Methacholine Research Articles

Molecular model of naphthalene-induced DNA damage in the murine lung

Airway epithelial damage and repair represents a novel therapeutic target in asthma and chronic obstructive pulmonary disease. An established mouse model of airway epithelial damage involves the Clara cell cytotoxicity of parenterally administered naphthalene, an important environmental toxicant with genotoxic and carcinogenic potential. The objective of the current study was to investigate naphthalene-induced toxicity and to identify and quantify DNA double-strand breaks in a murine naphthalene model of airway epithelial damage. Male C57/BL6 mice were injected with 200 mg/kg naphthalene and culled at 12-, 24-, 48- and 72-h time points. Lung function and bronchoalveolar lavage was performed and the lungs were dissected for histological analysis and for quantitation of DNA double-strand breaks using γH2AX as a molecular marker. Mice injected with naphthalene had increased epithelial denudation, bronchoalveolar lavage fluid cellularity and reactivity to nebulized methacholine chloride as compared to corn oil vehicle controls. Histological changes were most pronounced at the 12- and 24-h time points. DNA double-strand breaks, quantitated as the number of γH2AX foci per cell, were highest at the 24- and 48-h time points. All parameters had decreased at the 72-h time point, consistent with airway re-epithelization and cellular repair. Our findings indicate a time-dependent accumulation of γH2AX foci in mouse airway epithelial cells following administration of naphthalene. Naphthalene airway epithelial injury constitutes a model of DNA double-strand breaks in mice, which can be adapted as a suitable model for further investigation of genotoxic damage for evaluating the efficacy of potential therapeutics.

Aerosolized BIO-11006, a Novel MARCKS- Related Peptide, Improves Airway Obstruction in a Mouse Model of Mucus Hypersecretion

Rationale: Mucus hypersecretion has been shown to be an important cause of airway obstruction in COPD and asthma, and has been associated with fatal asthma. Previous in-vitro and in-vivo studies have provided direct evidence that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimu- lation at the goblet cell surface to mucin granule release. BIO-11006 is a novel, highly soluble, aerosolized peptide that inhibits MARCKS function and results in reduced mucus secretion and improved airway obstruction. BIO-11006 is being tested in Phase 2 clinical studies of COPD. Objective: To determine access of BIO-11006 to the intracellular compartment of normal human bronchial epithelial (NHBE) cells, and assess the effectiveness of BIO-11006 inhalation solution in inhibiting airway obstruction caused by mucus hypersecretion as a function of dose and time. Methods: Biotin labeled BIO-11006 was incubated with NHBE cells and visualized with fluorescein-labeled streptavidin. Ovalbumin-sensitized and challenged mice with airway inflammation and hyperresponsiveness to methacholine (MCh) were treated with aerosolized BIO-11006 solution prior to MCh challenge, and effects on airway obstruction and mucus secretion were measured. Results: A 30-minute aerosol exposure to a 10 mM BIO-11006 solution was maximally effective in inhibiting MCh- induced airway obstruction and mucus hypersecretion. There was a long-lasting effect on inhibition of airway obstruction after a single treatment with BIO-11006 (t 1/2 ~4 hrs). The duration of effect on inhibition of mucin secretion was shorter (t 1/2 ~2 hrs). Conclusion: The pharmacodynamic properties of aerosolized BIO-11006 Inhalation Solution are suitable for therapeutic use. Inhaled BIO-11006 effectively inhibits mucin secretion and improves airway obstruction.

Clinical research on the treatment of moderate bronchial asthma with inhaled inactivated-mycobacterium phlei

