Measurement of the fraction of exhaled nitric oxide (FENO) has been proposed as a noninvasive assessment of asthmatic airway inflammation. The influence of the expiratory flow rate during the collection maneuver on the ability of FENO to discriminate healthy subjects from those with asthma is unknown. We compared online and offline measurement of FENO at different flow rates. FENO was collected with expiratory flows of 50-500 ml/second in 34 patients with asthma (PC(20) of less than 8 mg/ml) and 28 healthy subjects (PC(20) of more than 10 mg/ml) using offline collection techniques. In a subgroup of 18 individuals with asthma and 17 healthy subjects, we additionally measured FENO at multiple expiratory flow rates (47-250 ml/second) using online methods. FENO fell with an increasing expiratory flow rate; FENO was higher in subjects with asthma as compared with healthy subjects at each flow rate studied with both techniques (p < 0.001). Receiver operating characteristic (ROC) curves for the diagnosis of asthma indicated that FENO is a robust discriminator between individuals with asthma and healthy subjects (area under the ROC curves 0.79 +/- 0.06 to 0.86 +/- 0.06, p for significant discrimination < 0.0001). Neither expiratory flow rate nor collection technique (online versus offline) significantly altered this discriminatory capacity (area under the ROC curves = 0.84 +/- 0.07 with the slowest online method versus 0.80 +/- 0.07 with the fastest offline method, p = 0.46). These data indicate that the choice of expiratory flow rate and collection method can be based on practicality and patient comfort without compromising the utility of this test for asthma.

Nakamura, H., A. D. Luster, et al. (2001). "Variant eotaxin: its effects on the asthma phenotype." J Allergy Clin Immunol 108(6): 946-53.

BACKGROUND: Eotaxin, a CC chemokine expressed in the asthmatic lung, has been associated with impaired lung function. The role of its variant form is unknown. OBJECTIVE: The purpose of this study was to detect the population frequency and effects of a known single-nucleotide polymorphism in the eotaxin gene in which a threonine residue (THR(23)) is substituted for the wild-type alanine (ALA(23)) at the 23rd amino acid at the terminus of the peptide leader sequence. METHODS: We measured eotaxin protein secretion in 293 cells transfected with expression vectors and in PBMCs obtained from individuals bearing the alternative forms of the gene. A case-control study of plasma eotaxin levels and eosinophil counts, a comparison of baseline lung function by genotype in a population of 806 subjects with asthma, and a comparison of the allele frequency with a nonasthmatic population were performed. RESULTS: Human 293 cells and PBMCs with THR(23) variant eotaxin secreted significantly less eotaxin protein than did ALA(23)-bearing cells. In the case-control study, THR(23)-THR(23) individuals had lower plasma levels of eotaxin (310 [240-350] vs 420 [270-700] pg/mL; P < .05) and eosinophil counts (120 [5-220] vs 190 [110-470] cells/microL; P < .05) than ALA(23)-ALA(23) subjects; heterozygous subjects had intermediate levels. Higher levels of lung function were associated with THR(23) eotaxin (percent of predicted FEV(1), 65% +/- 3.5% [THR(23)-THR(23)] vs 58% +/- 0.9% [THR(23)-ALA(23)] and 56% +/- 0.5% [ALA(23)-ALA(23)]; P < .05). CONCLUSION: The THR(23) variant is associated with both decreased eosinophil counts and higher levels of lung function in subjects with asthma.

Wechsler, M. E., H. Grasemann, et al. (2000). "Exhaled nitric oxide in patients with asthma: association with NOS1 genotype." Am J Respir Crit Care Med 162(6): 2043-7.

An increased concentration of nitric oxide (NO) in exhaled air (FENO) is now recognized as a critical component of the asthmatic phenotype. When we identified patients with asthma on the basis of a standard case definition alone, we found that they were remarkably heterogeneous with respect to their FENO. However, when we included genotype at a prominent asthma candidate gene (i.e., NOS1) in the case definition, and determined the number of AAT repeats in intron 20, we identified a remarkably homogeneous cohort of patients with respect to FENO. Both mean FENO (p = 0.00008) and variability around the mean (p = 0.000002) were significantly lower in asthmatic individuals with a high number (> or = 12) of AAT repeats at this locus than in those with fewer repeats. These data provide a biologically tenable link between genotype at a candidate gene in a region of linkage, NOS1, and an important component of the asthmatic phenotype, FENO. We show that addition of NOS1 genotype to the case definition of asthma allows the identification of a uniform cohort of patients, with respect to FENO, that would have been indistinguishable by other physiologic criteria. Our isolation of this homogeneous cohort of patients ties together the well-established associations among asthma, increased concentrations of NO in the exhaled air of asthmatic individuals, and variations of trinucleotide repeat sequences as identified in several neurologic conditions.

Grasemann, H., C. N. Yandava, et al. (2000). "A neuronal NO synthase (NOS1) gene polymorphism is associated with asthma." Biochem Biophys Res Commun 272(2): 391-4.

