This study examined the association between increased early oxidative stress, measured by F2-isoprostanes (IsoPs), and respiratory morbidity at term equivalent age and neurological impairment at 12 months of corrected age (CA). Plasma samples were collected from 136 premature infants on days 14 and 28 after birth. All participants were infants born at ⩽28 weeks of gestational age enrolled into the Prematurity and Respiratory Outcomes Program (PROP) study. Respiratory morbidity was determined at 40 weeks of postmenstrual age (PMA) by the Respiratory Severity Index (RSI), a composite measure of oxygen and pressure support. Neurodevelopmental assessment was performed using the Developmental Assessment of Young Children (DAYC) at 12 months of CA. Multivariable logistic regression models estimated associations between IsoP change, RSI and DAYC scores. Mediation analysis was performed to determine the relationship between IsoPs and later outcomes. Developmental data were available for 121 patients (90% of enrolled) at 12 months. For each 50-unit increase in IsoPs, regression modeling predicted decreases in cognitive, communication and motor scores of −1.9, −1.2 and −2.4 points, respectively (P<0.001). IsoP increase was also associated with increased RSI at 40 weeks of PMA (odds ratio=1.23; P=0.01). RSI mediated 25% of the IsoP effect on DAYC motor scores (P=0.02) and had no significant impact on cognitive or communication scores. In the first month after birth, increases in plasma IsoPs identify preterm infants at risk for respiratory morbidity at term equivalent age and worse developmental outcomes at 12 months of CA. Poor neurodevelopment is largely independent of respiratory morbidity.

This paper proposes a new algorithm to compute the residual risk of failure of an explosion protection system on an industrial process plant. A graph theoretic framework is used to model the process. Both the main reasons of failure are accounted for, viz hardware failure and inadequate protection even when the protection hardware functions according to specifications. The algorithm is shown to be both intuitive and Simple to implement in practice Its application is demonstrated with a realistic example of a protection system installation oil a spray drier. (C) 2009 Elsevier Ltd. All rights reserved

Process plant owners/operators have an obligation to assess and ascribe the residual risk of an unmitigated explosion occurrence under the ATEX Directives, and in doing so are making a key decision on the acceptability of that residual risk. All safety systems have a residual risk that they fail to achieve their mission, whether it be via hardware failures, personnel errors, errors in the theoretical and design assumptions, or inadequacies in the quantification of the prevailing hazard.It is essential that suppliers and users of explosion protection products and systems fully understand the efficacy and reliability upon demand of such products and systems. A systematic methodology for quantifying residual risk in the context of installed explosion mitigation has been described by the authors. This methodology explicitly accounts for the two principal mechanisms of failure:a) failure of the hardware;b) ineffective explosion protection (e.g. the reduced explosion pressure of a suppressed or vented explosion occurrence is greater than the pressure shock resistance of the vessel).This paper considers the challenges faced in determining a meaningful residual risk datum for a processing plant. In particular it sets out the importance of the implicit assumptions and shows, by reference to process industry examples, the benefits of electing a systematic means of ascribing explosion protection security.In order to quantify the residual risk of safety system failure in the practice, an overarching understanding of the efficacy of explosion mitigation means, system design, safety factors (both implicit and explicit) and the consequence of flame propagation between connected vessels, is of paramount importance. Existing explosion protection design guidance is invariably constructed around test data that have taken the premise that central ignition of a homogeneous and turbulent optimum fuel concentration in a closed vessel represents the worst case scenario. However this is not necessarily the most appropriate baseline for ascribing the risk of an unmitigated explosion occurrence.This work demonstrates that explosion protection "trade off" decisions, design safety factors, and the design premise itself all contribute to the "relied upon" safety integrity of an industrial process. With this understanding and the adoption of a systematic methodology to determine a residual risk datum, practitioners can make more informed and cost effective design decisions, leading to enhanced overall process safety.

