradually disappeared until five days. Spontaneous nociceptive behavior was induced on day 1 only, and mechanical allodynia persisted over day 3. Antibiotic pretreatment did not affect pain induction. Spontaneous nociceptive behavior was sensitive to indomethacin (cyclooxygenase inhibitor), ONO-8711 (prostanoid receptor EP1 antagonist), SB-366791, and HC-030031 (TRPV1 and TRPA1 antagonists, respectively). Prostaglandin E2 and 15-deoxyΔ12,14-prostaglandin J2 were upregulated only on day 1. In contrast, mechanical allodynia was sensitive to FSLLRY-NH2 (protease-activated receptor PAR2 antagonist) and RN-1734 (TRPV4 antagonist). Neutrophil elastase, which is known as a biased agonist for PAR2, was upregulated on days 1 to 2. These results suggest that prostanoids and PAR2 activation elicit TRPV1- and TRPA1-mediated spontaneous pain and TRPV4-mediated mechanical allodynia, respectively, independently of bacterial infection, following oral mucosal trauma. The pathophysiological pain mechanism suggests effective analgesic approaches for dental patients suffering from mucosal trauma-induced pain. PMID:28381109

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer

PubMed Central

Hamelin, Céline; Cornut, Emilie; Poirier, Florence; Pons, Sylvie; Beaulieu, Corinne; Charrier, Jean-Philippe; Haïdous, Hader; Cotte, Eddy; Lambert, Claude; Piard, Françoise; Ataman-Önal, Yasemin; Choquet-Kastylevsky, Geneviève

2011-01-01

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection. PMID:21973086

Graphical model for estimating oral bioavailability of drugs in humans and other species from their Caco-2 permeability and in vitro liver enzyme metabolic stability rates.

PubMed

Mandagere, Arun K; Thompson, Thomas N; Hwang, Kin-Kai

2002-01-17

This paper describes a graphical model for simplifying in vitro absorption, metabolism, distribution, and elimination (ADME) data analysis through the estimation of oral bioavailability (%F) of drugs in humans and other species. This model integrates existing in vitro ADME data, such as Caco-2 permeability (P(app)) and metabolic stability (percent remaining - %R) in liver S9 or microsomes, to estimate %F into groups of low, medium, or high regions. To test the predictive accuracy of our model, we examined 21 drugs and drug candidates with a wide range of oral bioavailability values, which represent approximately 10 different therapeutic areas in humans, rats, dogs, and guinea pigs. In vitro data from model compounds were used to define the boundaries of the low, medium, and high regions of the %F estimation plot. On the basis of the in vitro data, warfarin (93%), indomethacin (98%), timolol (50%), and carbamazepine (70%) were assigned to the high %F region; propranolol (26%) and metoprolol (38%) to medium %F region; and verapamil (22%) and mannitol (18%) to the low %F region. Similarly, the %F of 11 drug candidates from Elastase Inhibitor, NK1/NK2 antagonist, and anti-viral projects in rats, guinea pigs, and dogs were correctly estimated. This model estimates the oral bioavailability ranges of neutral, polar, esters, acidic, and basic drugs in all species. For a large number of drug candidates, this graphical model provides a tool to estimate human oral bioavailability from in vitro ADME data. When combined with the high throughput in vitro ADME screening process, it has the potential to significantly accelerate the processes of lead identification and optimization.

Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

PubMed

Rebordão, M R; Carneiro, C; Alexandre-Pires, G; Brito, P; Pereira, C; Nunes, T; Galvão, A; Leitão, A; Vilela, C; Ferreira-Dias, G

2014-12-01

Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Physiopathologic mechanisms involved in mare endometrosis.

PubMed

Rebordão, M R; Galvão, A; Szóstek, A; Amaral, A; Mateus, L; Skarzynski, D J; Ferreira-Dias, G

2014-10-01

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFβ and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1β, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition. © 2014 Blackwell Verlag GmbH.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill; Hook, Vivian Y H

2004-08-16

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Strain identification and quorum sensing inhibition characterization of marine-derived Rhizobium sp. NAO1

NASA Astrophysics Data System (ADS)

Chang, Hong; Zhou, Jin; Zhu, Xiaoshan; Yu, Shenchen; Chen, Lu; Jin, Hui; Cai, Zhonghua