Recent family-based studies have revealed evidence for linkage of chromosomal region 12q to both asthma and high total serum immunoglobulin E (IgE) levels. Among the candidate genes in this region for asthma is neuronal nitric oxide synthase (NOS1). We sought a genetic association between a polymorphism in the NOS1 gene and the diagnosis of asthma, using a case-control design. Frequencies for allele 17 and 18 of a CA repeat in exon 29 of the NOS1 gene were significantly different between 490 asthmatic and 350 control subjects. Allele 17 was more common in the asthmatics (0.83 vs 0.76, or 1.49 [95% CI 1.17-1.90], P = 0.013) while allele 18 was less common in the asthmatics (0.06 vs 0.12, or 0.49 [95% CI 0.34-0. 69], P = 0.0004). To confirm these results we genotyped an additional 1131 control subjects and found the frequencies of alleles 17 and 18 to be virtually identical to those ascertained in our original control subjects. Total serum IgE was not associated with any allele of the polymorphism. These findings provide support, from case-control association analysis, for NOS1 as a candidate gene for asthma.

Deykin, A., A. F. Massaro, et al. (2000). "Exhaled nitric oxide following repeated spirometry or repeated plethysmography in healthy individuals." Am J Respir Crit Care Med 161(4 Pt 1): 1237-40.

Subjects with asthma have higher concentrations of exhaled nitric oxide (NO) than normal individuals. It has been demonstrated that in asthmatics, repeated FVC maneuvers reduce NO. Although the cause of this phenomenon is not known, it has been hypothesized that deep breaths associated with FVC maneuvers reduce exhaled NO by affecting neural sources of NO, possibly via a mechanism related to the pathobiology of asthma. To establish whether FVC maneuvers influence NO concentrations in normal individuals, we measured exhaled NO at baseline values and after FVC maneuvers performed every 15 min for 1 h in subjects without asthma. To investigate the role of deep breaths in reducing exhaled NO, we compared these results with concentrations of exhaled NO after plethysmography. Repeated FVC maneuvers over 60 min produced a decrease in NO concentrations in mixed expired gas (F(E)NO; 24.6 +/- 5.1% decrease for F(E)NO, p < 0. 01 versus baseline). In contrast to the results after spirometry, repeated specific airway conductance (sGaw) maneuvers do not have a significant effect on F(E)NO (p = 0.16). These results, which demonstrate that in nonasthmatic subjects FVC maneuvers-but not panting maneuvers-produce a fall in NO, suggest that the mechanism responsible for the reduction in exhaled NO after FVC maneuvers is related to volume history of the lung rather than the pathobiology of asthma.

Deykin, A., O. Belostotsky, et al. (2000). "Exhaled nitric oxide following leukotriene E(4) and methacholine inhalation in patients with asthma." Am J Respir Crit Care Med 162(5): 1685-9.

Nitric oxide (NO) is a molecular gas that can be recovered in higher levels from the exhaled gas of subjects with asthma than from subjects without asthma. However, the precise mechanisms responsible of promoting increased fraction of expired nitric oxide (FE(NO)) in asthma are unknown. As leukotriene antagonism has been shown to reduce FE(NO) in patients with asthma, we hypothesized that leukotrienes mediate the increased FE(NO) encountered in this condition. Furthermore, because leukotriene antagonism stabilizes serum eosinophil markers during reductions in inhaled corticosteroid doses, and FE(NO) has been shown to correlate with sputum eosinophils in asthma, we reasoned that the effect of leukotrienes on FE(NO) might be mediated by eosinophils recruited to the airway by leukotrienes. To test this hypothesis, we performed methacholine and leukotriene (LT) E(4) bronchoprovocation challenges in 16 subjects with atopic asthma and measured FE(NO) and sputum differential counts before and after bronchoprovocation. We then compared FE(NO) in the seven subjects who developed increased sputum eosinophils following LTE(4) inhalation with values measured after methacholine inhalation in these seven subjects. Following LTE(4) inhalation, eosinophils rose from 4.01 +/- 0.89% pre-LTE(4) to 8.33 +/- 1.52% post-LTE(4). The mean change in sputum eosinophils from baseline after LTE(4) inhalation was larger than that after methacholine inhalation (+4.31 +/- 1.25% versus -1.14 +/- 0.93%). After LTE(4) inhalation, FE(NO) levels did not differ from prechallenge baseline or from levels following methacholine inhalation (ANOVA p > 0.05). These data indicate that neither LTE(4) nor recruitment of eosinophils into the airway by LTE(4) is a sufficient stimulus to acutely increase FE(NO) in subjects with asthma.

Attar, E. C., T. Ervin, et al. (2000). "Side effects of chemotherapy. Case 3. Acute interstitial pneumonitis related to gemcitabine." J Clin Oncol 18(3): 697-8.

Grasemann, H., J. M. Drazen, et al. (1999). "Simple tandem repeat polymorphisms in the neuronal nitric oxide synthase gene in different ethnic populations." Hum Hered 49(3): 139-41.

