Pharmacodynamic genome-wide association study identifies new responsive loci for glucocorticoid intervention in asthma.

Asthma is a chronic lung disease that has a high prevalence. The therapeutic intervention of this disease can be made more effective if genetic variability in patients' response to medications is implemented. However, a clear picture of the genetic architecture of asthma intervention response remains elusive. We conducted a genome-wide association study (GWAS) to identify drug response-associated genes for asthma, in which 909 622 SNPs were genotyped for 120 randomized participants who inhaled multiple doses of glucocorticoids. By integrating pharmacodynamic properties of drug reactions, we implemented a mechanistic model to analyze the GWAS data, enhancing the scope of inference about the genetic architecture of asthma intervention. Our pharmacodynamic model observed associations of genome-wide significance between dose-dependent response to inhaled glucocorticoids (measured as %FEV1) and five loci (P=5.315 × 10(-7) to 3.924 × 10(-9)), many of which map to metabolic genes related to lung function and asthma risk. All significant SNPs detected indicate a recessive effect, at which the homozygotes for the mutant alleles drive variability in %FEV1. Significant associations were well replicated in three additional independent GWAS studies. Pooled together over these three trials, two SNPs, chr6 rs6924808 and chr11 rs1353649, display an increased significance level (P=6.661 × 10(-16) and 5.670 × 10(-11)). Our study reveals a general picture of pharmacogenomic control for asthma intervention. The results obtained help to tailor an optimal dose for individual patients to treat asthma based on their genetic makeup.

A new perspective on concepts of asthma severity and control.

Concepts of asthma severity and control are important in the evaluation of patients and their response to treatment but the terminology is not standardised and the terms are often used interchangeably. This review, arising from the work of an American Thoracic Society/European Respiratory Society Task Force, identifies the need for separate concepts of control and severity, describes their evolution in asthma guidelines and provides a framework for understanding the relationship between current concepts of asthma phenotype, severity and control. "Asthma control" refers to the extent to which the manifestations of asthma have been reduced or removed by treatment. Its assessment should incorporate the dual components of current clinical control (e.g. symptoms, reliever use and lung function) and future risk (e.g. exacerbations and lung function decline). The most clinically useful concept of asthma severity is based on the intensity of treatment required to achieve good asthma control, i.e. severity is assessed during treatment. Severe asthma is defined as the requirement for (not necessarily just prescription or use of) high-intensity treatment. Asthma severity may be influenced by the underlying disease activity and by the patient's phenotype, both of which may be further described using pathological and physiological markers. These markers can also act as surrogate measures for future risk.

Predicting worsening asthma control following the common cold.

The asthmatic response to the common cold is highly variable, and early characteristics that predict worsening of asthma control following a cold have not been identified. In this prospective multicentric cohort study of 413 adult subjects with asthma, the mini-Asthma Control Questionnaire (mini-ACQ) was used to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models were used to examine demographic, physiological, serological and cold-related characteristics for their relationship to changes in asthma control following a cold. Clinically significant worsening of asthma control was observed following a cold (mean+/-SD increase in mini-ACQ score of 0.69+/-0.93). Univariate analysis demonstrated that season, centre location, cold duration and cold severity measurements were all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed that the day 2 and cumulative sum of day 1 and 2 WURSS-21 scores were significant predictors of the subsequent changes in asthma control. In asthmatic subjects, cold severity within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.

Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase.

Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens. In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, approximately 92-kD, Ca2+ - and Zn2+ -dependent proenzyme cleaved over time to smaller, active forms. Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products. The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments. To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors. Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme. In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast cells, it could play a role in tissue remodeling and repair.

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases.

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines.

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.

Role of inflammation and inflammatory mediators in airways disease.

