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Oropharyngeal dysphagia is characterized by impairments in airway protection and/or bolus clearance. When airway protection is inadequate, material such as food or liquid may be penetrated into the laryngeal vestibule [...], Purpose: Research remains equivocal regarding the links between hyoid movement and penetration-aspiration. The aims of this study were (a) to explore associations between hyoid parameters, laryngeal vestibule closure (LVC) parameters, and penetration-aspiration on thin liquids; and (b) to determine which of these parameters are the strongest predictors of penetration-aspiration. Method: This study involved retrospective analysis of an existing videofluoroscopy data set, collected in 305 participants (152 males) with noncongenital/ nonsurgical/non-oncological risk for dysphagia. We extracted data for six thin liquid swallows per participant, and obtained measures of hyoid movement (peak position, speed) and LVC (complete/incomplete, timing, duration). Resulting values were coded as typical/atypical relative to healthy reference data. Relationships were explored using chi-square tests and odds ratios (a) for the entire data set and (b) for the subset of data with complete LVC. Hierarchical logistic regression models determined the strongest predictors of penetration-aspiration. Results: Significant associations were found between penetration-aspiration and incomplete LVC, prolonged time-to-most-complete-LVC, short LVC duration, reduced anterior hyoid peak position, and reduced hyoid speed. Hyoid measures were also significantly associated with LVC parameters. In the first regression model, incomplete LVC and prolonged time-to-most-complete-LVC were the only significant predictors of penetration-aspiration. For cases with complete LVC, the only significant predictor was prolonged time-to-most-complete-LVC. Conclusions: Although reduced anterior hyoid peak position and speed are associated with penetration-aspiration on thin liquids, these measures do not independently account for penetration-aspiration when considered in conjunction with measures of LVC. When identifying mechanisms explaining penetration-aspiration, clinicians should focus on LVc (complete/incomplete) and timeliness of LVc.

Author(s): Swapna Naik [1]; Sujit Saraf [1]; Poonam Ghodki (corresponding author) [1]; Sunil Bapat [1] INTRODUCTION Laryngology is a unique speciality that deals with the diseases related to airway, voice [...], Background and Aims: Anaesthesia for endolaryngeal surgery is specialised to provide almost a tubeless surgical field. During the coronavirus disease-19 pandemic, when most of the surgeries were staggered, we being in a tertiary referral centre for airway surgery had to modify our existing techniques and observed an evolution in the anaesthesia management which we could continue even in the postpandemic period. Hence, we conducted this retrospective study to analyse the reliability of our locally developed apnoeic high-flow oxygenation technique (AHFO) for endolaryngeal procedures. Methods: We conducted this single-centric retrospective study from January 2020 to August 2021 to observe the choice of airway management techniques in endolaryngeal surgery and assess the feasibility and safety of AHFO. We also intend to propose an algorithm for airway management. We calculated the percentages of all necessary parameters to denote the trend in change of practices roughly classifying the study period as prepandemic, pandemic and postpandemic. Results: A total of 413 patients were analysed in our study. The changing trend over preference of AHFO from prepandemic (72) and dominance of AHFO (92.5) in the postpandemic period are the most significant observations of our study with 17 patients needing conversion to tube in-tube out technique due to desaturation which is comparable to 14 in prepandemic period. Conclusion: The tubeless field provided by AHFO replaced the conventional airway management techniques. Our study demonstrates the safety and feasibility of AHFO for endolaryngeal surgeries. We also propose an algorithm for anaesthetists involved in laryngology unit. Keywords: Apnoeic ventilation, endolaryngeal surgery, high flow, pandemic

Introduction The regulation of the mechanical properties of smooth muscle, such as the ability to generate force and maintain tensile stiffness, is fundamental to the proper functioning of the muscle. [...], Airway smooth muscle (ASM) plays a central role in the excessive narrowing of the airway that characterizes the primary functional impairment in asthma. This phenomenon is known as airway hyper-responsiveness (AHR). Emerging evidence suggests that the development and maintenance of ASM force involves dynamic reorganization of the subcellular filament network in both the cytoskeleton and the contractile apparatus. In this review, evidence is presented to support the view that regulation of ASM contraction extends beyond the classical actomyosin interaction and involves processes within the cytoskeleton and at the interfaces between the cytoskeleton, the contractile apparatus, and the extracellular matrix. These processes are initiated when the muscle is activated, and collectively they cause the cytoskeleton and the contractile apparatus to undergo structural transformation, resulting in a more connected and solid state that allows force generated by the contractile apparatus to be transmitted to the extracellular domain. Solidification of the cytoskeleton also serves to stiffen the muscle and hence the airway. Oscillatory strain from tidal breathing and deep inspiration is believed to be the counter balance that prevents hypercontraction and stiffening of ASM in vivo. Dysregulation of this balance could lead to AHR seen in asthma. Key words: asthma, Rho-kinase, cytoskeleton, cross-bridge, length adaptation, force adaptation. Le muscle lisse respiratoire (MLR) joue un role central dans le retrecissement excessif des voies respiratoires qui caracterise la principale deterioration fonctionnelle associee a l'asthme. Ce phenomene est connu sous le nom d'hyperreactivite bronchique (HRB). Une donnee nouvelle suggere que le developpement et le maintien de la force du MLR implique la reorganization dynamique du reseau de filaments subcellulaires du cytosquelette et de l'appareil contractile. Dans cet article de revue, les auteurs presentent une preuve qui appuie la vision que la contraction du MLR va au-dela de l'interaction classique de l'actomyosine et implique des processus a l'interieur du cytosquelette et a l'interface entre le cytosquelette, l'appareil contractile et la matrice extracellulaire. Ces processus sont inities lorsque le muscle est active, et ils provoquent collectivement une transformation structurale du cytosquelette et de l'appareil contractile resultant en un etat mieux connecte et solide qui permet a la force generee par l'appareil contractile de se transmettre au domaine extracellulaire. La solidification du cytosquelette sert aussi a durcir le muscle et, consequemment, les voies respiratoires. On croit que la pression oscillatoire de la respiration courante et de l'inspiration profonde constitue la contrebalance qui previent l'hyper-contraction et le durcissement du MLR in vivo. La deregulation de cette balance pourrait conduire a l'HRB observee dans l'asthme. [Traduit par la Redaction] Mots-cles: asthme, Rho-kinase, cytosquelette, ponts transversaux, adaptation de la longueur, adaptation de la force.

