| 1. |
- Janciauskiene, Sabina, et al.
(författare)
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{alpha}1-antitrypsin Inhibits the Activity of the Matriptase Catalytic Domain in vitro.
- 2008
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Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1535-4989. ; 39:6, s. 631-637
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Tidskriftsartikel (refereegranskat)abstract
- Matriptase is a type II transmembrane protease which is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of alpha1-antitrypsin with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate (SDS) electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x10(3) M(-1)s(-1). Notably, the oxidised form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiological processes including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.
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| 2. |
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| 3. |
- Lazrak, Ahmed, et al.
(författare)
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alpha(1)-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo
- 2009
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Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1535-4989. ; 41:3, s. 261-270
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Tidskriftsartikel (refereegranskat)abstract
- A variety of studies have shown that Na+ reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which alpha(1)-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na+ channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 mu M) decreased ENaC currents across Xenopus laevis oocytes injected with human alpha, beta, gamma-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. MAT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO-), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO--treated A1AT (1 mu M) in C57BL/6 mice also decreased Na+-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO--treated MAT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that MAT may be an important modulator of ENaC activity and of Na+-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.
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| 4. |
- Persson, Caroline, et al.
(författare)
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Do native and polymeric alpha1-antitrypsin activate human neutrophils in vitro?
- 2006
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Ingår i: Chest. - 1931-3543. ; 129:6, s. 1683-1692
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Tidskriftsartikel (refereegranskat)abstract
- Background: {alpha}1-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. Methods and results: The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (≤ 0.08 EU/mg protein) stimulated neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (≥ 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. Conclusions: The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.
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| 5. |
- Subramaniyam, Devipriya, et al.
(författare)
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C-36 peptide, a degradation product of alpha1-antitrypsin, modulates human monocyte activation through LPS signaling pathways.
- 2006
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Ingår i: International Journal of Biochemistry & Cell Biology. - : Elsevier BV. - 1878-5875 .- 1357-2725. ; 38:4, s. 563-575
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Tidskriftsartikel (refereegranskat)abstract
- alpha 1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products With novel biological activity. One Such molecule, the C-terminal fragment of AAT, corresponding to residues 359-394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LIPS), albeit with lower magnitude, by inducing monocyte cytokine (TNF alpha, IL-I beta) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-kappa B (NF-kappa B). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNF alpha release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit M/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways. (c) 2005 Elsevier Ltd. All rights reserved.
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| 6. |
- Subramaniyam, Devipriya, et al.
(författare)
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Effects of alpha 1-antitrypsin on endotoxin-induced lung inflammation in vivo
- 2010
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Ingår i: Inflammation Research. - : Springer Science and Business Media LLC. - 1420-908X .- 1023-3830. ; 59:7, s. 571-578
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Tidskriftsartikel (refereegranskat)abstract
- Previous in vitro experiments demonstrated that acute-phase protein, alpha 1-antitrypsin (AAT), could act either as an enhancer or as a suppressor of lipopolysaccharide (LPS)-induced cell activation depending on treatment time. Here we investigate how AAT regulates inflammatory responses in the short term when administrated post LPS challenge. Similar experimental setup was used both in vitro and in vivo: human monocytes and neutrophils were stimulated with LPS for 2 h followed by AAT for a total time of 4 h, and C57BL/6 mice were treated intranasally with LPS and 2 h later with AAT and sacrificed after 4 h. Bronchial lavage (BAL) and lung homogenates were analyzed using bio-plex cytokine assay. BAL cell counts were assessed. Within 4 h, AAT enhanced LPS-induced tumor necrosis factor-alpha (TNF alpha), interleukin (IL)-6, and IL-8 release from monocytes and neutrophils. Mice challenged for 4 h with LPS followed by AAT at 2 h showed no changes in BAL cell counts and higher levels of almost all measured cytokines, specifically RANTES in BAL and IL-12, IL-13, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-10 levels in lung homogenates, than in mice treated with LPS only. Within the short term, AAT enhances the magnitude of LPS-induced specific cytokine/chemokine production, which may play an important role in amplification and resolution of acute-phase inflammatory reactions in vivo.
