l-citrulline prevents asymmetric dimethylarginine-mediated reductions in nitric oxide and nitrosative stress in primary human airway epithelial cells.

BACKGROUND: Asthma is associated with reduced systemic levels of l-arginine and increased asymmetric dimethylarginine (ADMA). This imbalance leads to nitric oxide synthase (NOS) uncoupling with reduced nitric oxide (NO) formation and greater oxidative and nitrosative stress. Whether this imbalance also occurs in bronchial epitheliumof asthmatics is unknown. OBJECTIVES: We used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: (i) ADMA-mediated NOS uncoupling reduces epithelial production of NO and increases oxygen and nitrogen reactive species, and (ii) l-citrulline can reverse this mechanism by recoupling NOS, restoring NO production and reducing oxidative and nitrosative stress. RESULTS: In HBECsIL-13 and INFγ stimulated NOS2 and increased NOx levels. The addition of ADMA reduced NOx and increased H2 O2 levels (p<0.001). Treatment with l-citrulline (800, 1600 µm) rescued NOx when the l-arginine media concentration was 25 µm but failed to do so with higher concentrations (100 µm). Under reduced l-arginine media conditions, HBECs treated with l-citrulline increased the levels of argininosuccinate, an enzyme that metabolizes l-citrulline to l-arginine. l-citrulline prevented the ADMA-mediated increase in nitrotyrosine in HBECs in cells from asthmatics and controls. CONCLUSIONS AND CLINICAL RELEVANCE: Increasing ADMA reduces NO formation and increases oxidative and nitrosative stress in airway epithelial cells. l-citrulline supplementation restores NO formation, while preventing nitrosative stress. These results, suggest that l-citrulline supplementation may indeed be a powerful approach to restore airway NO production and may have a therapeutic potential in diseases in which there is a defective production of NO.

eQTL of bronchial epithelial cells and bronchial alveolar lavage deciphers GWAS-identified asthma genes.

BACKGROUND: Genome-wide association studies (GWASs) have identified various genes associated with asthma, yet, causal genes or single nucleotide polymorphisms (SNPs) remain elusive. We sought to dissect functional genes/SNPs for asthma by combining expression quantitative trait loci (eQTLs) and GWASs. METHODS: Cis-eQTL analyses of 34 asthma genes were performed in cells from human bronchial epithelial biopsy (BEC, n = 107) and from bronchial alveolar lavage (BAL, n = 94). RESULTS: For TSLP-WDR36 region, rs3806932 (G allele protective against eosinophilic esophagitis) and rs2416257 (A allele associated with lower eosinophil counts and protective against asthma) were correlated with decreased expression of TSLP in BAL (P = 7.9 × 10(-11) and 5.4 × 10(-4) , respectively) and BEC, but not WDR36. Surprisingly, rs1837253 (consistently associated with asthma) showed no correlation with TSLP expression levels. For ORMDL3-GSDMB region, rs8067378 (G allele protective against asthma) was correlated with decreased expression of GSDMB in BEC and BAL (P = 1.3 × 10(-4) and 0.04) but not ORMDL3. rs992969 in the promoter region of IL33 (A allele associated with higher eosinophil counts and risk for asthma) was correlated with increased expression of IL33 in BEC (P = 1.3 × 10(-6) ) but not in BAL. CONCLUSIONS: Our study illustrates cell-type-specific regulation of the expression of asthma-related genes documenting SNPs in TSLP, GSDMB, IL33, HLA-DQB1, C11orf30, DEXI, CDHR3, and ZBTB10 affect asthma risk through cis-regulation of its gene expression. Whenever possible, disease-relevant tissues should be used for transcription analysis. SNPs in TSLP may affect asthma risk through up-regulating TSLP mRNA expression or protein secretion. Further functional studies are warranted.

The complex relationship between inflammation and lung function in severe asthma.