Introduction Asthma affects more than 20 million people in the USA (~10% of the population), and more than 300 million people world-wide (GINA 2012). It is now recognized that inflammation [...], Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.cyt]) responses to agonist stimulation and [Ca.sup.2+] sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [[[Ca.sup.2+]].sub.cyt] induced by agonists leads to a transient increase in mitochondrial [Ca.sup.2+] ([[[Ca.sup.2+]].sub.mito]) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [[[Ca.sup.2+]].sub.mito] is blunted despite enhanced [[[Ca.sup.2+]].sub.cyt] responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial [Ca.sup.2+] buffering, mitochondrial fragmentation, and increased cell proliferation. Key words: inflammation, ER/SR stress, airway, asthma, unfolded protein response. L'inflammation de voies respiratoires est un aspect cle de maladies comme l'asthme. Plusieurs cytokines inflammatoires (ex. TNFα et IL-13) augmentent le [Ca.sup.2+] cytosolique ([[[Ca.sup.2+]].sub.cyt]) en reponse a une stimulation par un agoniste et la sensibilite de la generation de forces au calcium, augmentant ainsi la contractilite du muscle lisse respiratoire (MLR) (etat d'hyperreactivite). L'inflammation induit aussi la proliferation et le remodelage des cellules du MLR (etat de synthese). Chez le MLR, l'elevation transitoire de [[[Ca.sup.2+]].sub.cyt] induite par un agoniste mene a une augmentation transitoire du [Ca.sup.2+] mitochondrial ([[[Ca.sup.2+]].sub.mito]) qui peut etre importante pour arrimer la production d'ATP a sa consommation. Chez les cellules de MLR humain (hMLR) exposees au TNFα et a l'IL-13, l'augmentation transitoire de [[[Ca.sup.2+]].sub.mito] est attenuee malgre l'augmentation de [[[Ca.sup.2+]].sub.cyt]. Les auteurs trouvent aussi que le TNFα et l'IL-13 induisent la formation d'especes reactives d'oxygene (ERO) et un stress du reticulum endoplasmique/ sarcoplasmique (RE/RS) (reponse aux proteines depliees) chez les hMLR. Le stress RE/RS chez les hMLR est associe au decouplage de la mitochondrie a la membrane du RE/RS, phenomene relie a la reduction de l'expression de la mitofusine 2 (Mfn2). Ainsi, chez les hMLR, il semble que le TNFα et l'IL-13 induisent la formation d'ERO conduisant a un stress du RE/RS, a la reduction de l'expression de Mfn2, au decouplage de la mitochondrie au RE/RS, a la diminution du tamponnage du [Ca.sup.2+] mitochondrial, a la fragmentation mitochondriale et a l'augmentation de la proliferation cellulaire. [Traduit par la Redaction] Mots-cles: inflammation, stress du RE/RS, voies respiratoires, asthme, reponse aux proteines depliees.

Introduction The crucial step for the initiation of the ATP-dependent actomyosin interaction in smooth muscle is the phosphorylation of the myosin light chain 20 (MLC20) (Frearson et al. 1976; Sobieszek [...], The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10-6 mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate. Key words: urea-glycerol gel electrophoresis, MLC20 phosphorylation, supernatant and pellet, Western blots. La methode standard pour mesurer la phosphorylation de la chaine legere de la myosine regulatrice (MLC20) dans le muscle lisse consiste en une extraction de la chaine legere dans un tampon d'extraction comprenant de l'uree, une electrophorese sur gel d'uree-glycerol de la portion soluble de l'extrait (surnageant) et d'un buvardage Western. La portion tissulaire non dissoute lors de l'extraction (culot) est habituellement jetee. Parce que le culot contient une faible quantite de MLC20, l'omission du culot pourrait resulter en une mesure imprecise de la phosphorylation de la MLC20. Dans cette etude, les auteurs ont compare le niveau de phosphorylation de MLC20 du muscle lisse tracheal dans le surnageant seul a celui de l'homogenat tissulaire complet (surnageant et culot) a l'aide de la methode standard. La fraction du surnageant montrait les doubles bandes bien connues representant la MLC20 phosphorylee et non phosphorylee. La fraction dissoute du culot contenait des quantites variables de MLC20 phosphorylee et non phosphorylee. Il y avait une legere mais significative surestimation du pourcentage de MLC20 phosphorylee si le culot n'etait pas considere. La surestimation etait de7%±2% (moyenne ± ESM) (p < 0,05) dans le muscle non stimule et de 2%[+ or -]1%(p < 0,05) dans le muscle stimule a l'acetylcholine (10-6 mol/L). Ces donnees suggerent que l'estimation precise de la strechiometrie de la phosphorylation de la MLC20 necessite de considerer la contribution de la portion du culot issue de l'homogenat de tissu musculaire. [Traduit par la Redaction] Mots-cles: electrophorese sur gel uree-glycerol, phosphorylation de la MLC20, surnageant et culot, buvardages Western.

Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine [...]

During lung development, parabronchial SMC (PSMC) progenitors in the distal mesenchyme secrete fibro-blast growth factor 10 (Fgf10), which acts on distal epithelial progenitors to promote their proliferation. β-catenin signaling within [...]