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| 7. |
- Subramaniyam, Devipriya, et al.
(författare)
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Secretory Leukocyte Protease Inhibitor inhibits neutrophil apoptosis.
- 2011
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Ingår i: Respirology. - : Wiley. - 1440-1843 .- 1323-7799. ; Dec, s. 300-307
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Tidskriftsartikel (refereegranskat)abstract
- SUMMARY AT A GLANCE: Secretory leukocyte proteinase inhibitor (SLPI) is a major anti-elastase barrier at the epithelial surfaces of upper respiratory tract. Our findings indicate clear up-regulation of SLPI in response to endotoxin in nasal secretions. In addition, SLPI shows dose-dependent anti-apoptotic and chemotactic effects on primary human neutrophils. ABSTRACT: Background and objective: The Secretory Leukocyte Protease Inhibitor (SLPI) is a major anti-elastase barrier at the epithelial surfaces of upper respiratory tract. In addition to its anti-protease activity, SLPI has been shown to express anti-bacterial, anti-viral and anti-inflammatory properties. Methods: We measured SLPI concentration in nasal lavage fluid of healthy volunteers after challenge with endotoxin (LPS) and evaluated SLPI effects in vitro on neutrophil chemotaxis, adhesion, cytokine (IL-8) release and apoptosis. Results: SLPI concentration in nasal lavage (n = 9) 2, 6 and 24 hrs after the challenge with LPS (25 µg) increased from 32% to 238% compared to baseline (226 ± 71 ng/ml). In vitro, SLPI (20 to 80µg/ml) induced neutrophil chemotaxis (6-fold, p < 0.001) and decreased neutrophil apoptosis by 73% (p = 0.006), relative to controls. However, SLPI had no affect on IL-8 release or neutrophil adhesion to fibronectin. SLPI-positive immunoreactivity was co-localised with neutrophils in lung specimens from patients with chronic obstructive pulmonary disease. Conclusions: Our findings indicate up-regulation of SLPI in response to LPS in nasal secretions and show anti-apoptotic effects of SLPI in primary human neutrophils suggesting a new role of SLPI during neutrophilic inflammation.
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| 8. |
- Subramaniyam, Devipriya, et al.
(författare)
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TNF-alpha-induced self expression in human lung endothelial cells is inhibited by native and oxidized alpha 1-antitrypsin
- 2008
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Ingår i: International Journal of Biochemistry & Cell Biology. - : Elsevier BV. - 1878-5875 .- 1357-2725. ; 40:2, s. 258-271
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Tidskriftsartikel (refereegranskat)abstract
- Endothelial cells are among the main physiological targets of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In endothelial cells TNF-alpha elicits a broad spectrum of biological effects including differentiation, proliferation and apoptosis. alpha 1-antitrypsin (AAT), an endogenous inhibitor of serine proteases plays a vital role in protecting host tissue from proteolytic injury at sites of inflammation. Recently, it has been shown that AAT can be internalized by pulmonary endothelial cells, raising speculation that it may modulate endothelial cell function in addition to suppressing protease activity. Using Affymetrix microarray technology, real time PCR and ELISA methods we have investigated the effects of AAT on un-stimulated and TNF-alpha stimulated human primary lung microvascular endothelial cell gene expression and protein secretion. We find that AAT and TNF-alpha generally induced expression of distinct gene families with AAT exhibiting little activity in terms of inflammatory gene expression. Approximately 25% of genes up regulated by TNF-alpha were inhibited by co-administration of AAT including TNF-alpha-induced self expression. Surprisingly, the effects of AAT on TNF-alpha-induced self expression was inhibited equally well by oxidized AAT, a modified form of AAT, which lacks serine protease inhibitor activity. Overall, the pattern of gene expression regulated by native and oxidized AAT was similar with neither inducing pro-inflammatory gene expression. These findings suggest that inhibitory effects of native and oxidized forms of AAT on TNF-alpha stimulated gene expression may play an important role in limiting the uncontrolled endothelial cell activation and vascular injury in inflammatory disease.
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