Human Bronchi Research Articles

Effects of ouabain on human bronchial muscle in vitro.

The effects of ouabain, an inhibitor of the plasmalemmal Na(+)/K(+)-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with -logEC(50)=7.16+/-0.11 and maximal effect of 67+/-4% of the response to acetylcholine (1 mM). Ouabain (10 microM)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H(1)-receptors and alpha-adrenoceptors, or neuronal Na(+) channel blockade. The inhibition of ouabain contraction in tissues bathed in K(+)-free medium, and the inhibition by ouabain of the K(+)-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na(+)/K(+)-ATPase. Furthermore, depolarization (16.4+/-0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 microM)-induced contractions were abolished by a Ca(2+)-free solution but not by blockers of L-type Ca(2+) channels. In human cultured bronchial smooth muscle cells, ouabain (10 microM) produced a sustained increase in [Ca(2+)](i) (116+/-26 nM) abolished in Ca(2+)-free medium. Incubation with a Na(+)-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na(+)/Ca(2+) exchange in ouabain contraction while selective inhibitors of Na(+)/H(+)-antiport, Na(+)/K(+)/Cl(-)-antiport, or protein kinase C had no effect. Ouabain (10 microM) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 microM) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca(2+) entry unrelated to L-type channels and involving the Na(+)/Ca(2+)-antiporter.

Human fetal trachea xenografts recapitulate normal development: implications for fetal gene therapy using pseudotyped AAV vectors

Introduction: We have previously demonstrated efficient gene transfer with Adeno Associated Virus serotype2 (AAV2) to the respiratory epithelium (RE) and submucosal glands (SMG) in an in vitro human fetal trachea organ culture. To validate these results, we xenotransplanted human fetal tracheas into SCID mice to determine if they would recapitulate normal trachea development and evaluated the efficiency of pseudotyping AAV vectors on gene transfer. Methods: Human fetal tracheas (GA 15–24 wks, n = 12) were compared to gestational age matched fetal trachea xenografts (n = 9) at 4, 8, 12 weeks post transplantation using developmental staging and morphometric analysis. 1x1011 genome copies of AAV2/2 (n = 3/vector), 2/1, 2/5, 2/7 or 2/8 were injected into human fetal trachea and analyzed at 1 and 3 months for transgene expression. Adult human bronchi (n = 4) were xenografted as postnatal controls, injected with AAV 2/2 and 2/8 and analyzed at one month. Data expressed as mean ± SEM Results: Xenografted tracheal development was delayed following engraftment with a resumption of a normal developmental pattern with increased number of glands, gland size and decrease in gland density at 2 and 3 months respectively. AAV2/2 (93 ± 1.5% RE, 37 ± 2% SMG) and AAV2/8 (91 ± 1.9% RE, 54.4 ± 1.4% SMG) transduced significantly greater (p < 0.0001) RE and SMG cells compared to AAV2/1, 2/5, and 2/7(<0.01% RE and SMG). The efficiency of AAV2/8 in SMG transduction was significantly (p < 0.001) greater than AAV2 at one month. At 3 months, AAV2/2 and AAV 2/8 produced transgene expression in >99% of RE and SMG. Gene transfer with AAV 2/2 and 2/8 was significantly greater (p < 0.0001) in the RE and SMG in the fetal trachea compared to the postnatal xenografts. (2/2: RE 93 ± 2% vs.16 ± 2, SMG 37 ± 2% vs.12 ± 2; 2/8: RE 91 ± 2% vs.32 ± 4, SMG 54 ± 1% vs.20 ± 3) Conclusions: Fetal trachea xenografts recapitulate normal fetal tracheal development, but phase shifts 3–4 weeks following engraftment. AAV2/2 and 2/8 are the most efficient pseudotypes for gene transfer to the fetal RE and SMG. Gene transfer was more efficient to the fetal trachea than postnatal controls, suggesting the fetal trachea is a permissive environment highly receptive to gene transfer, and supports the concept of fetal gene therapy for Cystic Fibrosis.