Recent evidence suggests that airway inflammation is linked to hyper-responsiveness of airway smooth muscle. Increases in airway responsiveness after many stimuli are accompanied by increases in inflammatory cells in bronchoalveolar lavage fluid and in the airway epithelium. Airway epithelial cells may themselves be an important source of inflammatory mediators, producing metabolites that can cause chemotaxis of neutrophils and that can selectively activate other cells in the lungs. Mast cells produce a variety of enzymes and vasoactive, chemotactic, and bronchoconstrictor substances in response to non-immunologic as well as immunologic stimuli. The secretory profile of a mast cell may depend upon the specific stimulus applied. In addition, different populations of mast cells exist and distinct enzymatic pathways may predominate in different cell types. Mediators released by these cells may activate target cells by direct or indirect mechanisms. These inflammatory mediators, together with inflammatory cells, are important in the complex interactions involving airway epithelial cells, neutrophils, mast cells, smooth muscle, respiratory secretory cells, and nerves, which, in concert, are responsible for the pathophysiologic manifestations of obstructive lung disease.

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

The utility of peak flow, symptom scores, and beta-agonist use as outcome measures in asthma clinical research.

STUDY OBJECTIVES: Several methods of utilizing peak expiratory flow (PEF) and other markers of disease activity have been suggested as useful in the management of asthma. It remains unclear, however, as to which surrogate markers of disease status are discriminative indicators of treatment failure, suitable for use in clinical trials. DESIGN: We analyzed the operating characteristics of 66 surrogate markers of treatment failure using a receiver operating characteristic (ROC) curve analysis. PARTICIPANTS: Information regarding FEV(1), symptoms, beta(2)-agonist use, and PEF was available from 313 subjects previously enrolled in two Asthma Clinical Research Network trials, in which 71 treatment failures occurred (defined by a 20% fall in FEV(1) from baseline). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: None of the measures had an acceptable ability to discriminate subjects with a > or % fall in FEV(1) from those without, regardless of the duration of the period of analysis or the criteria for test positivity employed. Areas under the ROC curves generated ranged from 0.51 to 0.79, but none were statistically superior. Sensitivity and specificity combinations were generally poor at all cutoff values; true-positive rates could not be raised without unacceptably elevating false-positive rates concurrently. CONCLUSIONS: Studies that seek to detect treatment failure defined by a significant fall in FEV(1) should not use such individual surrogate measures to estimate disease severity.

In vitro histamine release induced by magnetic resonance imaging and iodinated contrast media.

RATIONALE AND OBJECTIVES: To investigate the mechanism of anaphylactoid reactions to contrast media, in vitro histamine release induced by magnetic resonance imaging, and iodinated contrast agents was examined in a dog mastocytoma cell line. METHODS: Two gadolinium (Gd)-based magnetic resonance contrast agents, Gd diethylenetriamine pentaacetic acid (Gd-DTPA), dimeglumine, and Gd-bismorpholide, and two iodinated contrast agents, diatrizoate meglumine and iohexol, were incubated with histamine-containing canine mastocytoma cells. Release of histamine into the supernatant was determined at various contrast-medium concentrations after incubation at 37 degrees C for 30 minutes. RESULTS: Iodinated and Gd-based contrast agents caused release of histamine from mastocytoma cells at similar concentrations (50-150 mM). Mannitol, an osmotic stimulus, caused release of histamine only at concentrations greater than 1,000 mM. CONCLUSIONS: Histamine release from canine mastocytoma cells does not appear to be solely due to osmotic effects, but results from direct stimulation by contrast media. For all agents examined, the concentration at which in vitro histamine release occurs far exceeds the serum contrast media concentration expected in routine clinical application. Direct release of histamine from mast cells does not completely explain the pathogenesis of idiosyncratic anaphylactoid responses to contrast media.

Antigen-induced airway hyperresponsiveness and pulmonary inflammation in allergic dogs.