Objective To introduce inhaled inactivated-mycobacterium phlei on prevention and treatment of moderate bronchial asthma to observe the clinical effect. Method This study was a prospective and controlled study. The patients diagnosed with asthma in our out-patient from March 2009 to December 2010 were collected, who met the following conditions were included in the study: age≥ 14 years; met the criteria of moderate chronic persistent bronchial asthma in Global Initiative for Asthma (GINA) in 2008; suspended receiving systemic corticosteroids, Montelukast, ketotifen and other anti-inflammatory and anti-allergic drugs in one month; no significant respiratory tract infections; and other serious illnesses or abnormalities known.A total of 100 patients with asthma were selected, including 37 males and 63 females, age (32.11 ± 12.95 )years. The patients were randomly(random number) divided into two groups: A group(treatment group; 16males and 34 females, age 33.56 ± 14.23 years) and B group (control group; 21 males and 29 females,age 30.66 ± 11.50 years); 50 in each group. No significant difference was noted between the two groups on age and gender composition. The patients in A group were treated with inhaled inactivated-mycobacterium phlei F. U. 36 Injection 1.72 μg/mL × 2 that adding 3 mL normal saline, once a day for 5 days. The patients in B group were treated with salmeterol xinafoate and fluticasone propionate powder for inhalation (50/100 μg), twice daily for sustainable use. The patients in the two groups were observed for one month. During this course, the patients in the two groups could inhale the salbutamol sulphate aerosol as need to relieve symptoms. And the number of using was recorded. Pulmonary function test and asthma provocative test were carried out on the Day O, 6 and 31. ACT scores were measured before and after the treatment. Results On Day 6 and 31 after treatment, the negative conversion rates of asthma provocative test of the patients in A group were 82% and 78% respectively, B group were 84% and 90% respectively. Provocative test of the patients in the two groups were negative conversion significantly before and after treatment. There was no significant difference between the two groups by chi-square test (P ＞ 0. 05 ). Completely random designed data was analyzed by analysis of variance. The analysis showed that the accumulated doses of methacholine of the patients in the two group increased significantly ( P ＜ 0. 05 ), but no difference between the two groups.There was a improvement trend on forced expiratory volume in one second( FEV1 )of the patients in A group after treatment, but no difference. FEV1 of the patients in B group increased significantly higher ( P ＜0.05), which was significantly higher than A group on the 31th day (P ＜0. 05); Peak expiratory flow (PEF) of the patients in the two group increased significantly on Day 6 and 31 after treatment (P ＜0.05 ).On Day 31, B group was significantly higher than A group ( P ＜ 0. 05 ); Scores of asthma control test (ACT)of the patients in the two group were significantly increased, and the number of using of salbutamol sulfate aerosol was significantly reduced (P ＜0.01 ). B group was obvious than group A (P ＜0.05 ). During treatment, there were only two adverse reaction cases of transient low fever; most obvious was on the third day.Conclusions Inhaled inactivated-mycobacterium phlei would inhibit the airway hyperresponsiveness of the patients with moderate bronchial asthma in short time, improve the symptoms, reduce the acute exacerbation, and reduce the use of rescue medication, which has the roles of prevention and treatment of moderate asthma in a certain period of time. Key words: Asthma; Mycobaterium phlei; Airway hyperresponsiveness; Aerosol inhalation; Asthma control test; Forced expiratory volume in one second; Peak expiratory flow

Pulmonary effects of inhaled oil dispersant (COREXIT® EC9500A) in rats

COREXIT® EC9500A (CE) was used to disperse crude oil during the 2010 Deepwater Horizon oil spill response. While the environmental impact of CE has been examined, the pulmonary effects of CE are unknown. Here we investigated whether inhaled CE elicits airway inflammation, alters pulmonary function, or affects airway reactivity to methacholine (MCh). Rats were exposed to CE (mean 27 mg/m3, 5 h) and examined 1 and 7 d post‐exposure. Under anesthesia, the lung lavage fluid was harvested. Lactate dehydrogenase (LDH) and albumin (A) were measured as indices of lung injury, recovered lung cells were differentiated (macrophages, neutrophils, lymphocytes, and eosinophils) to evaluate inflammation, and oxidant production by macrophages was measured. There were no significant differences in LDH, A, inflammatory cell levels or oxidant production between air‐ and CE‐exposed groups at either time point. Pulmonary function assessed in conscious animals indicated that neither breathing frequency nor specific airway resistance were altered at 1 and 7 d post‐exposure. Airway resistance responses to MCh aerosol in anesthetized animals were unaffected at 1 and 7 d post‐exposure, while dynamic compliance responses were decreased at 1 d but not at 7 d post‐exposure. Our findings suggest that CE inhalation does not initiate lung inflammation, but may transiently increase the difficulty of breathing.