Thromboxane prostanoid receptor in human airway smooth muscle cells: a relevant role in proliferation

Thromboxane A 2 has been implicated as a mediator of bronchial hyperresponsiveness in asthma. Modulating agents are currently marketed in Japan and under clinical evaluation in the US, but full characterization of the thromboxane A 2 receptor and the signaling pathways that link it to the proliferative events taking place during airways structural remodeling has not been achieved. Here, we report that the presence of mRNA for both α and β isoforms of the thromboxane A 2 receptor in smooth muscle cells from human bronchi correlates with protein expression evaluated by radioligand binding of the antagonist, SQ29,548 ([1 S-[1α,2α( Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic-acid) ( K d=3.4 nM±44%CV, coefficient of variation, B max=41 fmol/mg prot±38%CV). The receptor is functional, as the agonist, U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5 Z,13 E-dien-1-oic-acid), induced a concentration-dependent Ca 2+ transient (EC 50=0.12 μM±27%CV). Furthermore, U46619 concentration dependently increased DNA synthesis and markedly potentiated the epidermal growth factor mitogenic effect. Both events were specifically inhibited by SQ29,548, independently from transactivation of the epidermal growth factor receptor and partially sensitive to pertussis toxin.

Effects of cysteinyl leukotrienes in small human bronchus and antagonist activity of montelukast and its metabolites.

Evidence suggests that small airways contribute to clinically significant processes in asthma. Cysteinyl leukotrienes (CysLTs) are considered to be pivotal mediators in the pathogenesis of asthma. Montelukast (MK), a specific CysLT1 receptor antagonist, is metabolized in two main hydroxylated metabolites (termed M5 and M6, respectively). The aims of this study were to compare the responsiveness of small and large human bronchi to the three CysLTs, to evaluate the antagonist activity of MK, M5 and M6 in these preparations of human bronchi, and to characterize the CysLT receptors involved in the contractile response. In isolated small bronchus (i.d. 0.5-2 mm), the potencies (-log molar EC50) of LTC4, LTD4 and LTE4 were 9.3 (n=11), 9.1 (n=30) and 8.4 (n=14), respectively. The three CysLTs were about 30-fold more potent in small bronchi than in larger bronchi (i.d. 4-6 mm). In small bronchi, MK significantly shifted to the right the CysLT concentration-effect curves with pA2 values against LTC4, LTD4 and LTE4 of 9.1 (n=3), 9.0 (n=11) and 8.7 (n=5), respectively. The antagonist potencies of M6 and M5 were similar to MK and fivefold lower, respectively. A similar activity of MK against the three CysLTs suggested that CysLT1 receptors are involved in the contraction of human bronchus. Analysis by RT-PCR also indicated that human bronchus mainly expressed CysLT1 receptors. MK exerts a potent antagonist activity against the particularly potent constricting effects of CysLTs in isolated human small bronchi, which only expressed the CysLT1 receptor subtype. The metabolites of MK are also potent in vitro antagonists, but may not participate in the therapeutic activity of MK due to their low plasma concentrations in patients treated with the recommended dose of MK.

T cells of atopic asthmatics preferentially infiltrate into human bronchial xenografts in SCID mice.