We studied whether antigen-induced airway hyperresponsiveness was associated with pulmonary inflammation in 11 anesthetized ragweed-sensitized dogs. Airway responsiveness to acetylcholine aerosol was determined before and at 2, 6, and 24 h after ragweed or sham aerosol challenge. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) performed at either 2 or 6 h. Total pulmonary resistance increased 11-fold at 5 min after ragweed. Airway responsiveness was unchanged at 2 h but was increased 6.6-fold at 6 h in 8 of 11 dogs (P less than 0.001); hyperresponsiveness persisted from 4 days to 4 mo. Airway responsiveness was unchanged by aerosols of diluent. Neutrophils in BAL fluid increased approximately sixfold at 2 h (P less than 0.02) and at 6 h (P less than 0.02) after antigen challenge. There were fewer eosinophils in fluid recovered at 6 h after antigen compared with 2 h lavages (P less than 0.05). In three nonresponders, BAL showed no significant changes in neutrophils and eosinophils after antigen. Thus antigen-induced hyperresponsiveness is associated with the presence of pulmonary inflammation, presumably arising from the airways and involving both neutrophils and eosinophils.

Inflammation, inflammatory mediators, and mediator antagonists in asthma.

Asthma is an inflammatory disease that involves mast cells, antigen-presenting cells, eosinophils, neutrophils, and TH2-lymphocytes. These cells produce a broad array of mediators and cytokines that lead to the bronchoconstriction, mucosal edema, mucus secretion, and bronchial hyperresponsiveness that characterize asthma. Current guidelines for therapy recommend that all patients whose asthma is more severe than mild intermittent receive chronic treatment with drugs that interrupt this inflammatory cascade. Corticosteroids have been the gold standard for treatment, but a greater understanding of the specific cells and mediators involved in the pathogenesis of asthma has led to more focused, specific therapy. Pharmacologic agents that interrupt the synthesis of action of leukotrienes, and monoclonal antibodies directed against intracellular adhesion molecules or immunoglobulin E are examples of the new generation of specific targeted therapy for use in asthma.

Safety and clinical efficacy of zileuton in patients with chronic asthma.

Zileuton, a leukotriene pathway inhibitor used to treat asthma, improves lung function, relieves symptoms, and is well tolerated. The purpose of this 12-month, parallel-group, open-label study was to assess the efficacy of zileuton and evaluate liver function in patients treated with this drug (approximately 2% of patients treated with zileuton in controlled trials had reversible liver enzyme elevations). A total of 2,947 patients at 233 centers in the United States were randomly assigned in a 5:1 ratio to treatment with zileuton plus usual asthma care or usual asthma care alone. Efficacy variables included asthma exacerbations; need for alternative treatment, steroid rescue, emergency care, and hospitalizations; forced expiratory volume in 1 second (FEV1); and asthma symptom scores. The safety evaluation included measurement of alanine aminotransferase levels. Patients treated with zileuton had significantly fewer corticosteroid rescues (P < 0.001), required less emergency care (P < 0.05), had fewer hospitalizations, and had greater increases in FEV1 (P = 0.048). They also had significantly greater improvements in asthma symptoms. Increases in alanine aminotransferase levels to three times or more the upper limit of normal occurred in 4.6% of patients treated with zileuton and 1.1% of those receiving usual care (P < 0.001); most increases occurred during the first 2 to 3 months. Alanine aminotransferase levels decreased to less than two times the upper limit of normal or to baseline levels during zileuton treatment or after drug cessation. Jaundice or chronic liver disease did not develop in any patient. Adding zileuton to the therapeutic regimens of patients with asthma is likely to improve asthma control and lower utilization of healthcare resources.

Rational therapy of acute asthma.

Despite effective pharmacologic therapy, asthma remains a common cause of emergency room and hospital admissions, and mortality due to asthma is increasing. Asthma deaths often are related to failure on the part of patients or their physicians to appreciate the severity of their illness. Subjective assessment of the severity of airflow obstruction is unreliable and actual measurement of airflow and arterial blood gases should be utilized initially, as well as during therapy. An increasing awareness of the role of inflammation in the pathogenesis of asthma has led to pharmacologic therapy aimed both at producing bronchodilation and at reducing inflammation. Aggressive therapy with inhaled beta-adrenergic agonists and systemic corticosteroids remains the mainstay of treatment for acute asthma. Some patients may benefit from the addition of anticholinergic therapy and/or theophylline. If mechanical ventilation is required, controlled hypoventilation may decrease the incidence of pulmonary barotrauma. Although inhaled corticosteroids, cromolyn sodium, antihistamines and calcium-channel blockers are not effective in the treatment of acute asthma, inhaled steroids should probably be used in the recovery phase of most hospitalized asthmatics to decrease the bronchial hyperreactivity associated with airway inflammation.