Asthma is a common respiratory disease affecting ∼300 million people worldwide. Airway inflammation is thought to contribute to asthma pathogenesis, but the direct relationship between inflammation and airway hyperresponsiveness (AHR) remains unclear. This study investigates the role of inflammation in a steroid-insensitive, severe allergic airway disease model and in severe asthmatics stratified by inflammatory profile. First, we used the T-helper (T(H))-17 cells adoptive transfer mouse model of asthma to induce pulmonary inflammation, which was lessened by tumor necrosis factor (TNF)-α neutralization or neutrophil depletion. Although decreased airspace inflammation following TNFα neutralization and neutrophil depletion rescued lung compliance, neither intervention improved AHR to methacholine, and tissue inflammation remained elevated when compared with control. Further, sputum samples were collected and analyzed from 41 severe asthmatics. In severe asthmatics with elevated levels of sputum neutrophils, but low levels of eosinophils, increased inflammatory markers did not correlate with worsened lung function. This subset of asthmatics also had significantly higher levels of T(H)17-related cytokines in their sputum compared with severe asthmatics with other inflammatory phenotypes. Overall, this work suggests that lung compliance may be linked with cellular inflammation in the airspace, whereas T-cell-driven AHR may be associated with tissue inflammation and other pulmonary factors.

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma.

BACKGROUND: Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics' both large and small airways has not been investigated. OBJECTIVE: To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. METHODS: Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. RESULTS: Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

TLR4/MyD88-induced CD11b+Gr-1 int F4/80+ non-migratory myeloid cells suppress Th2 effector function in the lung.

In humans, environmental exposure to a high dose of lipopolysaccharide (LPS) protects from allergic asthma, the immunological underpinnings of which are not well understood. In mice, exposure to a high LPS dose blunted house dust mite-induced airway eosinophilia and T-helper 2 (Th2) cytokine production. Although adoptively transferred Th2 cells induced allergic airway inflammation in control mice, they were unable to do so in LPS-exposed mice. LPS promoted the development of a CD11b(+)Gr1(int)F4/80(+) lung-resident cell resembling myeloid-derived suppressor cells in a Toll-like receptor 4 and myeloid differentiation factor 88 (MyD88)-dependent manner that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells. LPS effects switched from suppressive to stimulatory in MyD88(-/-) mice. Suppression of Th2 effector function was reversed by anti-interleukin-10 (IL-10) or inhibition of arginase 1. Lineage(neg) bone marrow progenitor cells could be induced by LPS to develop into CD11b(+)Gr1(int)F4/80(+)cells both in vivo and in vitro that when adoptively transferred suppressed allergen-induced airway inflammation in recipient mice. These data suggest that CD11b(+)Gr1(int)F4/80(+) cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.

IL-13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells.

BACKGROUND: Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T-helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells. OBJECTIVES: We hypothesized that primary human airway epithelial cells in air-liquid interface (ALI) culture would increase iNOS expression and activity in response to IL-13, while decreasing arginase expression. METHODS: iNOS and arginase mRNA (real-time PCR) and protein expression (Western blot and immunofluorescence) as well as iNOS activity (nitrite levels) were measured in ALI epithelial cells cultured from bronchial brushings of normal and asthmatic subjects following IL-13 stimulation. RESULTS: IL-13 up-regulated iNOS mRNA primarily at a transcriptional level in epithelial cells. iNOS protein and activity also increased, arginase1 protein expression decreased while arginase 2 expression did not change. The changes in iNOS protein correlated strongly with changes in nitrites, and inclusion of arginase (1 or 2) did not substantially change the relationship. Interestingly, iNOS mRNA and protein were not correlated. CONCLUSIONS: These results contrast with many previous results to confirm that Th2 stimuli enhance iNOS expression and activity. While arginase 1 protein decreases in response to IL-13, neither arginase appears to substantially impact nitrite levels in this system.

Transbronchial biopsy as a tool to evaluate small airways in asthma.