T cells play an important role in the pathogenesis of bronchial asthma. However, it is not completely known how circulating lymphocytes infiltrate into the airways of asthmatic patients. Because SCID mice are unable to reject xenogenic transplants, many xenotransplant models using various human tissues have been developed. Therefore, to examine the interaction between bronchi and T lymphocytes of asthma, it may be possible to use the human bronchial xenograft and PBMC xenograft in SCID mice. We transplanted human bronchi into the subcutaneum of SCID mice and i.p. injected PBMCs that were obtained from patients with atopic asthma, atopic dermatitis and rheumatoid arthritis, and normal subjects (asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice). There was no difference in the percentage of CD3-, CD4-, CD8-, CD25-, CD45RO-, CD103-, and cutaneous lymphocyte Ag-positive cells in PBMCs among the patients with asthma, dermatitis, rheumatoid arthritis, and normal subjects, and CD3-positive cells in peripheral blood of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. The number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice. IL-4 mRNA and IL-5 mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than those in the xenografts of normal huPBMC-SCID mice, but there were no significant differences in the expressions of IL-2 mRNA or IFN-gamma mRNA between them. These findings suggest that T cells, especially Th2-type T cells, of asthmatics preferentially infiltrate into the human bronchi.

Immuno affinity purification combined with mass spectrometry detection for the monitoring of VEGF isoforms in patient tumor tissue

AbstractVascular endothelial growth factor (VEGF) is a key mediator of tumor growth and metastasis. At least 7 isoforms of the protein exist due to differential splicing, but little is known about the biological activities of these different isoforms. We purified VEGF from resected human bronchial carcinoma tissue and normal bronchus by immuno affinity chromatography (IAC) and immuno capture (IC) on a ProteinChip® Array, identifying the isoforms by MS. Both methods proved to be useful for the analysis of protein isoforms, which are present in situ in tumor tissue. Different expression patterns of VEGF were demonstrated in tumor tissue compared to healthy tissue. The possibility to analyse VEGF protein isoforms in situ creates new opportunities to study the regulation of angiogenesis in healthy and malignant tissues.

Lactoperoxidase and human airway host defense.

The lactoperoxidase (LPO) antibiotic system is a well-characterized component of mammary and salivary gland secretions. Because LPO has been shown to function in ovine airways, human airway tissue and secretions were examined for the presence of LPO and its substrate, the anion thiocyanate (SCN-). In addition, human airway secretions were tested for LPO-mediated antibacterial activity, and LPO's activity was assessed against some human airway pathogens. The data showed that normal human airway secretions contained LPO enzyme activity (0.65 +/- 0.09 microg/mg secreted protein; n = 17), and Western blots of secretions demonstrated bands of the expected sizes for LPO. LPO mRNA was detected in trachea by sequencing PCR-amplified cDNA. SCN-, LPO's substrate, was present in undiluted airway secretions at concentrations sufficient for LPO catalysis (0.46 +/- 0.19 mM; n = 8), and diluted secretions contained antibacterial activity with LPO-like properties. Immunocytochemistry localized LPO to submucosal glands in human bronchi. Finally, as expected based on the known antibacterial spectrum of the LPO system, airway secretions showed LPO-dependent activity against Pseudomonas aeruginosa. In addition, the airway LPO system was shown to be effective against Burkholderia cepacia and Haemophilus influenzae. Thus, a functional LPO system exists in human airways and may contribute to airway host defense against infection.

Spectroscopic imaging and the characterization of the autofluorescence properties of human bronchus tissues using uv laser diodes

Ultraviolet laser diodes (UV-LD) were used for the excitation source of autofluorescence (AF) measurements and spectroscopic imaging of the AF originating from the human bronchus was obtained. The AF spectra from normal bronchus tissues were measured and a clear AF spectrum was obtained by using a short wavelength (400 nm) laser diode; the overlap of the AF signal and excitation source could be substantially eliminated. In order to study the origin of AF intensity deterioration from bronchus tissue due to the formation of tumor tissues, the fluorescence spectrum was measured for various AF substances under various conditions. The blue AF signal of elastin and NADH solutions, which could not be easily studied by conventional excitation light sources, as well as the green AF became weak by adding lactic acid. The AF spectrum was measured for 512/spl times/512 pixels and the intensity mapping as a function of emission wavelength was obtained. Two-dimensional information of the AF signal intensity distribution for a certain wavelength component was measured. The feature originating from a region as small as about 100 /spl mu/m could be recognized. Numerical calculations of the data were performed and precise features of the AF were revealed.