Sexual dimorphism in lung function responses to acute influenza A infection

Please cite this paper as: Larcombe et al. (2011) Sexual dimorphism in lung function responses to acute influenza A infection. Influenza and Other Respiratory Viruses 5(5), 334–342. Background Males are generally more susceptible to respiratory infections; however, there are few data on the physiological responses to such infections in males and females. Objectives To determine whether sexual dimorphism exists in the physiological/inflammatory responses of weanling and adult BALB/c mice to influenza. Methods Weanling and adult mice of both sexes were inoculated with influenza A or appropriate control solution. Respiratory mechanics, responsiveness to methacholine (MCh), viral titre and bronchoalveolar lavage (BAL) cellular inflammation/cytokines were measured 4 (acute) and 21 (resolution) days post‐inoculation. Results Acute infection impaired lung function and induced hyperresponsiveness and cellular inflammation in both sexes at both ages. Males and females responded differently with female mice developing greater abnormalities in tissue damping and elastance and greater MCh responsiveness at both ages. BAL inflammation, cytokines and lung viral titres were similar between the sexes. At resolution, all parameters had returned to baseline levels in adults and weanling males; however, female weanlings had persisting hyperresponsiveness. Conclusions We identified significant differences in the physiological responses of male and female mice to infection with influenza A, which occurred in the absence of variation in viral titre and cellular inflammation.

Effects of fudosteine, a new mucoactive drug, on the increase in mucus secretion produced by secretagogues in human pulmonary mucoepidermoid carcinoma cells

The purpose of this study was to clarify the effects of fudosteine ((—)-(R)-2-amino-3-(3-hydroxypropylthio)propionic acid), a new mucoactive drug, on mucus secretion in the human pulmonary mucoepidermoid carcinoma cell line NCI-H292. NCI-H292 cells produced hyaluronidase-resistant high-molecular-weight glycoconjugates (HMWG), which were eluted in the void volume on Superose 6HR column chromatography. ATP, bradykinin, and methacholine increased the basal secretion of [14C]glucosamine-labelled HMWG in NCI-H292 cells. Fudosteine significantly suppressed the HMWG secretion induced by ATP or bradykinin, but not that induced by methacholine. Other mucoactive drugs, such as ethylcysteine, ambroxol and carboxymethylcysteine, did not affect the increase in HMWG. These results indicate that fudosteine suppresses the mucin secretion induced by ATP and bradykinin in airway mucus-producing cancer cells.

Comparison of Exhaled Nitric Oxide Analyzers in Childhood Asthma

목 적: 호기산화질소(exhaled nitric oxide, eNO)는 천식환자의 기도염증을 반영하는 생물학적 지표로 잘 알려져 있지만, 검사 방법과 환자의 상태에 따라서 검사 결과가 달라질 수 있다. 본 연구에서는 소아 천식 환자에서 호기산화질소 측정기기 CLD88(R) (Ecomedics; Durnten, Switzerland) 과 Niox mino(R) (Aerocrine; Solna, Sweden) 의 호기산화질소 측정치를 비교하고 그 임상적 의의를 알아보고자 하였다. 방법: 세브란스 어린이병원에 내원한 천식환자 104명, 대조군 59명을 대상으로 하였으며 Niox mino(R)와 CLD88(R)을 이용하여 호기산화질소를 측정하고, 기관지확장제 투여 전후 폐기능검사, 메타콜린 유발시험, 유도객담검사를 시행하였다. 결과: Niox mino(R)로 측정한 호기산화질소는 천식군(38.4±27.9 ppb)에서 대조군(17.2±9.0 ppb, P<0.001)에 비해 유의하게 높았고, CLD88(R)을 이용한 경우에도 천식군(47.8±31.8 ppb)에서 대조군(22.2±12.7 ppb, P<0.001)보다 높았다. 측정치는 Niox mino(R)로 측정한 경우약 9.0 ppb 낮았다. 측정치의 급내상관계수(intraclass correlation coefficient)는 0.786(P<0.001)이었다. 호기산화질소 농도는 폐기능의 지표 중 FEV1/FVC, 유도 객담 내 호산구 비율과 유의한 상관관계를 보였으며, PC20 농도와는 전체를 대상으로 분석하는 경우 유의한 상관관계를 보였으나, 천식환자만을 대상으로 분석하였을 때에는 유의한 상관관계를 보이지 않았다. 결론: 호기산화질소 측정치는 Niox mino(R)와 CLD88(R)로 측정한 모두에서 천식군에서 대조군에 비하여 유의하게 높으므로 천식의 보조적인 진단도구로 사용될 수 있을 것으로 생각된다. 그러나 측정 기기에 따라 호기산화질소 농도의 측정치가 다를 수 있음을 유의해야 한다.