Small airway (SA) inflammation in asthmatics is poorly understood. Surgical biopsies to obtain peripheral lung tissue are seldom justified in asthmatics. Therefore, the authors hypothesised that transbronchial biopsy could be an alternative approach to evaluate SA in asthma. Transbronchial and endobronchial biopsy tissue samples (TBBX and EBBX) from 12 severe asthmatics were evaluated for airway and parenchymal total inflammatory cell count expressed as the sum of immunostained T-cells (CD3), macrophages (CD68), mast cells (tryptase AAI), neutrophils (neutrophil elastase) and eosinophils (EG2) per mm2. The large airways (LA) were evaluated in EBBXs, while SA, medium airways (MA) and alveolar tissue (AT) were evaluated in TBBXs. When cell counts from SA, MA, LA and AT were compared, SA had a significantly higher cell count than MA or LA (SA 1011 x mm(-2) (539-1,290), MA 346 x mm(-2) (223-415), LA 332 x mm(-2) (189-416), AT 464 x mm(-2) (298-834)). The cell density and pattern of the inflammatory cell distribution in subjects with TBBXs appeared similar to those in three severe asthmatics whose inflammatory cells were analysed in surgical tissue samples. This study suggests that small airway may be identified and analysed in transbronchial biopsy tissue samples and therefore transbronchial biopsy tissue samples could expand the analysis of inflammation and tissue remodelling in asthma.

Expression and activation of 15-lipoxygenase pathway in severe asthma: relationship to eosinophilic phenotype and collagen deposition.

BACKGROUND: Although 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), a product of 15-lipoxygenase (15-LO), may be involved in mild to moderate asthma, little is known about its potential roles in severe asthma. OBJECTIVES: This study was performed to evaluate 15(S)-HETE levels in bronchoalveolar lavage fluid (BALF) from severe asthmatics with and without airway eosinophils and from the control groups. In addition, 15-LO protein expression was examined in endobronchial biopsy, while its expression and activation were evaluated in BAL cells. RESULTS: While 15(S)-HETE levels in BALF were significantly higher in all severe asthmatics than normal subjects, severe asthmatics with airway eosinophils had the highest levels compared with mild, moderate asthmatics and normal subjects. 15(S)-HETE levels were associated with tissue eosinophil numbers, sub-basement membrane thickness and BALF tissue inhibitor of metalloproteinase-1 levels, and were accompanied by increased 15-LO expression in bronchial epithelium. In addition, activation of 15-LO was suggested by the increased proportion of 15-LO in the cytoplasmic membrane of alveolar macrophages from severe asthmatics. CONCLUSION: The data suggest that severe asthmatics with persistent airway eosinophils manifest high levels of 15(S)-HETE in BALF, which may be associated with airway fibrosis. It is likely that 15-LO expression and activation by airway cells explain the increased 15(S)-HETE levels.

Persistent wheezing in very young children is associated with lower respiratory inflammation.

Despite advances in understanding the pathophysiology of asthma, morbidity and mortality in pediatrics continue to rise. Little is known about the initiation and chronicity of inflammation resulting in asthma in this young population. We evaluated 20 "wheezing" children (WC) (median age 14.9 mo) with a minimum of two episodes of wheezing or prolonged wheezing > or = 2 mo in a 6-mo period with bronchoscopy and bronchoalveolar lavage (BAL). Comparisons were made with six normal controls (NC) (median age 23.3 mo) undergoing general anesthesia for elective surgery. BAL fluid cell counts and differentials were determined. The eicosanoids, leukotriene (LT) B(4), LTE(4), prostaglandin (PG)E(2), and 15-hydroxyeicosatetraenoic acid (HETE) and the mast cell mediators, beta-tryptase and PGD(2), were evaluated by enzyme immunoassay (EIA). WC had significant elevations in total BAL cells/ml (p = 0.01), as well as, lymphocytes (LYMPH, p = 0.007), macrophages/monocytes (M&M, p = 0.02), polymorphonuclear cells (PMN, p = 0.02), epithelial cells (EPI, p = 0.03), and eosinophils (EOS, p = 0.04) compared with NC. Levels of PGE(2) (p = 0.0005), 15-HETE (p = 0.002), LTE(4) (p = 0.04), and LTB(4) (p = 0.05) were also increased in WC compared with NC, whereas PGD(2) and beta-tryptase were not. This study confirms that inflammation is present in the airways of very young WC and may differ from patterns seen in adults with asthma.