16HBE14o- human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1.

To study the expression of P-glycoprotein (P-gp), lung resistance-related protein (LRP), and caveolin-1 (cav-1) in the human bronchial epithelial cell line 16HBE14o-. The presence of P-gp, LRP, and cav-1 in 16HBE14o- cell layers was evaluated using immunocytochemical staining and visualization with confocal laser scanning microscopy (CLSM). Functionality of P-gp was determined by bidirectional transport of rhodamine-123 with and without a P-gp inhibitor, verapamil. Caveolae were visualized using transmission electron microscopy (TEM). Flux of fluorescein-Na was also studied as a paracellular transport marker. Immunocytochemical staining showed expression of P-gp localized at the apical membrane of 16HBE14o- cell layers. The flux of rhodamine 123 across cell layers exhibited a greater Papp value for the secretory (i.e., basolateral-to-apical) direction. This asymmetry disappeared in the presence of verapamil. CLSM provided evidence for the expression of LRP and cav-1. TEM further showed typically shaped caveolae at the apical and basolateral membranes. Cell layers of 16HBE14o- express drug transport systems that are also present in the human bronchus in vivo, indicating that the 16HBE14o- cell line may be a suitable candidate for an in vitro model for mechanistic studies of drug transport processes involved in the smaller airways.

SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]- 4-piperidinyl)-2-methylpropanamide], a centrally active nonpeptide antagonist of the tachykinin neurokinin-1 receptor: I. biochemical and pharmacological characterization.

The biochemical and pharmacological properties of a novel antagonist of the tachykinin neurokinin 1 (NK1) receptor, SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], were evaluated. SSR240600 inhibited the binding of radioactive substance P to tachykinin NK1 receptors in human lymphoblastic IM9 cells (K(i) = 0.0061 nM), human astrocytoma U373MG cells (K(i) = 0.10 nM), and human brain cortex (IC50 = 0.017 nM). It also showed subnanomolar affinity for guinea pig NK1 receptors but was less potent on rat and gerbil NK1 receptors. SSR240600 inhibited [Sar(9),Met(O2)(11)]substance P-induced inositol monophosphate formation in human astrocytoma U373MG cells with an IC50 value of 0.66 nM (agonist concentration of 100 nM). It also antagonized substance P-induced contractions of isolated human small bronchi with a pIC50 value of 8.6 (agonist concentration of 100 nM). The compound was >100- to 1000-fold more selective for tachykinin NK1 receptors versus tachykinin NK2 or NK3 receptors as evaluated in binding and in vitro functional assays. In vivo antagonistic activity of SSR240600 was demonstrated on tachykinin NK1 receptor-mediated hypotension in dogs (3 and 10 microg/kg i.v.), microvascular leakage (1 and 3 mg/kg i.p.), and bronchoconstriction (50 and 100 microg/kg i.v.) in guinea pigs. It also prevented citric acid-induced cough in guinea pigs (1-10 mg/kg i.p.), an animal model in which central endogenous tachykinins are suspected to play a major role. In conclusion, SSR240600 is a new, potent, and centrally active antagonist of the tachykinin NK1 receptor, able to antagonize various NK1 receptor-mediated pharmacological effects in the periphery and in the central nervous system.

Nerve growth factor levels and localisation in human asthmatic bronchi.