Individuals with chronic asthma show evidence of structural remodeling of the airways, including accumulation of extracellular matrix proteins such as collagen and thickening of smooth muscle. Current therapies for asthma [...], Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

We present a systematic quantitative analysis of power-law force relaxation and investigate logarithmic superposition of force response in relaxed porcine airway smooth muscle (ASM) strips in vitro. The term logarithmic superposition describes linear superposition on a logarithmic scale, which is equivalent to multiplication on a linear scale. Additionally, we examine whether the dynamic response of contracted and relaxed muscles is dominated by cross-bridge cycling or passive dynamics. The study shows the following main findings. For relaxed ASM, the force response to length steps of varying amplitude (0.25-4% of reference length, both lengthening and shortening) are well-fitted with power-law functions over several decades of time ([10.sup.-2] to [10.sup.3] s), and the force response after consecutive length changes is more accurately fitted assuming logarithmic superposition rather than linear superposition. Furthermore, for sinusoidal length oscillations in contracted and relaxed muscles, increasing the oscillation amplitude induces greater hysteresivity and asymmetry of force-length relationships, whereas increasing the frequency dampens hysteresivity but increases asymmetry. We conclude that logarithmic superposition is an important feature of relaxed ASM, which may facilitate a more accurate prediction of force responses in the continuous dynamic environment of the respiratory system. In addition, the single power-function response to length changes shows that the dynamics of cross-bridge cycling can be ignored in relaxed muscle. The similarity in response between relaxed and contracted states implies that the investigated passive dynamics play an important role in both states and should be taken into account. smooth muscle dynamics; power-law force relaxation doi: 10.1152/ajplung.00023.2010.

Allogeneic human mesenchymal stem cells (hMSCs) introduced intravenously can have profound anti-inflammatory activity resulting in suppression of graft vs. host disease as well as regenerative events in the case of stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of these diseases, hMSCs produce bioactive factors that provide molecular cuing for: 1) immunosuppression of T cells; 2) antiscarring; 3) angiogenesis; 4) antiapoptosis; and 5) regeneration (i.e., mitotic for host-derived progenitor cells). Studies have shown that hMSCs have profound effects on the immune system and are well-tolerated and therapeutically active in immunocompetent rodent models of multiple sclerosis and stroke. Furthermore, intravenous administration of MSCs results in pulmonary localization. Asthma is a major debilitating pulmonary disease that impacts in excess of 150 million people in the world with uncontrolled asthma potentially leading to death. In addition, the socioeconomic impact of asthma-associated illnesses at the pediatric and adult level are in the millions of dollars in healthcare costs and lost days of work. hMSCs may provide a viable multiaction therapeutic for this inflammatory lung disease by secreting bioactive factors or directing cellular activity. Our studies show the effectiveness and specificity of the hMSCs on decreasing chronic airway inflammation associated with the murine ovalbumin model of asthma. In addition, the results from these studies verify the in vivo immunoeffectiveness of hMSCs in rodents and support the potential therapeutic use of hMSCs for the treatment of airway inflammation associated with chronic asthma. models of asthma; stem cell therapy doi: 10.1152/ajplung.00182.2009.

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidohate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using [16HBE14o.sup.-] sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using [cftr.sup.-/-] transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, [PGE.sub.2], and [PGF.sub.2[alpha]] secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation, [cftr.sup.-/-] Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators. fatty acid metabolism; prostaglandins; inflammation doi: 10.1152/ajplung.00346.2009.

It is widely accepted that atopic asthma depends on an allergic response in the airway, yet the immune mechanisms that underlie the development of airway hyperresponsiveness (AHR) are poorly understood. Mouse models of asthma have been developed to study the pathobiology of this disease, but there is considerable strain variation in the induction of allergic disease and AHR. The aim of this study was to compare the development of AHR in BALB/c, 129/Sv, and C57BL/6 mice after sensitization and challenge with ovalbumin (OVA). AHR to methacholine was measured using a modification of the forced oscillation technique in anesthetized, tracheostomized mice to distinguish between airway and parenchymal responses. Whereas all strains showed signs of allergic sensitization, BALB/c was the only strain to develop AHR, which was associated with the highest number of activated ([CD69.sup.+]) [CD4.sup.+] T cells in the airway wall and the highest levels of circulating OVA-specific [IgG.sub.1]. AHR did not correlate with total or antigen-specific IgE. We assessed the relative contribution of [CD4.sup.+] T cells and specific [IgG.sub.1] to the development of AHR in BALB/c mice using adoptive transfer of OVA-specific [CD4.sup.+] T cells from DO11.10 mice. AHR developed in these mice in a progressive fashion following multiple OVA challenges. There was no evidence that antigen-specific antibody had a synergistic effect in this model, and we concluded that the number of antigen-specific T cells activated and recruited to the airway wall was crucial for development of AHR. mouse models of asthma; forced oscillation technique; BALB/c; 129/Sv; C57BL/6

Airway smooth muscle (ASM) is cyclically stretched during breathing, even in the active state, yet the factors determining its dynamic force-length behavior remain incompletely understood. We developed a model of the activated ASM strip and compared its behavior to that observed in strips of rat trachealis muscle stimulated with methacholine. The model consists of a nonlinear viscoelastic element (Kelvin body) in series with a force generator obeying the Hill force-velocity relationship. Isometric force in the model is proportional to the number of bound crossbridges, the attachment of which follows first-order kinetics. Crossbridges detach at a rate proportional to the rate of change of muscle length. The model accurately accounts for the experimentally observed transient and steady-state oscillatory force-length behavior of both passive and activated ASM. However, the model does not predict the sustained decrement in isometric force seen when activated strips of ASM are subjected briefly to large stretches. We speculate that this force decrement reflects some mechanism unrelated to the cycling of crossbridges, and which may be involved in the reversal of bronchoconstriction induced by a deep inflation of the lungs in vivo. crossbridge kinetics; viscoelasticity; deep lung inflation; computational model; rat trachealis; deep inspiration