The measurement of cysteinyl leukotrienes in urine.

The cysteinyl leukotrienes, comprising leukotriene (LT) C4 and its major metabolites LTD4 and LTE4, are inflammatory lipid mediators derived from metabolism of arachidonic acid by 5-lipoxygenase. These leukotrienes have received considerable attention for their potential role in asthma and other inflammatory diseases. Since there is a potential role for these lipid mediators in both health and disease, the analysis of leukotrienes in biological fluids, especially urine, has generated significant interest.

Peripheral blood and airway tissue expression of transforming growth factor beta by neutrophils in asthmatic subjects and normal control subjects.

BACKGROUND: Airway remodeling may play an important role in asthma pathophysiology. Transforming growth factor beta (TGF-beta) has a critical role in the remodeling process. Although cellular sources for TGF-beta have been previously investigated in asthma airways, the expression, release, or both of TGF-beta from asthmatic airways and blood neutrophils has not been reported. OBJECTIVE: The current study evaluated the TGF-beta protein and messenger (m)RNA expression by airway and peripheral blood neutrophils in asthmatic and normal subjects. METHODS: TGF-beta protein expression by airway and peripheral blood neutrophils was detected by using immunocytochemistry. TGF-beta protein levels in blood neutrophil supernatant were measured by using an enzyme immunoassay. TGF-beta mRNA expression was evaluated by using reverse transcription-PCR. RESULTS: Higher numbers of TGF-beta(+) cells and neutrophils were found in airway tissue of asthmatic (n = 15) compared with normal subjects (n = 10). Although neutrophils in both asthmatic and normal airway tissue expressed TGF-beta protein and the percentage of neutrophils expressing TGF-beta was similar between the two groups, the total number of TGF-beta(+) neutrophils was higher in the asthmatic subjects (P =.01). Peripheral blood neutrophils from asthmatic (n = 5) and normal subjects (n = 7) also expressed TGF-beta protein and mRNA. Blood neutrophils from asthmatic subjects spontaneously released significantly higher levels of TGF-beta than those from normal subjects (P =.007). CONCLUSION: These data suggest that airway and blood neutrophils from both asthmatic and normal subjects can express and release TGF-beta. Higher levels of TGF-beta expression-release from asthmatic neutrophils indicate that neutrophils may be involved in the airway remodeling process of asthmatic subjects.

Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics.

The mechanisms associated with the development of severe, corticosteroid (CS)-dependent asthma are poorly understood, but likely heterogenous. It was hypothesized that severe asthma could be divided pathologically into two inflammatory groups based on the presence or absence of eosinophils, and that the inflammatory subtype would be associated with distinct structural, physiologic, and clinical characteristics. Thirty-four severe, refractory CS-dependent asthmatics were evaluated with endobronchial biopsy, pulmonary function, allergy testing, and clinical history. Milder asthmatic and normal control subjects were also evaluated. Tissue cell types and subbasement membrane (SBM) thickness were evaluated immunohistochemically. Fourteen severe asthmatics [eosinophil (-)] had nearly absent eosinophils (< 2 SD from the normal mean). The remaining 20 severe asthmatics were categorized as eosinophil (+). Eosinophil (+) severe asthmatics had associated increases (p < 0.05) in lymphocytes (CD3+, CD4+, CD8+), mast cells, and macrophages. Neutrophils were increased in severe asthmatics and not different between the groups. The SBM was significantly thicker in eosinophil (+) severe asthmatics than eosinophil (-) severe asthmatics and correlated with eosinophil numbers (r = 0.50). Despite the absence of eosinophils and the thinner SBM, the FEV(1) was marginally lower in eosinophil (-) asthmatics (p = 0.05) with no difference in bronchodilator response. The eosinophil (+) group (with a thicker SBM) had more intubations than the eosinophil (-) group (p = 0.0004). Interestingly, this group also had a decreased FVC/slow vital capacity (SVC). These results suggest that two distinct pathologic, physiologic, and clinical subtypes of severe asthma exist, with implications for further research and treatment.