Nerve growth factor (NGF) has recently been suggested to be an important mediator of inflammation. In support of this, serum levels of NGF have been shown to be enhanced in asthmatics. However, it has not yet been shown whether the levels of NGF are also altered locally in asthmatic airways, when compared with healthy subjects, and the localisation of potential sources of NGF in the human bronchus have not yet been described. The aim of the present study was to assess NGF levels in bronchoalveolar lavage fluid (BALF) from asthmatics and to compare them to those of control subjects. Furthermore, the authors wanted to localise potential sources of NGF in bronchial tissue, and to number NGF-immunopositive infiltrating cells in the bronchial submucosa. BALF and bronchial biopsies were obtained from seven control subjects and seven asthmatic patients by fibreoptic bronchoscopy. NGF protein levels were quantified by enzyme-linked immunosorbent assay in BALF. NGF localisation was examined by immunohistochemistry on bronchial biopsy sections. The asthmatics exhibited significantly enhanced NGF levels in BALF. Intense NGF-immunoreactivity was observed in bronchial epithelium, smooth muscle cells and infiltrating inflammatory cells in the submucosa, and to a lesser extent in the connective tissue. The asthmatics exhibited a higher number of NGF-immunoreactive infiltrating cells in the bronchial submucosa than control subjects. This study provides evidence that nerve growth factor is locally produced in the airways, and shows that this production is enhanced in asthmatics. These findings suggest that nerve growth factor is produced by both structural cells and infiltrating inflammatory cells in human bronchus in vivo, and the authors suggest that the increase in nerve growth factor protein in bronchoalveolar lavage fluid observed in asthmatic patients may originate both from structural cells, producing increased nerve growth factor levels in inflammatory conditons, and from the increase in nerve growth factor-immunopositive cells determined in the bronchial submucosa.

In vitro sensitization of human bronchus by beta2-adrenergic agonists.

Incubation of human distal bronchi from 48 patients for 15 h with 10(-7) M fenoterol induced sensitization characterized by an increase in maximal contraction to endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human bronchi with 10(-6), 3 x 10(-6), and 10(-5) M forskolin (an adenyl cyclase activator) reproduced sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was inhibited by coincubation with gliotoxine (a nuclear factor-kappaB inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor), GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor antagonists) with or without HOE-140 (a bradykinin B(2) receptor antagonist), SB-203580 (an inhibitor of the 38-kDa mitogen-activated protein kinase, p38(MAPK)), or calphostin C (a protein kinase C blocker). Our results suggest that chronic exposure to fenoterol induces proinflammatory effects mediated by nuclear factor-kappaB and pathways involving leukotrienes, prostanoids, bradykinin, tachykinins, protein kinase C, and p38(MAPK), leading to the regulation of smooth muscle contraction to ET-1 and ACh.

Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus.

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.

Abnormal Re-epithelialization and Lung Remodeling in Idiopathic Pulmonary Fibrosis: The Role of ΔN-p63

Products of the p63 gene, a recently described member of the p53 family, are constitutively expressed in the basal cells of human bronchi and bronchioli. The truncated isoforms of the p63 gene (ΔN-p63 proteins) counteract the apoptotic and cell cycle inhibitory functions of p53 after DNA damage, and this property is likely to be central in the cell renewal strategy of stratified epithelial tissues. To investigate the dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we immunohistochemically analyzed the expression of the transactivating and dominant-negative isoforms of the p63 gene on 16 tissue samples obtained from patients suffering from this disorder. In most IPF cases herein investigated, epithelial cells expressing ΔN-p63 were observed at sites of abnormal proliferation at the bronchiolo-alveolar junctions, characterized by epithelial hyperplasia, squamous metaplasia, bronchiolization, and abnormal p53 nuclear accumulation. Similar features were not observed in normal lung and in samples taken from other pulmonary diseases used as controls, including acute interstitial pneumonia, idiopathic bronchiolitis obliterans organizing pneumonia, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia. On the basis of these findings, we can hypothesize a new model for UIP pathogenesis, involving a deregulated development of mesenchymal-epithelial interactions and abnormal proliferation of epithelial cells at the bronchiolo-alveolar junction after cell injury. In our view, the progressive loss of alveolar tissue and lung remodeling after injury in IPF/UIP is concomitantly produced by pneumocyte loss and alveolar collapse on one hand and by progressive bronchiolar proliferation and architectural distortion on the other.