The relative contribution of inositol 1,4,5-trisphosphate ([IP.sub.3]) receptors ([IP.sub.3]Rs) and ryanodine receptors (RyRs) to agonist-induced [Ca.sup.2+] signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phasecontrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptarnine (5-HT) induced [Ca.sup.2+] oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an [IP.sub.3]R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the [Ca.sup.2+] oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited [Ca.sup.2+] waves generated by the photolytic release of [IP.sub.3]. However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or [IP.sub.3]-induced [Ca.sup.2+] oscillations or [Ca.sup.2+] wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced [Ca.sup.2+] oscillations in a concentration-dependent manner. However, tetracaine did not affect [IP.sub.3]-induced [Ca.sup.2+] release or wave propagation nor the [Ca.sup.2+] content of SMC [Ca.sup.2+] stores as evaluated by [Ca.sup.2+]-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow [Ca.sup.2+] oscillations induced by KC1. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or [Ca.sup.2+] oscillations of SMCs were also observed at 37[degrees]C. In [Ca.sup.2+]-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular [Ca.sup.2+] levels indicating that relaxation also resulted from a reduction in [Ca.sup.2+] sensitivity. These results indicate that agonist-induced [Ca.sup.2+] oscillations in mouse small airway SMCs are primary mediated via [IP.sub.3]Rs and that tetracaine induces relaxation by both decreasing [Ca.sup.2+]sensitivity and inhibiting agonist-induced [Ca.sup.2+] oscillations via an [IP.sub.3]-dependent mechanism. lung slices; [Ca.sup.2+]-induced [Ca.sup.2+] release; membrane depolarization; tetracaine

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia. To investigate the paracellular pathway, we used primary cultures of differentiated human airway epithelia and assessed expression of claudins, the primary determinants of paracellular permeability, and measured transepithelial electrical properties, ion fluxes, and [La.sup.3+] movement. Like many other tissues, airway epithelia expressed multiple claudins. Moreover, different cell types in the epithelium expressed the same pattern of claudins. To evaluate tight junction regulation, we examined the response to histamine, an acute regulator of airway function. Histamine stimulated a rapid and transient increase in the paracellular [Na.sup.+] conductance, with a smaller increase in [CI.sup.-] conductance. The increase was mediated by histamine H1 receptors and depended on an increase in intracellular [Ca.sup.2+] concentration. These results suggest that ion flow through the paracellular pathway can be acutely regulated. Such regulation could facilitate coupling of the passive flow of counter ions to active transcellular transport, thereby controlling net transepithelial salt and water transport. claudin | histamine | paracellular pathway

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces ''mucus' with a protein composition similar to that of in vivo. induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products. mucus; mucin; innate immunity; proteomics; human tracheobronchial epithelial cell culture

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a [Cl.sup.-] channel. Its dysfunction limits [Cl.sup.-] secretion and enhances [Na.sup.+] absorption, leading to viscous mucus in the airway. [Ca.sup.2+]-activated [Cl.sup.-] channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic [Ca.sup.2+] activate the epithelial CaCCs, which provides an alternative [Cl.sup.-] secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic [Ca.sup.2+], activate the CaCC, and increase [Cl.sup.-] secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular [Ca.sup.2+] enhancer and demonstrated that it stimulates intracellular [Ca.sup.2+], not by acting in its well-known role as an antagonist of serotonin [5-HT.sub.2] or dopamine [D.sub.2] receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates [Cl.sup.-] secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective [Cl.sup.-] secretion in CF via a pathway independent of CFTR. cystic fibrosis therapy; calcium-activated chloride channel

Regulation of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]]i) in airway smooth muscle (ASM) during agonist stimulation involves .sub.sarcoplasmic reticulum (SR) [Ca.sup.2+] release and reuptake. The sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) is key to replenishment of SR [Ca.sup.2+] stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CAM)CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the [Ca.sup.2+] indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [[[Ca.sup.2+]].sub.i] responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [[[Ca.sup.2+]].sub.i] transients to 1 [micro]M ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [[[Ca.sup.2+]].sub.i] oscillations (which were previously shown to result from repetitive SR [Ca.sup.2+] release/reuptake). However, when ACh-induced [[[Ca.sup.2+]].sub.i] oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [[[Ca.sup.2+]].sub.i] transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [[[Ca.sup.2+]].sub.i] oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR [Ca.sup.2+] release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR [Ca.sup.2+] reuptake, potentially through altered PLN phosphorylation. sarco(endo)plasmic reticulum calcium-ATPase; phospholamban; calmodulin; calmodulin kinase

Proteoglycans (PG) are altered in the asthmatic airway wall. Because PGs are known to affect cell proliferation and apoptosis, we hypothesized that alterations in PG might influence the airway smooth muscle (ASM) hyperplasia observed in the asthmatic airway. Human ASM cells were seeded on plastic or plates coated with decorin (Dcn), biglycan (Bgn), or collagen type I (Col I) (1, 3, and 10 [micro]g/ml). Cells were stimulated with platelet-derived growth factor (PDGF), and cell number was assessed at 0, 48, and 96 h. Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation and apoptosis by annexin V and propidium iodide staining at 48 h post-PDGF stimulation. A significant decrease in cell number was observed with cells seeded on Dcn (10 [micro]g/ml) at 0, 48, and 96 h (P < 0.01). Dcn induced both decreases in BrdU incorporation and increases in annexin V staining (P < 0.05). Bgn decreased cell number at time 0 only (P < 0.05) and affected neither proliferation nor apoptosis. Col I (10 [micro]g/ml) caused a significant increase in cell number at 48 and 96 h (P < 0.01). Adding exogenous Dcn (1-30 [micro]g/ml) to the medium had no effect on cell number. Exposing Dcn-coated matrices to chondroitinase ABC, an enzyme that degrades glycosaminoglycan side chains, reversed the Dcn-induced decrease in cell number. These studies demonstrate that different PGs have variable effects on ASM cell proliferation and apoptosis. Recently described decreases in Dcn in the asthmatic airway wall could potentially permit more exuberant ASM growth. decorin; biglycan; collagen; asthma; airway remodeling