Background: Eotaxin is implicated in asthmatic eosinophilia. Oncostatin M (OSM) causes eotaxin release from fibroblasts. Objective: We sought to examine the effects and mechanism of action of OSM and other IL-6 family cytokines on eotaxin release from human airway smooth muscle cells.Methods: Eotaxin 1 release was measured by means of ELISA. Western blotting was used to examine mitogen-activated protein kinase and signal transducer and activator of transcription 3 (STAT-3) phosphorylation. Eotaxin promoter activity was analyzed in cells transfected with wild-type STAT-3, a mutant form of STAT-3 that cannot be phosphorylated, and a constitutively active form of STAT-3. The mRNA and protein expression of IL-4R alpha, the signaling receptor for IL-4 and IL-13, was evaluated by means of real-time PCR and flow cytometry, respectively.Results: OSM increased eotaxin I release and augmented IL-4-or IL-13-induced eotaxin release, whereas other IL-6 family cytokines did not. OSM caused a greater increase in STAT-3 phosphorylation and STAT-3-mediated gene transcription than other IL-6 family cytokines. OSM increased eotaxin promoter activity and augmented IL-13- and IL-4-induced increases in promoter activity. The constitutively active form of STAT-3 increased eotaxin promoter activity, whereas the mutant form of STAT-3 that cannot be phosphorylated significantly reduced eotaxin promoter activity induced by OSM or IL-4 plus OSM. OSM increased IL-4R alpha mRNA and protein levels.Conclusions: OSM induces eotaxin 1 expression in human airway smooth muscle cells by a mechanism involving STAT-3. OSM synergizes with IL-13 and 11-4 to increase eotaxin I expression, possibly as a result of effects on IL-4Ra expression.

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of > or =30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-alpha. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 micro M), which inhibits ERK phosphorylation, abolished the synergism between TNF-alpha and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of > or =10 ng/ml caused an approximately 50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE(2) release and did not augment the COX-2 expression that was induced by IL-1beta. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.

The chemokine thymus- and activation-regulated chemokine (TARC) induces selective migration of Th2, but not Th1, lymphocytes and is upregulated in the airways of asthmatic patients. The purpose of this study was to determine whether human airway smooth muscle (HASM) cells produce TARC. Neither IL-4, IL-13, IL-1beta, IFN-gamma, nor TNF-alpha alone stimulated TARC release into the supernatant of cultured HASM cells. However, both IL-4 and IL-13 increased TARC protein and mRNA expression when administered in combination with TNF-alpha but not IL-1beta or IFN-gamma. Macrophage-derived chemokine was not expressed under any of these conditions. TARC release induced by TNF-alpha + IL-13 or TNF-alpha + IL-4 was inhibited by the beta-agonist isoproterenol and by other agents that activate protein kinase A, but not by dexamethasone. To determine whether polymorphisms of the IL-4Ralpha have an impact on the ability of IL-13 or IL-4 to induce TARC release, HASM cells from multiple donors were genotyped for the Ile50Val, Ser478Pro, and Gln551Arg polymorphisms of the IL-4Ralpha. Our data indicate that cells expressing the Val50/Pro478/ Arg551 haplotype had significantly greater IL-13- or IL-4-induced TARC release than cells with other IL-4Ralpha genotypes. These data indicate that Th2 cytokines enhance TARC expression in HASM cells in an IL-4Ralpha genotype-dependent fashion and suggest that airway smooth muscle cells participate in a positive feedback loop that promotes the recruitment of Th2 cells into asthmatic airways.

We examined the ability of the corticosteroid dexamethasone to inhibit short-term (1 h) and long-term (24 h) β2-adrenergic desensitization in cultured human airway smooth muscle (HASM) cells. In cells from 19 individuals studied on 47 experimental days, pretreatment with isoproterenol at 10−7 mol/L for 1 h or 24 h caused approximately 21% and 77% decreases in the ability of subsequent isoproterenol (10−6 mol/L) stimulation to reduce HASM cell stiffness as measured by magnetic twisting cytometry, similar to results we have previously reported. 1 Moore PE Laporte PD Abraham JH et al. Polymorphism of the β2-adrenergic receptor gene and desensitization in human airway smooth muscle. Am J Respir Crit Care Med. 2000; 162: 2117-2124 Crossref PubMed Scopus (90) Google Scholar We examined the ability of dexamethasone (10−6 mol/L) administered 6 h or 48 h prior to isoproterenol pretreatment, and stratified our results by presence or absence of the Arg19 allele of the β2AR upstream peptide, located in the 5′-leader cistron of the β2AR gene. This polymorphism has been associated with decreased β2AR expression in HASM cells. In cells containing at least one Arg19 allele, dexamethasone did not significantly reverse short-term or long-term desensitization, but in cells without the Arg19 allele, dexamethasone significantly reversed short-term and long-term desensitization (35% and 31%, respectively; p < 0.05 for each). Our results suggest that the Arg19 allele, or other polymorphisms of the promoter of the β2AR gene that are in linkage disequilibrium, may affect the ability of dexamethasone to upregulate β2AR expression in HASM cells. In addition, our results suggest that β2AR genotype may be predictive of corticosteroid responsiveness in individuals with asthma.