The role of mast cell-derived mediators in airway function.

The airway mast cell appears to play an important role in a number of pathophysiologic responses. Mast cells may be activated directly (e.g., by specific antigen) or indirectly (e.g., by an epithelial cell product such as 15-HETE). When activated, the mast cell releases mediators that may have direct effects or indirect effects. Mast cells and mediators undoubtedly play a role in specific airway responses. The data presented here are intended to serve only as examples of the complex way in which various cells and inflammatory mediators may interact to produce a physiologic response in the airways. Possibilities exist for numerous interactions between additional mediators as well as additional cell types. Because of the complexity of the system, characterization of activation and secretion by isolated pure cells, and studies of cell-to-cell interactions in vitro appear to be important first steps toward understanding the even more complex interactions that occur in vivo.

Mast cell-derived mediators and their role in cell-to-cell interactions in the lung.

Mast cells produce a variety of enzymes and vasoactive, chemotactic, and bronchoconstrictor substances in response to nonimmunologic as well as immunologic stimuli. These mast cell-derived mediators may act on airway smooth muscle, submucosal glands, epithelial cells, circulating blood cells, vascular cells, and other cell types. The secretory profile of a mast cell appears to depend on the specific stimulus applied. In addition, different populations of mast cells exist, and distinct enzymatic pathways may predominate in different cell types. The effect of mast cell-derived mediators on the various target cells may be direct or indirect. For example, mediators released by immunologic challenge of sensitized lung fragments or by nonimmunologic challenge of canine mastocytoma cells stimulate the transport of ions and water across the tracheal epithelium. This effect, however, is indirect, is blocked by indomethacin, and therefore appears to be dependent upon an intact cyclooxygenase pathway in the tracheal epithelium. Canine mastocytoma cells resemble normal mast cells in dog lung and skin, and can be propagated to provide a continuous source of large numbers of pure, identical mast cells for biochemical and physiologic studies. Studies of these cells and their inflammatory mediators will increase our understanding of the complex series of cell-to-cell interactions which are responsible for the pathophysiologic manifestations of obstructive lung disease.

Mast cells and cell-to-cell interactions in airways.

Dog mastocytomas (anatomic and biochemical features comparable to normal dog and human mast cells) were used to study actions of mast cell mediators on several airway effector systems. We showed mastocytoma cell adherence to both cultured tracheal epithelial cells and tracheal tissue sections for greater than 48 h that was abolished completely by pretreatment of mast cells with proteases. This mast cell-epithelial cell adhesion-interaction reaction is probably mediated by a mast cell plasma membrane protein. Mast cell mediators stimulate short circuit current and ion flux across dog tracheal epithelia mounted in Ussing chambers. Pretreatment of epithelia with indomethacin blocks this effect, probably by inhibiting LTC4-induced activation of epithelial cyclooxygenases. Mastocytoma cells also increase secretion from cultured serous submucosal gland cells. Blockade of cyclooxygenase and lipoxygenase pathways in mastocytoma cells activated by calcium ionophore does not alter secretion of the serous cells induced by mastocytoma supernatant, but secretion induced by mastocytoma supernatant or purified mast cell chymase is markedly reduced by an inhibitor of chymase. These results suggest that mast cells can alter airway secretions not only by actions on ion flux in epithelial cells but also by actions on submucosal gland secretion; this latter action appears to be mediated by mast cell chymase. Finally, supernatants from mastocytoma cells stimulated by calcium ionophore greatly increase the sensitivity and magnitude of the contractile response of dog bronchial smooth muscle to histamine. These effects are blocked by an inhibitor of mast cell tryptase.(ABSTRACT TRUNCATED AT 250 WORDS)