The signal transducers and activators of transcription (STAT) family of transcription factors regulates a variety of biological functions including cellular proliferation, transformation, apoptosis, and differentiation. We have previously determined that PDGF activates the STAT pathway in human airway smooth muscle cells (HASMC) and that the Jak and Src kinases are required for both PDGF-induced STAT activation and HASMC proliferation. As increased airway smooth muscle (ASM) volume is associated with airflow obstruction and disease severity in patients with asthma, it is important to elucidate the cellular and molecular pathways that regulate ASM accumulation. In this paper, we investigated the requirement of STAT3 for PDGFinduced HASMC proliferation. We demonstrate that knockdown of STAT3 expression in HASMC resulted in a significant decrease in mitogen-induced cellular proliferation. Additionally, PDGF-induced activation of STAT3 required the small GTP-binding protein Rac1, and Rac1 was also required for PDGF-induced HASMC proliferation. Furthermore, PDGF treatment induced STAT3 and Rac1 to physically associate and translocate to the nucleus, identifying one mechanism by which STAT3 is regulated in response to PDGF in HASMC. Finally, we determined that STAT3 expression is required for PDGF-mediated regulation of cell cycle targets cyclin D3 and p27. These data define a novel mitogenic signaling pathway in airway smooth muscle cells leading from PDGF to Rac1 and STAT3 and subsequent cell cycle gene regulation. Thus, targeting STAT3 may prove to be a novel therapeutic approach for patients with severe asthma and significant airway wall remodeling, as manifested by ASM accumulation. asthma; airway remodeling; human airway smooth muscle cells

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component ([p47.sup.phox]) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in [p47.sup.phox-/-] than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in [p47.sup.phox-/-] compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 [+ or -] 2.4 and 9.2 [+ or -] 2.3% in null and wild-type mice (P < 0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the [p47.sup.phox] subunit of NAD(P)H oxidase plays a role in ASM relaxation. airways; airway responsiveness; contractility; reactive oxygen species

Airway smooth muscle (ASM) cells are characterized by phenotypic plasticity and can switch between differentiated and proliferative phenotypes. In rabbit tracheal ASM cells that had been differentiated in vitro by serum starvation, readdition of FBS caused initiation of proliferation and induction of nuclear and transcriptionally active hypoxia-inducible factor (HIF)-1[alpha]. In addition, FBS stimulated the induction of HIF-1[alpha] by the hypoxia mimetic cobalt. Treatment with actinomycin D, cycloheximide, the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin or the reactive oxygen species scavenger diphenyleneiodonium inhibited the FBS-dependent induction of HIF-1[alpha]. These data indicate that, in differentiated ASM cells, FBS upregulates HIF-1[alpha] by a transcription-, translation-, phosphatidylinositol 3-kinase-, and reactive oxygen species-dependent mechanism. Interestingly, addition of FBS and cobalt also induced HIF-l[alpha] in organ cultures of rabbit trachea strips and synergistically increased their contractile response to ACh, suggesting that HIF-1[alpha] might be implicated in airway hypercontractility. cobalt; fetal bovine serum

5-HT2 receptor activity in the hypoglossal nucleus and hypercapnia is associated with airway dilation. 5-HT neurons in the medullary raphe and hypercapnia are responsible for tidal volume change. In this study, the effects of 5-HT2 receptors in the dorsomedial medulla oblongata (DMM), which receives projections from the medullary raphe, and hypercapnia on airway resistance and respiratory variables were studied in mice while monitoring 5-HT release in the DMM. A microdialysis probe was inserted into the DMM of anesthetized adult mice. Each mouse was placed in a double-chamber plethysmograph. After recovery from anesthesia, the mice were exposed to stepwise increases in C[O.sub.2] inhalation (5%, 7%, and 9% C[O.sub.2] in [O.sub.2]) at 8-min intervals with a selective serotonin reuptake inhibitor, fluoxetine, or fluoxetine plus a 5-HT2 receptor antagonist, LY-53857 in the DMM. In response to fluoxetine plus LY-53857 coperfusion, specific airway resistance was increased, and tidal volume and minute ventilation were decreased. C[O.sub.2] inhalation with fluoxetine plus LY-53857 coperfusion in the DMM largely decreased airway resistance and additively increased minute ventilation. Thus, 5-HT2 receptor activity in the DMM increases basal levels of airway dilation and ventilatory volume, dependent on central inspiratory activity and the volume threshold of the inspiratory off-switch mechanism. Hypercapnia with low 5-HT2 receptor activity in the DMM largely recovers airway dilation and additively increases ventilatory volume. Interaction between 5-HT2 receptor activity in the DMM and C[O.sub.2] drive may elicit a cycle of hyperventilation with airway dilation and hypoventilation with airway narrowing. serotonin; hypoglossal nucleus; airway resistance; solitary tract nucleus; respiration

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP-/-) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP-/- mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA 1) to more acidic isoforms in CCSP-/- mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP-/- tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense. Cara cell secretory protein; chronic lung disease doi:10.1152/ajplung.00014.2007

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-[beta] is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-[beta] mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-[beta]1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-[beta] 1 on ASM cells was inhibited by selective inhibitors of TGF-[beta] receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-[kappa]B (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-[beta]1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-[beta]1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-[beta]1-induced proliferation. Our findings suggest that TGF-[beta]1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-[kappa] B is also involved in the TGF-[beta] 1 mitogenic signaling, but Smad pathway does not appear important. laser capture microdissection; transforming growth factor-[beta]1 expression; airway smooth muscle cells; asthma; corticosteroids doi:10.1152/ajplung.00068.2007