2017-03-01

A novel strategy for combating pathogens is through the ongoing development and use of anti-quorum sensing (QS) treatments such as therapeutic bacteria or their anti-QS substances. Relatively little is known about the bacteria that inhabit the open ocean and of their potential anti-pathogenic attributes; thus, in an initiative to identify these types of therapeutic bacteria, planktonic microbes from the North Atlantic Ocean were collected, isolated, cultured and screened for anti-QS activity. Screening analysis identified one such strain, Rhizobium sp. NAO1. Extracts of Rhizobium sp. NAO1 were identified via ultra-performance liquid chromatography (UPLC) analysis. They were shown to contain N-acyl homoserine lactone (AHL)-based QS analogues (in particular, the N-butyryl homoserine lactone (C4-AHL) analogue) and could disrupt biofilm formation by Pseudomonas aeruginosa PAO1. QS inhibition was confirmed using confocal scanning laser microscopy and growth curves, and it was shown to occur in a dose-dependent manner without affecting bacterial growth. Secondary metabolites of Rhizobium sp. NAO1 inhibited PAO1 pathogenicity by downregulating AHL-mediated virulence factors such as elastase activity and siderophore production. Furthermore, as a result of biofilm structure damage, the secondary metabolite products of Rhizobium sp. NAO1 significantly increased the sensitivity of PAO1 to aminoglycoside antibiotics. Our results demonstrated that Rhizobium sp. strain NAO1 has the ability to disrupt P. aeruginosa PAO1 biofilm architecture, in addition to attenuating P. aeruginosa PAO1 virulence factor production and pathogenicity. Therefore, the newly identified ocean-derived Rhizobium sp. NAO1 has the potential to serve as a QS inhibitor and may be a new microbial resource for drug development.

Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway.

PubMed

Simon, D I; Ezratty, A M; Francis, S A; Rennke, H; Loscalzo, J

1993-10-15

Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator-deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac-1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium-dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac-1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via Mac-1 in the absence of plasmin, thereby providing an alternative fibrinolytic pathway. Thus, in addition to the function of cell adhesion, integrins may also act as receptors that mediate the internalization and degradation of bound ligands.

Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

1988-07-01

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

A canine model for production of severe unilateral panacinar emphysema.

PubMed

Chino, Kimiaki; Choong, Cliff K; Toeniskoetter, P Diane; Cooper, Joel D; Lausberg, Henning F; Bae, Kyongtae T; Pierce, John A; Hogg, James C

2004-06-01

The authors created a canine model of severe emphysema using whole lung lavage to deliver repeated porcine pancreatic elastase solution to the terminal airways and alveoli of the right lung. This model produces extreme unilateral panacinar emphysema closely resembling that encountered in patients with alpha1-antitrypsin deficiency. Because the contralateral lung remains functional, the animals can be maintained indefinitely. The model will be of value in developing imaging techniques capable of safely evaluating the effect of treatment on panacinar emphysema in alpha1-antitrypsin-deficient patients.

Discovery of New Eunicellins from an Indonesian Octocoral Cladiella sp.

PubMed Central

Chen, Yung-Husan; Tai, Chia-Ying; Su, Yin-Di; Chang, Yu-Chia; Lu, Mei-Chin; Weng, Ching-Feng; Su, Jui-Hsin; Hwang, Tsong-Long; Wu, Yang-Chang; Sung, Ping-Jyun

2011-01-01

Two new 11-hydroxyeunicellin diterpenoids, cladieunicellin F (1) and (–)-solenopodin C (2), were isolated from an Indonesian octocoral Cladiella sp. The structures of eunicellins 1 and 2 were established by spectroscopic methods, and eunicellin 2 was found to be an enantiomer of the known eunicellin solenopodin C (3). Eunicellin 2 displayed inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. The previously reported structures of two eunicellin-based compounds, cladielloides A and B, are corrected in this study. PMID:21747739

Krempfielins Q and R, Two New Eunicellin-Based Diterpenoids from the Soft Coral Cladiella krempfi

PubMed Central

Tai, Chi-Jen; Chokkalingam, Uvarani; Cheng, Yang; Shih, Shou-Ping; Lu, Mei-Chin; Su, Jui-Hsin; Hwang, Tsong-Long; Sheu, Jyh-Horng

2014-01-01

Two new eunicellin-based diterpenoids, krempfielins Q and R (1 and 2), and one known compound cladieunicellin K (3) have been isolated from a Formosan soft coral Cladiella krempfi. The structures of these two new metabolites were elucidated by extensive spectroscopic analysis. Anti-inflammatory activity of new metabolites to inhibit the superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (FMLP/CB)-induced human neutrophil cells and cytotoxicity of both new compounds toward five cancer cell lines were reported. PMID:25437917

Krempfielins Q and R, two new eunicellin-based diterpenoids from the soft coral Cladiella krempfi.