X선 CT영상으로부터 인체의 기관지 모델의 개발

In this paper, we investigate the potential for retrieval of morphometric data from three dimensional images of conducting bronchus obtained by X-ray Computerized Tomography (CT) and to explore the potential for the use of rapid prototype machine to produce physical hollow bronchus casts for mathematical modeling and experimental verification of particle deposition models. We segment the bronchus of lung by mathematical morphology method from obtained images by CT. The surface data representing volumetric bronchus data in three dimensions are converted to STL(streolithography) file and three dimensional solid model is created by using input STL file and rapid prototype machine. Two physical hollow cast models are created from the CT images of bronchial tree phantom and living human bronchus. We evaluate the usefulness of the rapid prototype model of bronchial tree by comparing diameters of the cross sectional area bronchus segments of the original CT images and the rapid prototyping-derived models imaged by X-ray CT.

SCH 206272: a potent, orally active tachykinin NK 1, NK 2, and NK 3 receptor antagonist

Experiments were performed to characterize the pharmacology of SCH 206272 [( R, R)-1′[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4( Z)-(methoxyimino)pentyl]- N-methyl-2-oxo-[1,4′bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK 1, NK 2, and NK 3 receptors. SCH 206272 inhibited binding at human tachykinin NK 1, NK 2, and NK 3 receptors ( K i =1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca 2+] i mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK 1, NK 2, or NK 3 receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (p K b=7.7±0.3) induced by the tachykinin NK 1 receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (p K b=8.2±0.3) induced by the tachykinin NK 2 receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (p K b=7.6±0.2), NKA-induced contraction of the bronchus (p K b=7.7±0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1–10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1–3 mg/kg, p.o.) inhibited NK 1 and NK 2 activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK 1, NK 2, and NK 3 receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease.

Relaxation of proximal and distal isolated human bronchi by halothane, isoflurane and desflurane.

Volatile anaesthetics relax airway smooth muscle in vitro. The amount of relaxation might depend on the type and concentration of volatile anaesthetics, the calibre and precontraction level of the bronchi, and also on the species considered. These effects were investigated on isolated human bronchi. Isometric relaxations produced by halothane, isoflurane and desflurane bubbled on human bronchial rings precontracted with carbachol were recorded and compared with time controls. Volatile anaesthetics induced a concentration-dependent relaxation at 0.66, 1.33 and 2 minimum alveolar concentration (MAC). The relaxation was greater in mildly (carbachol 3x10(-7) M) than in highly (carbachol 2x10(-6) M) precontracted bronchi. Halothane was more potent in relaxing distal as compared to proximal bronchi; this differential effect was less pronounced with isoflurane and not observed with desflurane. While the three volatile anaesthetics induced similar relaxation on proximal bronchi, halothane was significantly more potent than desflurane on distal bronchi, with isoflurane being intermediate. The relaxation induced by 1.33 MAC of halothane, isoflurane and desflurane on moderately precontracted distal bronchi (carbachol 1x10(-6) M) was attenuated by pretreatment with glibenclamide 1x10(-5) M. In conclusion, halothane, isoflurane and desflurane exert direct but differential relaxant effects on human isolated bronchial smooth muscle. This may provide supplemental bronchodilation during anaesthesia. Although adenosine triphosphate-sensitive K+ channels are involved in these relaxant effects, they are unlikely to explain the observed differences between the three volatile anaesthetics.

Biotransformation of Formaldehyde in Cultured Human Bronchus

Biotransformation of formaldehyde to formic acid was studied to investigate the capacity of human bronchial epithelial cells to detoxicate formaldehyde. Normal human bronchial explants and normal primary bronchial epithelial cells were grown in medium containing 0.5 to 5 mM formaldehyde for up to 48 h. Formic acid was quantitated by analytical isotachophoresis. Explants were cultured with up to 5 mM formaldehyde for 48 h with approximately linear turnover, but at 5 mM the cells showed reduced biotransformation relative to the lower concentrations. The mean Km values for explants were 1.4 and 5.1 mM for cells and the mean Vmax values were 3.3 nmol/mg protein·min for the explants and 6.1 nmol/mg protein·min for the cells. By using the same technique with hepatocytes we found Km 1.25 mM and Vmax 4.2 nmol/mg protein·min, indicating that human bronchial epithelium cells have formaldehyde biotransforming activity similar to that of hepatocytes. Our results indicate that human bronchial epithelial cells oxidize formaldehyde at a relatively fast rate at concentrations up to 3 mM formaldehyde in the medium over a period up to 48 h.