Maturational changes have been noted in neurally mediated contractile and relaxant responses in airways from New Zealand White rabbits. In this study, we focused on prostaglandins with bronchoprotective properties as potential modulators of airway tone in maturing rabbits. Tracheal rings from 1-, 2-, and 13-wk-old rabbits were assessed for neurally mediated contractile and relaxant responses produced by electrical field stimulation (EFS) of nerves in the presence and absence of the prostaglandin inhibitor, indomethacin (Indo). We also measured EFS-induced release of prostaglandin [E.sub.2] ([PGE.sub.2]) and the stable metabolite of prostacyclin, 6-keto-prostaglandin [F.sub.1[alpha]] (6-keto-[PGF.sub.1[alpha]]). In the presence of Indo, EFS produced significant increases in contractile responses in segments from 1- and 2-wk-old animals but not in segments from 13-wk adult rabbits. Tracheal tings from 1- and 2-wk-old animals precontracted with neurokinin A (NKA) relaxed 100% in response to EFS when Indo was not in the bath. In rings from 13-wk-old animals, relaxation was 40%. With Indo, relaxation was abolished in 1-wk-old animals and reduced to 30% in the 2- and 13-wk-old groups. Buffer from baths collected after EFS had significant increases in [PGE.sub.2] and 6-keto-[PGF.sub.1[alpha] released from tissues from 1- vs. 2- and 13-wk-old animals. Dose response curves to [PGE.sub.2] using tissues precontracted to NKA showed significant increases in relaxant responses in 1- and 2-vs. 13-wk-old rabbits. In rabbit airways, this study demonstrates enhanced modulation of airway tone by [PGE.sub.2] and greater release of the bronchoprotective prostaglandins [PGE.sub.2] and prostacyclin early in life. airway development; bronchoprotection; prostacyclin; prostaglandin [E.sub.2]; relaxant responses of airways doi:10.1152/ajplung.00413.2006

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic [Ca.sup.2+]. In this work, we found that the contraction caused by histamine depends on external [Na.sup.+], possibly involving nonselective cationic channels (NSCC) and the [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal tings stimulated by histamine. We observed that force reached 53 [+ or -] 1% of control during external [Na.sup.+] substitution by [N-methyl-D-glucamine.sup.+], whereas substitution by [Li.sup.+] led to no significant change (91 [+ or -] 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 [+ or -] 5.6%), whereas preincubation with nifedipine did not (89.7 [+ or -] 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 [+ or -] 3%, significantly different from nifedipine alone (49 [+ or -] 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 [+ or -] 1% and 19 [+ or -] 7%, respectively. Intracellular [Ca.sup.2+] measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 [+ or -] 0.1 and 0.19 [+ or -] 0.09 for KB-R, 0.43 [+ or -] 0.16 and 0.47 [+ or -] 0.18 for SKF, expressed as mean of differences). Moreover, [Na.sup.+]-free solution only inhibited the sustained response (0.54 [+ or -] 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces [Na.sup.+] influx through NSCC that promotes the [Ca.sup.2+] entry mode of NCX and [Ca.sub.v] 1.2 channel activation, thereby causing contraction. airway smooth muscle; KB-R7943; SKF-96365 doi:10.1152/ajplung.00485.2006

Chronic airway rejection, characterized by lymphocytic bronchitis, epithelial cell damage, and ohliterative bronchiolitis (OB), limits long-term survival after lung transplantation. The transcription factor early growth response gene-1 (Egr-1) induces diverse inflammatory mediators, some involved in OB pathogenesis. An orthotopic mouse tracheal transplant model was used to determine whether Egr-1 promotes development of airway allograft rejection. Significantly higher Egr-1 mRNA levels were seen in allografts (3.2-fold increase vs. isografts, P = 0.012). Allografts revealed thickening of epithelial and subepithelial airway layers (51 [+ or -] 4% luminal encroachment for allografts vs. 20 [+ or -] 3% for isografts, P < 0.0001) marked by significant lymphocytic infiltration. Absence of the Egr-1 gene in donor (but not recipient) tissue resulted in significant reduction in luminal narrowing (34 [+ or -] 4%, P = 0.0001) with corresponding diminution of T cell infiltration. Egr-1 null allografts exhibited a striking reduction in inducible nitric oxide synthase (iNOS) expression. Effector cytokines previously implicated in OB pathogenesis with known Egr- 1 promoter motifs (IL- 1[beta] and JE/monocyte chemoattractant protein-1) were reduced in Egr-1 null allografts. These data suggest a paradigm wherein local induction of Egr-1 in tracheal allografts drives expression of inflammatory mediators responsible for lymphocyte recruitment and tissue destruction characteristic of airway rejection. transplantation; inflammation; lymphocytic bronchiolitis; obliterative bronchiolitis doi:10.1152/ajplung.00285.2006

Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-[alpha]-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-[alpha] were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-[kappa]B, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-[alpha]-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 [+ or -] 0.01964, [t.sub.1/2] = 12.0 h), compared with vehicle (-0.01030 [+ or -] 0.007963, [t.sub.1/2] = 67.3 h) [results are expressed as decay constant (means [+ or -] SE) and half-life ([t.sub.1/2] in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-[alpha]-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells. inflammation; cytokines; messenger ribonucleic acid stability; neutrophils; structural cells; airway smooth muscle: interleukin; mitogenactivated protein kinase; tumor necrosis factor; interleukin 17A