PubMed

Tai, Chi-Jen; Chokkalingam, Uvarani; Cheng, Yang; Shih, Shou-Ping; Lu, Mei-Chin; Su, Jui-Hsin; Hwang, Tsong-Long; Sheu, Jyh-Horng

2014-11-27

Two new eunicellin-based diterpenoids, krempfielins Q and R (1 and 2), and one known compound cladieunicellin K (3) have been isolated from a Formosan soft coral Cladiella krempfi. The structures of these two new metabolites were elucidated by extensive spectroscopic analysis. Anti-inflammatory activity of new metabolites to inhibit the superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (FMLP/CB)-induced human neutrophil cells and cytotoxicity of both new compounds toward five cancer cell lines were reported.

Exocrine Dysfunction Correlates with Endocrinal Impairment of Pancreas in Type 2 Diabetes Mellitus.

PubMed

Prasanna Kumar, H R; Gowdappa, H Basavana; Hosmani, Tejashwi; Urs, Tejashri

2018-01-01

Diabetes mellitus (DM) is a chronic abnormal metabolic condition, which manifests elevated blood sugar level over a prolonged period. The pancreatic endocrine system generally gets affected during diabetes, but often abnormal exocrine functions are also manifested due to its proximity to the endocrine system. Fecal elastase-1 (FE-1) is found to be an ideal biomarker to reflect the exocrine insufficiency of the pancreas. The aim of this study was conducted to assess exocrine dysfunction of the pancreas in patients with type-2 DM (T2DM) by measuring FE levels and to associate the level of hyperglycemia with exocrine pancreatic dysfunction. A prospective, cross-sectional comparative study was conducted on both T2DM patients and healthy nondiabetic volunteers. FE-1 levels were measured using a commercial kit (Human Pancreatic Elastase ELISA BS 86-01 from Bioserv Diagnostics). Data analysis was performed based on the important statistical parameters such as mean, standard deviation, standard error, t -test-independent samples, and Chi-square test/cross tabulation using SPSS for Windows version 20.0. Statistically nonsignificant ( P = 0.5051) relationship between FE-1 deficiency and age was obtained, which implied age as a noncontributing factor toward exocrine pancreatic insufficiency among diabetic patients. Statistically significant correlation ( P = 0.003) between glycated hemoglobin and FE-1 levels was also noted. The association between retinopathy ( P = 0.001) and peripheral pulses ( P = 0.001) with FE-1 levels were found to be statistically significant. This study validates the benefit of FE-1 estimation, as a surrogate marker of exocrine pancreatic insufficiency, which remains unmanifest and subclinical.

Piperine ameliorates oxidative stress, inflammation and histological outcome in collagen induced arthritis.

PubMed

Umar, Sadiq; Golam Sarwar, Abu Hasnath Md; Umar, Khalid; Ahmad, Niyaz; Sajad, Mir; Ahmad, Sayeed; Katiyar, Chandra Kant; Khan, Haider A

2013-01-01

Piperine, a main component of Piper species, is a plant alkaloid with a long history of medical use in a variety of inflammatory disorders like rheumatoid arthritis. Due to side effects in current treatment modalities of rheumatoid arthritis, the interest in alternative, well tolerated anti-inflammatory remedies has re-emerged. The aim of this work was to evaluate the anti-inflammatory and antiarthritic effects of piperine. Arthritis was induced in male Wistar rats by collagen induced arthritis (CIA) method. Piperine was administered at a dose of 100mgkg(-1) and indomethacin at 1mgkg(-1) body weight once daily for 21days. The effects of treatment in the rats were assessed by biochemical (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO), inflammatory mediators (IL-1β, TNF-α, IL-10 and PGE2) and histological studies in joints. Piperine was effective in bringing significant changes on all the parameters (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO) studied. Oral administration of piperine resulted in significantly reduced the levels of pro-inflammatory mediators (IL-1β, TNF-α and PGE2) and increased level of IL-10. The protective effects of piperine against RA were also evident from the decrease in arthritis scoring and bone histology. In conclusion, the fact that piperine alter a number of factors known to be involved in RA pathogenesis indicates that piperine can be used similar to indomethacin as a safe and effective therapy for CIA and may be useful in the treatment of rheumatoid arthritis. Copyright © 2013 Elsevier Inc. All rights reserved.