Leukotriene receptor antagonists and related compounds.

Leukotrienes (LTs), lipid mediators of inflammation, have proved to be important biochemicals involved in the symptoms and physiological changes of asthma. In the past year and a half, the development of three new drugs that modulate the LT pathway has been completed. The first subclass of these drugs, leukotriene receptor antagonists (LTRA) (zafirlukast and montelukast), blocks the interaction of the cysteinyl form of the LTs with the cell type bearing the receptor. The second subclass, the 5-lipoxygenase (5-LO) inhibitors (zileuton) inhibits the 5-LO enzyme, which prevents the formation of both cysteinyl LTs and LTB4. The LT modulators have shown efficacy in inhibiting the physiological changes occurring after allergen, acetylsalicylic acid and exercise challenge in asthmatics. In addition, they have shown efficacy in improving symptoms, beta-agonist use and forced expiratory volume in 1 s (FEV1) in chronic, 'day-to-day' asthma in patients with mild disease. Comparison studies with low doses of inhaled corticosteroids suggest that LT modulators may have similar effects on symptom scores and beta-agonist use, but have lesser effects on FEV1. Finally, emerging data suggest that these drugs are beneficial in decreasing the dose of inhaled corticosteroids necessary to control more moderate to severe asthma. While long term studies will be helpful in determining the 'disease modifying' effects of these drugs, data suggest that these drugs are useful in the treatment of a broad range of asthmatic patients.

Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease.

Chronic airway inflammation and remodeling, including fibrosis, have been proposed as important contributors to asthma pathophysiology. Previous studies of airway fibrosis have been performed mainly in mild and moderate asthmatics at the subepithelial "basement membrane" (SBM) level. The current study was designed to evaluate the large airway SBM thickness and submucosal collagen deposition, as measured by three different collagen staining methods, in endobronchial biopsies from 17 severe, nine moderate, and seven mild asthmatics, as well as eight normal control subjects. Tissue eosinophils and transforming growth factor-beta (TGF-beta) immunoreactivity were also examined. There were no statistically significant differences in the SBM thickness, submucosal collagen deposition, eosinophil numbers, or TGF-beta positive cells among the three groups of asthmatics and the normal control subjects. It was only when examining all asthmatics (n = 33) together, that a modestly thickened SBM (p = 0.04), as evaluated by collagen type III immunostaining, was observed as compared with normal control subjects. Despite this difference, no significant differences were found in the amount of submucosal collagen deposition and the number of eosinophils or TGF-beta expressing cells when comparing total asthmatics and normal control subjects. Additionally, no significant correlations were found between collagen deposition and eosinophil count, TGF-beta expression level, FEV1, or duration of asthma. These results suggest that although increased collagen deposition in the SBM at the large airway level is a characteristic of asthma, it may not explain the differences in severity of asthma.

Immunoaffinity resin for purification of urinary leukotriene E4.

Urinary leukotriene E4 (LTE4) has been used as an index of total leukotriene synthesis. A wide variety of methods have been applied to measure LTE4 which has made direct comparison of urinary levels reported by different laboratories difficult. A new peptidoleukotriene immunoaffinity resin was utilized for urinary LTE4 purification in a method that is easy and inexpensive, utilizing commercially available reagents. This method is described and compared to other methods. LTE4 (50-250 pg/mL) added to a urine extract was quantitatively recovered using the immunoaffinity resin. Similarly, LTE4 (50-400 pg/mL) added to urine was recovered between 63 and 76%. The coefficient of variation of samples purified and quantified on the same or on different days ranged from 8-10%. There was a strong correlation (r2 = 0.95) between LTE4 concentrations determined after immunofiltration and immunoaffinity purification. Although there was a good correlation between urinary LTE4 levels measured without purification compared to after immunoaffinity purification, the high y-intercept of 179 indicates the presence of interfering substances in unpurified urine. Urinary LTE4 in normal healthy adults was 80 +/- 7 pg/mg creatinine, similar to that previously reported following HPLC or immunofiltration purification. Urinary LTE4 was also measured in healthy children (age 3-12) and found to be 103 +/- 9.