Antibody-antigen interactions in the airway initiate inflammation in acute asthma exacerbations. This inflammatory response is characterized by the recruitment of granulocytes into the airways. In murine models of asthma, granulocyte recruitment into the lung contributes to the development of airway hyperresponsiveness (AHR), mucus production, and airway remodeling. Leukotriene [B.sub.4] is a mediator released following antigen challenge that has chemotactic activity for granulocytes, mediated through its receptor, BLT1. We investigated the role of BLT1 in granulocyte recruitment following antigen challenge. Wild-type mice and BLT[1.sup.-/-] mice were sensitized and challenged with ovalbumin (OVA) to induce acute allergic airway inflammation. In addition, to explore the relevance to antibody-antigen interactions, we injected OVA bound to anti-OVA Ig[G.sub.1] or anti-OVA IgE intra-tracheally into naive wild-type and BLT[1.sup.-/-] mice. Cell composition of the lungs, cytokine levels, histology, and AHR were determined. After sensitization and challenge with ovalbumin, there was significantly reduced neutrophil and eosinophil recruitment into the airways of BLT[1.sup.-/-] mice compared with wild-type animals after one or two daily antigen challenges, but this difference was not seen after three or four daily antigen challenges. Mucus production and AHR were not affected. Intratracheal injection of OVA bound to Ig[G.sub.1] or IgE induced neutrophil recruitment into the airways in wild-type mice but not in the BLT[1.sup.-/-] mice. We conclude that BLT1 mediates early recruitment of granulocytes into the airway in response to antigen-antibody interactions in a murine model of acute asthma. lung inflammation; asthma; lipid mediator

Airway remodeling describes the structural changes that occur in the asthmatic airway that include airway smooth muscle hyperplasia, increases in vascularity due to angiogenesis, and thickening of the basement membrane. Our aim in this study was to examine the effect of transforming growth factor-[beta] on the release of connective tissue growth factor and vascular endothelial growth factor from human airway smooth muscle cells derived from asthmatic and nonasthmatic patients. In addition we studied the immunohistochemical localization of these cytokines in the extracellular matrix after stimulating bronchial rings with transforming growth factor-[beta]. Connective tissue growth factor and vascular endothelial growth factor were released from both cell types and colocalized in the surrounding extracellular matrix. Prostaglandin [E.sub.2] inhibited the increase in connective tissue growth factor mRNA but augmented the release of vascular endothelial growth factor. Matrix metalloproteinase-2 decreased the amount of connective tissue growth factor and vascular endothelial growth factor, but not fibronectin deposited in the extracellular matrix. This report provides the first evidence that connective tissue growth factor may anchor vascular endothelial growth factor to the extracellular matrix and that this deposition is decreased by matrix metalloproteinase-2 and prostaglandin [E.sub.2]. This relationship has the potential to contribute to the changes that constitute airway remodeling, therefore providing a novel focus for therapeutic intervention in asthma. transforming growth factor-[beta]; airway remodeling; matrix metalloproteinase; prostaglandin [E.sub.2]

CFTR channels conduct HC[O.sup.-.sub.3] in addition to [Cl.sup.-] in airway epithelial cells. A defective HC[O.sup.-.sub.3]-transporting function of CFTR may underlie the pathogenesis of cystic fibrosis. In the present study, we have investigated whether a HC[O.sup.-.sub.3]-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for HC[O.sup.-.sub.3] transport in human airway epithelial Calu-3 cells. A reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. HC[O.sup.-.sub.3] stimulated a modest increase in cAMP production, and the increase was sensitive to 2-hydroxyestradiol (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggest that sAC is functional in Calu-3 cells. Adding 25 mM HC[O.sup.-.sub.3] to the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM HC[O.sup.-.sub.3] in the bath markedly increased the product of channel number and open probability of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium and thereby provides a link between the level of intracellular HC[O.sup.-.sub.3]/C[O.sub.2] and the modulation of HC[O.sup.-.sub.3]-conductive CFTR function by cAMP/PKA. cystic fibrosis transmembrane conductance regulator; bicarbonate; adenosine 3',5'-cyclic monophosphate; 2-hydroxyestradiol

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX3C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, C[X.sub.3]CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1[beta], TNF-[alpha], and IFN-[gamma], the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-[beta]. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-[gamma] and TNF-[alpha] induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-[beta] had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone ([10..sup.-8]-[10.sup.-6] M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun N[H.sub.2]terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 [micro]M) and SB-203580 (20 [micro]M), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-[gamma]- and TNF-[alpha]-induced JNK phosphorylation remained unaltered in the presence of TGF-[beta] but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-[beta]- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors. chemokine; airway inflammation; asthma; chronic obstructive pulmonary disease

Byline: Y. Riou (1), F. Leclerc (2), V. Neve (1), L. Dupuy (2), O. Noizet (2), S. Leteurtre (2), A. Sadik (2) Keywords: Dead space; Mechanical ventilation; Children Abstract: Objectives To assess the reproducibility of respiratory dead space measurements in ventilated children. Design Prospective study. Setting University pediatric intensive care unit. Patients Thirty-two mechanically ventilated children (0.13--15.4 years) who were clinically stable. Methods The single-breath CO.sub.2 test (SBT-CO.sub.2) was recorded using the C[O.sub.2]SMO Plus from the mean of 30 ventilatory cycles during 1 h (at T0, T15, T30, T45, and T60). Airway dead space was determined automatically (Novametrix Medical Systems, USA), and manually by Bohr- Enghoff equations using data obtained by SBT-CO.sub.2. At the end of the study period, arterial blood gas was sampled in order to calculate alveolar and physiologic dead space. Intrasubject reproducibility of measurements was evaluated by the intraclass correlation coefficient. Two-way analysis of variance was used to evaluate the relationships between time and measurements. The two methods for calculating airway dead space were compared by using two-tailed Student's t-test and Bland-Altman analysis. Results Airway dead space measurement had a good reproducibility during the 1-h period, whatever the method used (intraclass correlation coefficient: 0.84 to 0.87). No significant difference was observed with time. Airway dead space values from the SBT-CO.sub.2 method were smaller than those from Bohr-Enghoff equations. Physiologic dead space values from the SBT-CO2 method were similar to those from Bohr-Enghoff equations. Conclusion The measurement of airway dead space by the C[O.sub.2]SMO Plus was reproducible over a 1-h period in children requiring mechanical ventilation, provided ventilatory parameters were constant throughout the study. SBT-CO.sub.2 analysis may provide a bedside non-invasive monitoring of volumetric capnography. Author Affiliation: (1) Department of Respiratory Physiology, University Hospital of Lille, Lille, France (2) Service de Reanimation infantile, Hopital Jeanne de Flandre, Centre Hospitalier Regional et Universitaire, 59037, Lille, France Article History: Registration Date: 23/03/2004 Received Date: 16/11/2003 Accepted Date: 18/03/2004 Online Date: 11/05/2004