Human airway smooth muscle (HASM) cells express interleukin (IL)-13 and IL-4 receptors and respond to these cytokines with signal transducer and activator of transcription-6 and extracellular signal-regulated kinase (ERK) activation. The purpose of this study was to determine whether IL-13 and/or IL-4 influence eotaxin release in HASM cells and whether the ERK mitogen-activated protein (MAP) kinase pathway is involved in these events. Eotaxin release into HASM cell supernatants was assayed by ELISA, and eotaxin mRNA expression was determined by Northern blot analysis. Pretreatment with either IL-13 or IL-4 resulted in a concentration- and time-dependent release of eotaxin, although IL-4 was more effective. Eotaxin release was approximately twice baseline after treatment with 50 ng/ml IL-13 or IL-4 (P < 0.001). IL-13 and IL-4 also acted synergistically with tumor necrosis factor (TNF)-alpha to induce eotaxin release: TNF-alpha alone (10 ng/ml for 24 h) resulted in an approximately fourfold increase in eotaxin release, whereas TNF-alpha in combination with IL-13 or IL-4 resulted in 10- or 20-fold increases (P < 0.05). Similar results were obtained for eotaxin mRNA expression. Pretreatment with either U-0126 (10 microM) or PD-98059 (30 microM), both inhibitors of MAP/ERK kinase, the enzyme upstream of ERK, inhibited IL-13- or IL-4-induced eotaxin release (P < 0.05). U-0126 also inhibited IL-13, and TNF-alpha induced mRNA expression. Our results indicate that IL-13 and IL-4 cause eotaxin release in HASM cells through a mechanism that, in part, involves ERK activation and suggest that the smooth muscle may be an important source of chemokines leading to eosinophil recruitment in asthma.

Decreased responsiveness to beta-adrenergic receptor agonists is a characteristic feature of human asthma. This review summarizes data regarding the impact of chronic beta agonist stimulation, cytokines, prostanoids and other factors on beta-adrenergic responses in human airway smooth muscle, as well as the impact of polymorphisms of the beta(2)-adrenergic receptor on these responses. Effects of beta-agonists on both airway smooth muscle relaxation and gene expression are considered. Understanding the regulation of beta-adrenergic responses in airway smooth muscle cells may prove to be an important step in improving the efficacy of beta-agonists for the treatment of asthma.

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.

Numerous studies have suggested an important role for the Th2 cytokines interleukin (IL)-13 and IL-4 in the development of allergic asthma. We tested the hypothesis that IL-13 and IL-4 have direct effects on cultured airway smooth muscle cells (HASM). Using RT-PCR, we showed that HASM cells express transcripts for IL-4alpha, IL-13RalphaI, and IL-13RalphaII, but not for the common IL-2Rgamma chain. We then analyzed the capacity of the two cytokines to activate signaling pathways in HASM cells. Both IL-13 and IL-4 caused STAT-6 phosphorylation, but the time course was different between the two cytokines, with peak effects occurring 15 min after addition of IL-4 and 1 h after addition of IL-13. Effects on signaling were observed at cytokine concentrations as low as 0.3 ng/ml. IL-4 and IL-13 also caused phosphorylation of ERK MAP kinase. As suggested by the signaling studies, the biological responses of the two cytokines were also different. We used magnetic twisting cytometry to measure cell stiffness of HASM cells and tested the capacity of IL-4 and IL-13 to interfere with the reductions in cell stiffness induced by the beta-agonist isoproterenol (ISO). IL-13 (50 ng/ml for 24 h), but not IL-4, significantly reduced beta-adrenergic responsiveness of HASM cells, and the MEK inhibitor U0126 significantly reduced the effects of IL-13 on ISO-induced changes in cell stiffness. We propose that these direct effect of IL-13 on HASM cells may contribute at least in part to the airway narrowing observed in patients with asthma.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.