Ex vivo development of functional human lymph node and bronchus-associated lymphoid tissue

We introduce a novel in vivo model of human mucosal immunity, based on the implantation of human fetal bronchial mucosa and autologous peribronchial lymph node (PLN) in the severe combined immunodeficiency (SCID) mouse. In the SCID host, human fetal bronchi implanted alone retain macrophages and mast cells but lose T cells. In contrast, fetal bronchi co-implanted with PLN contain, in addition to macrophages and mast cells, numerous T cells and B cells, often clustered in intramucosal bronchus-associated lymphoid tissue (BALT). Functionally, bronchus-PLN cografts are able to mount robust alphabeta and gammadelta T-cell-mediated immune responses to Pseudomonas aeruginosa and 3,4-epoxy-3-methyl-1-butyl-diphosphate challenges. No other autologous lymphoid organ (bone marrow, thymus, liver) allows for BALT development in co-implanted bronchi, which suggests special ontogenetic and functional relations between extramucosal PLN and intramucosal BALT. Overall, the bronchus-PLN cograft appears as a promising model for human bronchial immune development and function. Our study is the first to document long-term ex vivo maintenance of functional human lymph nodes as native appendices to mucosal tissue. Our results, therefore, suggest a simple strategy for developing similar experimental models of human immune function in other mucosae.

The IL-5 receptor on human bronchus selectively primes for hyperresponsiveness

Background: The role of IL-5–induced eosinophilia in airway hyperresponsiveness has been questioned. In addition, eosinophil-independent IL-5–induced airway hyperresponsiveness has been demonstrated in animals. Objective: In this study, IL-5 was investigated for direct effects on human bronchial responsiveness. Methods: Human muscle preparations were isolated from organ donor and surgical tissue. Bronchus, jejunum, and saphenous vein were incubated for 24 hours in vitro with recombinant human (rh) IL-5. Contractility to acetylcholine (bronchus, jejunum) and phenylephrine (saphenous vein) was then investigated. RT-PCR was used to evaluate IL-5 receptor α (IL-5Rα) expression in various tissues and to assess bronchus and saphenous vein eosinophils through use of CCR3 expression. Results: rhIL-5 primed bronchus for an exaggerated contraction to acetylcholine. The acetylcholine concentration that produced 50% of the control maximum response was reduced 17- to 20-fold in bronchus treated with 1 and 10 nmol/L rhIL-5. The lower concentration of 0.1 nmol/L rhIL-5 had no effect. The rhIL-5 effect on bronchial contractility was attenuated by antibodies to IL-5 (TRFK-5; 100 nmol/L) and human IL-5Rα (100 nmol/L). rhIL-5 (10 nmol/L) did not enhance contractility of saphenous vein or jejunum. When RT-PCR was used, IL-5Rα expression was strong in bronchus muscle, weak in trachealis, saphenous vein, and atrial muscle, and undetectable in jejunum, urinary bladder, and pulmonary and renal artery muscle. Comparable weak expression of CCR3 was identified in bronchus and saphenous vein. Conclusion: The findings are consistent with an airway tissue–selective expression of the IL-5 receptor that mediates IL-5–induced airway hyperresponsiveness independent of eosinophils. In asthma, in which IL-5 expression is elevated, IL-5 might directly induce bronchial hyperresponsiveness. (J Allergy Clin Immunol 2002;109:404-9.)