Secretion of HC[O.sup.-.sub.3] by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HC[O.sup.-.sub.3]-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HC[O.sup.-.sub.3]-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHC[O.sup.-.sub.3] (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 [+ or -] 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when [pH.sub.LBs] was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 [micro]M) but was inhibited by 100 nM bafilomycin [A.sub.1] (by 76 [+ or -] 13%), suggesting involvement of vacuolar-[H.sup.+] ATPase. Addition of ACh (10 [micro]M) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HC[O.sup.-.sub.3]-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 [micro]M), an inhibitor of [Cl.sup.-] secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 [micro]M) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HC[O.sup.-.sub.3] and [Cl.sup.-]. Isolated distal airways thus secrete both acid and base equivalents. airway epithelium; bicarbonate transport; pH

In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-[kappa]B promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-KB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity. cystic fibrosis transmembrane conductance regulator; human papillomavirus type 16; human airway epithelial cells; telomerase

Byline: L. Uttman (1), L. Beydon (2), B. Jonson (1) Keywords: Respiratory insufficiency Acute lung injury Respiratory dead space Pulmonary gas exchange Mechanics Artificial ventilation Abstract: Objective. We examined whether computer simulation predicts airway pressures after increments of positive end-expiratory pressure (PEEP) in acute respiratory failure. Design and setting. Prospective, nonrandomized comparative trial in an intensive care unit of a university hospital. Patients. Twelve consecutive acute respiratory failure patients. Interventions. PEEP increments from 0 to 2.5, 5, 7.5, 10, and 15 cm[H.sub.2]O. Measurements and results. A physiological profile comprising values for compliance, respiratory resistance and C[O.sub.2 ]elimination as a function of tidal volume was established from a recording of ordinary breaths prior to increments of PEEP. Airway pressures and C[O.sub.2 ]elimination were measured 30 s after resetting, pressures also after 10 min. Values from simulation of the resetting, based on the profile, were compared to measured values. The profiles indicated vast differences in physiology between the 12 subjects. Errors of simulation of airway pressures were nonsignificant or trivial up to PEEP levels of 10 cm[H.sub.2]O (95% of errors Conclusions. On the basis of a simple lung model, simulation predicted effects of moderate increments of PEEP on airway pressures in patients with complex physiology. Author Affiliation: (1) Department of Clinical Physiology, University Hospital, 22185, Lund, Sweden (2) Department of Anaesthesia, University Hospital, Angers, France Article History: Received Date: 21/02/2002 Accepted Date: 21/11/2002 Article note: Electronic Publication

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular [Ca.sup.2+] mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater [Ca.sup.2+] mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher [Ca.sup.2+] transients in Fischer than in Lewis ASM cells. ASM cell [B.sub.2] receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize [Ca.sup.2+] in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated [Ca.sup.2+] transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect [Ca.sup.2+] transients in Lewis cells. Enhanced [Ca.sup.2+] mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR. bradykinin; protein kinase C

Asthma, a chronic inflammatory disease of the airways, involves the increased expression of inflammatory mediators, including granulocyte-monocyte colony-stimulating factor (GM-CSF). Heme oxygenase-1 (HO-1), a stress-response protein, confers protection against oxidative stress. We hypothesized that carbon monoxide (CO), a byproduct of HO-1-dependent heme catabolism, regulates GM-CSF synthesis in human airway smooth muscle cells (HASMC). IL-1[beta] treatment induced a time-dependent induction of GM-CSF in HASMC. Furthermore, IL-1[beta] stimulated the major MAPK pathways, including ERK1/ERK2, JNK, and p38 MAPK. Exposure of HASMC to CO at low concentration (250 ppm) markedly inhibited IL-1[beta]-induced GM-CSF synthesis (>90%) compared with air-treated controls. CO treatment inhibited IL-1[beta]-induced ERK1/2 activation but did not inhibit JNK and p38 MAPK. Furthermore, CO increased cGMP levels in HASMC. Inhibition of guanylate cyclase by IH-[1,2,4] oxadiazolo[4,3a]quinoxalin-1-1 (ODQ) abolished the inhibitory effects of CO on GM-CSF synthesis and ERK1/2 activation. Collectively, these data demonstrate that the inhibitory effect of CO on GM-CSF synthesis depends on ERK1/2 MAPK and guanylate cyclase/cGMP-dependent pathways. cyclic guanosine monophosphate; granulocyte-monocyte colony-stimulating factor; mitogen activated protein kinase; interleukin-1[beta]

Interferon [gamma] (IFN-[gamma]), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-[gamma] concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-[gamma] is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-[gamma] stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-[gamma]-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-[gamma]-responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-[gamma]-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3[beta]. hepatocyte nuclear factor-3; CCAAT/enhancer-binding protein; mouse transformed Clara cells; thyroid transcription factor-1; interferon regulatory factor-1; Clara cell secretory protein

To model the competition between capillary and elastic forces in controlling the shape of a small lung airway and its interior liquid lining, we compute the equilibrium configurations of a liquid-lined, externally pressurized, buckled elastic tube. We impose axial uniformity and assume that the liquid wets the tube wall with zero contact angle. Non-zero surface tension has a profound effect on the tube's quasi-steady inflation-deflation characteristics. At low liquid volumes, hysteresis arises through two distinct mechanisms, depending on the buckling wavenumber. Sufficient compression always leads to abrupt and irreversible collapse and flooding of the tube; flooding is promoted by increasing liquid volumes or surface tension. The model captures mechanisms whereby capillary-elastic instabilities can lead to airway closure. [DOI: 10.1115/1.1516811]