Biochanin A, a Phytoestrogenic Isoflavone with Selective Inhibition of Phosphodiesterase 4, Suppresses Ovalbumin‐Induced Airway Hyperresponsiveness

The present study investigated the potential of biochanin A, a phytoestrogenic isoflavone of red clover (Triflolium pratense), for use in treating asthma or chronic obstructive pulmonary disease (COPD). Biochanin A (100 μmol/kg, orally (p.o.)) significantly attenuated airway resistance (R L), enhanced pause (P enh), and increased lung dynamic compliance (C dyn) values induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed an increase in the number of total inflammatory cells, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) of the mice. However, it did not influence interferon (IFN)-γ levels. Biochanin A (100 μmol/kg, p.o.) also significantly suppressed the total and ovalbumin (OVA)-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the total IgG2a level in the serum of these mice. The PDE4H/PDE4L value of biochanin A was calculated as >35. Biochanin A did not influence xylazine/ketamine-induced anesthesia. Biochanin A (10~30 μM) significantly reduced cumulative OVA (10~100 μg/mL)-induced contractions in the isolated guinea pig trachealis, suggesting that it inhibits degranulation of mast cells. In conclusion, red clover containing biochanin A has the potential for treating allergic asthma and COPD.

S‐Petasin, the Main Sesquiterpene of Petasites formosanus, Inhibits Phosphodiesterase Activity and Suppresses Ovalbumin‐Induced Airway Hyperresponsiveness

S-Petasin is the main sesquiterpene of Petasites formosanus, a traditional folk medicine used to treat hypertension, tumors and asthma in Taiwan. The aim of the present study was to investigate its inhibitory effects on phosphodiesterase (PDE) 1–5, and on ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) in a murine model of allergic asthma. S-Petasin concentration-dependently inhibited PDE3 and PDE4 activities with 50% inhibitory concentrations (IC50) of 25.5, and 17.5 μM, respectively. According to the Lineweaver-Burk analysis, S-petasin competitively inhibited PDE3 and PDE4 activities with respective dissociation constants for inhibitor binding (K i) of 25.3 and 18.1 μM, respectively. Both IC50 and K i values for PDE3 were significantly greater than those for PDE4. S-Petasin (10–30 μmol/kg, administered subcutaneously (s.c.)) dose-dependently and significantly attenuated the enhanced pause (P enh) value induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed the increases in total inflammatory cells, lymphocytes, neutrophils, eosinophils and levels of cytokines, including interleukin (IL)-2, IL-4 and IL-5, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in bronchoalveolar lavage fluid (BALF) of these mice. In addition, S-petasin (10–30 μmol/kg, s.c.) dose-dependently and significantly attenuated total and OVA-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the IgG2a level in serum of these mice. The PDE4H value of S-petasin was >300 μM; therefore, its PDE4H/PDE4L value was calculated to be >17. In conclusion, the present results for S-petasin at least partially explain why Petasites formosanus is used as a folk medicine to treat asthma in Taiwan.

Fetal nicotine exposure increases airway responsiveness and alters airway wall composition in young lambs

To test the hypotheses that fetal nicotine exposure alters airway wall composition and enhances the airway response to inhaled methacholine (MCh), lambs were exposed during the last fetal trimester to (1) a low dose (LN) (n=13, 0.5mg/kg/d (maternal weight) of free base nicotine, (2) a moderate dose (MN) (n=10, 1.5mg/kg/d) or (3) saline (n=14). Studies were performed at postnatal days 12, 26 and 52. Prenatal nicotine exposure induced a dose- and age-related hyper-responsiveness to MCh in the proximal airways. Moment analysis of nitrogen decay curves showed no nicotine or MCh effects on ventilation homogeneity or gas-mixing efficiency in the distal airways during MCh inhalations suggesting a bimodal response. Fetal nicotine exposure increased epithelial mucosubstance volume in central (LN, MN) and distal bronchi (LN), increased smooth muscle volume in distal bronchi and bronchioles (LN) and decreased bronchiolar diameter (MN). In conclusion, third trimester nicotine exposure causes hyperreactive proximal airways and alters proximal airway wall composition associated with airflow limitation.