Bronchoscopic evaluation of severe asthma. Persistent inflammation associated with high dose glucocorticoids.

The role of inflammation in the pathogenesis of severe asthma chronically treated with high doses of glucocorticoids is poorly understood. Despite this, treatment has been aimed at advancing anti-inflammatory and immunomodulator therapy. This study was designed to evaluate both the presence and type of airway inflammation in patients with severe asthma. A prospective bronchoscopic study evaluated 14 severe, high-dose oral glucocorticoid dependent asthmatics. Bronchoalveolar lavage fluid was analyzed for cytology and inflammatory mediators. Endobronchial and transbronchial biopsies were performed in selected patients for morphometric evaluation of macrophage/monocytes, neutrophils, eosinophils and lymphocytes. These results were compared with lavage and endo- and transbronchial biopsy studies in normal controls and patients with moderate asthma. The concentration of eosinophils in bronchoalveolar lavage fluid was highest in the moderate asthmatics not on glucocorticoids, with very little difference between normal controls and severe asthmatics (significant difference among the groups, p = 0.007). In contrast, the severe asthmatics demonstrated a twofold higher concentration of neutrophils in lavage than either the mild-moderate asthmatics, or the normal controls (p = 0.032 among the groups, p < 0.05 between the severe asthmatics and both controls). Similar results were obtained in the endobronchial and transbronchial biopsy specimens, which consistently showed significantly higher numbers of neutrophils in the severe asthmatics than in the control groups. The eicosanoid mediators, thromboxane and leukotriene B4, were also highest in the severe asthma group (differences among the groups, p = 0.019 and p = 0.023, respectively). These findings suggest that inflammation remains in severe symptomatic asthmatics despite treatment with high dose glucocorticoids which may be due to the severity of disease, glucocorticoid treatment, or other as yet undefined factors.

Immunofiltration purification for urinary leukotriene E4 quantitation.

Leukotriene E4 (LTE4) is a major leukotriene metabolite in urine. Urinary LTE4 concentration is often utilized as an index of total leukotriene synthesis. A novel method employing immunofiltration for the purification of urinary LTE4 was developed. This immunofiltration method is based upon the addition of excess anti-LTE4 antibody to urine which binds LTE4. Separation of bound LTE4 (high M(r)) from high levels of unbound contaminants (low M(r)) is then accomplished by filtration through a 10,000 M(r) cut-off filter. The LTE4-antibody complex is separated by precipitation of the antibody with methanol which is subsequently removed by centrifugation. Following evaporation of the methanol, enzyme immunoassay is utilized for quantitation. This methodology was validated by determining the recovery of tritiated and unlabeled LTE4 added to urine and buffer and by comparison of results obtained with urine samples measured after HPLC purification (correlation r2 = 0.72). Reproducibility of the assay was assessed by analyzing the same sample on two different days (standard deviation of 18%). The mean urinary LTE4 levels in healthy subjects and asthmatics measured utilizing this method were found to be identical to levels determined by HPLC/immunoassay. The ease and accuracy of this assay make it amenable for the analysis of large numbers of samples.

The effect of salmeterol on nocturnal symptoms, airway function, and inflammation in asthma.

STUDY OBJECTIVE: To determine the efficacy of salmeterol alone in a group of patients with moderate asthma with nocturnal worsening of symptoms. DESIGN: Double-blind, randomized, placebo-controlled crossover study. SETTING: Tertiary care hospital specializing in respiratory diseases. PARTICIPANTS: Ten patients with nocturnal asthma. INTERVENTIONS: Subjects were randomized to salmeterol, 100 micrograms twice daily, or placebo for 6 weeks with a 1-week washout between treatment periods. Symptoms, nocturnal awakenings, and beta 2-agonist use were recorded daily. Spirometry was performed at weeks 1 and 6 of each period at bedtime and at 4 AM, and methacholine challenge was performed at 4 AM followed by bronchoscopy with BAL. BAL fluid analysis included cell count and differential count, eosinophil cationic protein, Charcot-Leyden crystal protein, leukotriene B4, and thromboxane B2. RESULTS: The percentage of nights with awakenings decreased significantly with salmeterol (69.8 +/- 8.7% vs 30.6 +/- 10.8% for placebo and salmeterol, respectively; p = 0.02). The percentage of 24-h days with supplemental inhaled beta 2-agonist use significantly decreased with salmeterol (85.9 +/- 9.4% vs 70.4 +/- 10.1% for placebo and salmeterol, respectively; p = 0.04). There were no significant differences in bronchial reactivity, 4 AM FEV1, overnight percentage change in FEV1, or indexes of airway inflammation. CONCLUSIONS: Salmeterol alone improves the number of nocturnal awakenings and supplemental 24-h beta 2-agonist use in nocturnal asthma without significantly altering lung function and airway inflammation.