Leukotrienes play a critical role In promoting bronchoconstriction in asthma. The purpose of this study was to examine whether interferon (IFN)-gamma, a cytokine upregulated in asthmatic airways, modulates leukotriene (LT)D-4 receptor expression and contractile responses In cultured human airway smooth muscle (HASM) cells. Treatment of HASM cells with IFN-gamma (10 to 1,000 U/ml) stimulated a dose-dependent increase In cell-surface expression of cysteinyl leukotriene receptor 1 (CysLT(1)) as determined by flow cytometry, CysLT(1) messenger RNA (mRNA) levels were also significantly enhanced by IFN-gamma, as demonstrated by reverse transcription-polymerase chain reaction. To determine the functional relevance of increased CysLT(1) expression in HASM, cell stiffness responses to LTD4 were measured with magnetic twisting cytometry. IFN-gamma (1,000 U/ml for 24 h) markedly increased LTD4-induced changes In cell stiffness, from 4.6 +/-1 [mean +/- SEM]% to 24.4 +/-3.7% (n=8, p<0.05). Montelukast, a CysLT(1) antagonist, completely inhibited LTD4-induced increases In cell stiffness. IFN- had no effect on the cell stiffness responses to bradykinin, another contractile agonist. Collectively, these data suggest that IFN-gamma increases LTD4 responses in HASM cells by increasing cell-surface expression of CysLT(1). Our data suggest that increased levels of IFN-gamma in asthmatic Individuals may promote airway hyperresponsiveness and asthma exacerbations by directly modulating contractile responses of HASM.

In human cultured airway smooth muscle cells, interleukin (IL)-1 beta increases cyclooxygenase (COX)-2 expression and PGE(2) release, ultimately resulting in decreased beta -adrenergic responsiveness. In this study, we aimed to determine whether tumor necrosis factor-alpha (TNF-alpha) synergizes with IL-1 beta in the induction of these events. TNF-alpha alone, at concentrations up to 10 ng/ml, had no effect on COX-2 protein expression; at concentrations as low as 0.1 ng/ml, it significantly enhanced the ability of IL-1 beta (0.2 ng/ml) to induce COX-2 and to increase PGE(2) release. IL-1 beta and TNF-alpha in combination also significantly enhanced COX-2 promoter activity, indicating that synergism between the cytokines is mediated at the level of gene transcription. Although IL-1 beta and TNF-alpha each increased nuclear factor-kappaB activation and induced extracellular regulated kinase and p38 phosphorylation, combined administration of the cytokines did not enhance either nuclear factor-kappaB or mitogen-activated protein kinase activation. Combined administration of IL-1 beta (0.2 ng/ml) and TNF-alpha (0.1 or 1.0 ng/ml) reduced the ability of isoproterenol to decrease human airway smooth muscle cell stiffness, as measured by magnetic twisting cytometry, even though individually these cytokines, at these concentrations, had no effect on isoproterenol responses. Treatment with the selective COX-2 inhibitor NS-398 abolished the synergistic effects of TNF-alpha and IL-1 beta on beta -adrenergic responsiveness. Our results indicate that low concentrations of IL-1 beta and TNF-alpha synergize to promote beta -adrenergic hyporesponsiveness and that effects on COX-2 expression and PGE(2) are responsible for these events. The data suggest that the simultaneous release in the airway, of even very small amounts of cytokines, can have important functional consequences.

Interleukin (IL)-1beta induces cyclooxygenase (COX)-2 expression and prostanoid formation in cultured human airway smooth muscle (HASM) cells. In other cell types, IL-6 family cytokines induce COX-2 or augment IL-1beta-induced COX-2 expression. The purpose of this study was to determine whether IL-6 family cytokines were involved in COX-2 expression in HASM cells. RT-PCR was used to demonstrate that the necessary receptor components for IL-6-type cytokine binding are expressed in HASM cells. IL-6 and oncostatin M (OSM) each caused a dose-dependent phosphorylation of signal transducer and activator of transcription-3, whereas IL-11 did not. IL-6, IL-11, and OSM alone had no effect on COX-2 expression. However, OSM caused dose-dependent augmentation of COX-2 expression and prostaglandin (PG) E(2) release induced by IL-1beta. In contrast, IL-6 and IL-11 did not alter IL-1beta-induced COX-2 expression. IL-6 did increase IL-1beta-induced PGE(2) formation in unstimulated cells but not in cells stimulated with arachidonic acid (AA; 10(-5) M), suggesting that IL-6 effects were mediated at the level of AA release. Our results indicate that IL-6 and OSM are capable of inducing signaling in HASM cells. In addition, OSM and IL-1beta synergistically cause COX-2 expression and PGE(2) release.