Allelic frequencies of a CA dinucleotide repeat in exon 29 and an intronic AAT trinucleotide repeat in the neuronal nitric oxide synthase (NOS1) gene were determined by simple sequence length polymorphism (SSLP) in 305 American-Caucasian and 105 African-American healthy subjects. There were highly significant differences in allele frequencies between the two ethnically diverse study populations.

Deykin, A. and E. Israel (1999). "Newer therapeutic agents for asthma." Adv Intern Med 44: 209-37.

The past decade has seen significant advances in the available treatments for asthma. These include longer-acting bronchodilating agents, high topical potency inhaled corticosteroids, and agents that interfere with leukotriene production or action. Table 3 summarizes the clinical effects of the newer therapeutic agents reviewed. Experimental therapies for the steroid-dependent patient have also been discussed. Although clinical trials to date have established many of these as effective in asthma, the results of ongoing, large, multicenter studies investigating the relative merits of these therapies, alone and in combination, will further clarify how to maximize the utility of these agents in the treatment of asthma.

Deykin, A. and E. Israel (1999). "Newer therapeutic agents for asthma." Dis Mon 45(4): 117-44.

The past decade has seen significant advances in the available treatments for asthma. These include longer-acting bronchodilating agents, high topical potency inhaled corticosteroids, and agents that interfere with leukotriene production or action. Table 3 summarizes the clinical effects of the newer therapeutic agents reviewed. Experimental therapies for the steroid-dependent patient have also been discussed. Although clinical trials to date have established many of these as effective in asthma, the results of ongoing, large, multicenter studies investigating the relative merits of these therapies, alone and in combination, will further clarify how to maximize the utility of these agents in the treatment of asthma.

Lara-Marquez, M. L., A. Deykin, et al. (1998). "Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma." J Allergy Clin Immunol 101(5): 699-708.

BACKGROUND: T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified. OBJECTIVE: To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma. METHODS: We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge. RESULTS: At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mean +/- SE, baseline, 12.5% +/- 1% versus 2 hours, 18.1% +/- 1% and 24 hours, 17.8% +/- 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% +/- 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO. CONCLUSIONS: We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.

Deykin, A., O. Halpern, et al. (1998). "Expired nitric oxide after bronchoprovocation and repeated spirometry in patients with asthma." Am J Respir Crit Care Med 157(3 Pt 1): 769-75.

Compared with normal individuals, subjects with asthma have elevated levels of expired nitric oxide (NO). These levels are hypothesized to reflect the degree of airway inflammation. Expired NO levels rise during the late phase of allergen challenge and decrease in asthmatics after steroid treatment. Isocapnic cold air hyperventilation (ISH) is believed to cause airway narrowing through noninflammatory mechanisms. We measured mixed expired NO in 10 individuals with atopic asthma who underwent both ISH challenge and allergen challenge, and compared these measurements with the change in expired NO that occurred after serial spirometry alone. We found that ambient NO levels affected mixed expired NO. Controlling for inspired NO, we found that repeated spirometry alone produced a significant fall in mixed expired NO (p < 0.01) that was maximal after 30 min (36.6 +/- 8.5% fall). After allergen and ISH challenges, expired NO was elevated relative to levels after repeated spirometry (p < 0.01 and p = 0.065, respectively). In addition, we found that prechallenge expired NO levels were significantly correlated with the magnitude of the late fall in FEV1 following allergen challenge (r = 0.80, p < 0.01). These data demonstrate that repeated spirometry results in reduced mixed expired NO and suggest that both ISH and allergen-induced bronchoconstriction share pathobiologic mechanisms that produce increases in mixed expired NO.

Davar, G., A. Hama, et al. (1991). "MK-801 blocks the development of thermal hyperalgesia in a rat model of experimental painful neuropathy." Brain Res 553(2): 327-30.

Loose ligation of the sciatic nerve in the rat can produce behavioral signs of hyperalgesia in the hindpaw. This study examined the effect of an NMDA (N-methyl-D-aspartate) receptor antagonist (MK-801) on the development of hyperalgesia in this model. Rats received i.p. injections of saline or MK-801 (1.0 mg/kg) prior to and then for 7 days after a unilateral sciatic nerve ligation. Testing of each hindpaw for latency to withdrawal from a standardized thermal stimulus was performed prior to ligation and then at 10, 12, 17, 27, and 37 days postoperatively. Hyperalgesia of the operated hindpaw developed in saline-treated animals as measured by a decrease in withdrawal latency. Hyperalgesia did not develop in animals treated with MK-801. MK-801 may therefore prevent the development of hyperalgesia following experimental nerve injury, possibly through an NMDA receptor-mediated effect.

Kramer, R. M., G. C. Checani, et al. (1986). "Solubilization and properties of Ca2+-dependent human platelet phospholipase A2." Biochim Biophys Acta 878(3): 394-403.

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.