Arachidonate-induced eicosanoid synthesis in RBL-2H3 cells: stimulation with antigen or A23187 induces prolonged activation of 5-lipoxygenase.

We studied the ability of rat basophilic leukemia (RBL-2H3) cells stimulated with either IgE/antigen or calcium ionophore, A23187, to synthesize LTC4 and PGD2 after addition of exogenous arachidonic acid. RBL-2H3 cells preferentially synthesized PGD2 in response to stimulation with low concentrations of antigen or A23187 while higher concentrations also resulted in a marked synthesis of LTC4. The synthesis of LTC4 was dependent upon initial activation of 5-lipoxygenase by IgE/antigen or A23187, since arachidonic acid lone failed to induce LTC4 synthesis. Following the addition of IgE/antigen or A23187 alone, the synthesis of PGD2 and LTC4 was essentially complete by 10 min. To determine whether a limitation of substrate precluded further eicosanoid synthesis, exogenous arachidonic acid was added to washed cells 15-145 min following the initial stimulation with IgE/antigen or A23187, PGD2 and LTC4 synthesis was resumed following the addition of arachidonic acid to washed prestimulated cells, demonstrating that the termination of eicosanoid synthesis in RBL-2H3 cells was nor caused by the inactivation of cyclooxygenase and 5-lipoxygenase. DNP-lysine was added to cells previously stimulated with IgE/antigen to stop receptor aggregation and this greatly inhibited subsequent production of LTC4 following the addition of arachidonic acid, suggesting that ongoing stimulation of Fc epsilon XsRI was required for LTC4 synthesis in this setting. These results indicate that the magnitude of a physiologic stimulus (IgE/antigen) can profoundly affect the arachidonate metabolites produced by mast cells and that the synthesis of these metabolites quickly becomes limited by substrate availability rather than the activity of cyclooxygenase or 5-lipoxygenase.

Peripheral airways responsiveness to cool, dry air in normal and asthmatic individuals.

Peripheral airways resistance (Rp) has been shown to be increased in asymptomatic asthmatic patients with normal spirometric values, and to be correlated with airways hyperresponsiveness to methacholine. We investigated whether Rp in asthmatic subjects with exercise-induced bronchospasm (EIB) would rise in response to cool, dry air. Using a wedged bronchoscope technique, we challenged an isolated lung segment with high flows (500 to 1,000 ml/min) of cool (22 degrees C) dry 5% CO2 in air for 5 min in eight asthmatic subjects with EIB and eight normal subjects. Baseline Rp and Rp following challenge were measured with saturated air at 37 degrees C at a flow rate of 100 ml/min. Baseline Rp was significantly greater in the asthmatic (0.09; [0.05 to 0.23] cm H2O/ml/min; median [interquartile range]) than in the normal subjects (0.05; [0.03 to 0.07] cm H2O/ml/min) (p = 0.04). The asthmatic, but not the normal subjects, had a significant absolute maximal increase in Rp following cool, dry air (0.10 [0.03 to 0.15] cm H2O/ml/min) (p < 0.01). In the asthmatic subjects, baseline Rp correlated with airways hyperresponsiveness to exercise (r = -0.76, p = 0.03). We conclude that the peripheral airways of asthmatic individuals with EIB are responsive to cool, dry air, and may play an important role in EIB.