Comparison of central and peripheral airway involvement before and during methacholine, mannitol and eucapnic hyperventilation challenges in mild asthmatics

Testing for airway hyperresponsiveness with indirect stimuli as exercise or mannitol has been proposed to better reflect underlying airway inflammation, as compared with methacholine (MCh), believed to act directly on airway smooth muscle cells. To investigate whether different direct and indirect stimuli induces different patterns of obstruction, recorded as central and peripheral resistance, and to see whether baseline resistance could predict a positive response to direct or indirect provocation. Thirty-four mild asthmatics and 15 controls underwent MCh, mannitol and eucapnic voluntary hyperventilation (EVH) challenge tests. The response was evaluated with spirometry and impulse oscillometry (IOS). Twenty-three out of 34 asthmatics were positive to either EVH (22) or mannitol (13). Those positive to mannitol had a significant increased baseline value of IOS parameters such as ΔR5-R20 and AX. Twelve of the asthmatics had a 10% fall or more in forced expiratory volume in 1 s (FEV(1)) in all three challenge tests. However, the response pattern measured by IOS did not differ between the tests. When the limit for a positive mannitol provocation was set to 10% fall in FEV(1), 16 out of 19 mannitol-positive patients were also positive to EVH. Even in mild asthmatics, a substantial number had a positive indirect test. Mannitol FEV(1) provocative dose to decrease FEV(1) by 10% from baseline (PD10) was closely associated to EVH10%. No difference in bronchoconstrictive pattern could be seen between the different provocation tests, but those positive to mannitol had more peripheral airway involvement at baseline. This supports the idea that peripheral airway involvement is an important predictor of asthma airway reactivity.

Amphetamine modulates cellular recruitment and airway reactivity in a rat model of allergic lung inflammation

Asthma is characterized by pulmonary cellular infiltration, vascular exudation and airway hyperresponsiveness. Several drugs that modify central nervous system (CNS) activity can modulate the course of asthma. Amphetamine (AMPH) is a highly abused drug that presents potent stimulating effects on the CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12 h prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium–leukocyte interaction mechanisms, cytokine release and mast cell degranulation.

Bronchodilation response to deep inspirations in asthma is dependent on airway distensibility and air trapping

In healthy individuals, deep inspirations (DIs) have a potent bronchodilatory ability against methacholine (MCh)-induced bronchoconstriction. This is variably attenuated in asthma. We hypothesized that inability to bronchodilate with DIs is related to reduced airway distensibility. We examined the relationship between DI-induced bronchodilation and airway distensibility in 15 asthmatic individuals with a wide range of baseline lung function [forced expired volume in 1 s (FEV(1)) = 60-99% predicted]. After abstaining from DIs for 20 min, subjects received a single-dose MCh challenge and then asked to perform DIs. The effectiveness of DIs was assessed by the ability of the subjects to improve FEV(1). The same subjects were studied by two sets of high-resolution CT scans, one at functional residual capacity (FRC) and one at total lung capacity (TLC). In each subject, the areas of 21-41 airways (0.8-6.8 mm diameter at FRC) were matched and measured, and airway distensibility (increase in airway diameter from FRC to TLC) was calculated. The bronchodilatory ability of DIs was significantly lower in individuals with FEV(1) <75% predicted than in those with FEV(1) ≥75% predicted (15 ± 11% vs. 46 ± 9%, P = 0.04) and strongly correlated with airway distensibility (r = 0.57, P = 0.03), but also with residual volume (RV)/TLC (r = -0.63, P = 0.01). In multiple regression, only RV/TLC was a significant determinant of DI-induced bronchodilation. These relationships were lost when the airways were examined after maximal bronchodilation with albuterol. Our data indicate that the loss of the bronchodilatory effect of DI in asthma is related to the ability to distend the airways with lung inflation, which is, in turn, related to the extent of air trapping and airway smooth muscle tone. These relationships only exist in the presence of airway tone, indicating that structural changes in the conducting airways visualized by high-resolution CT do not play a pivotal role.

Tidal Airway Closure During Bronchoconstriction in Asthma: Usefulness of Lung Volume Measurements

Background. The presence and extent of tidal airway closure is not routinely assessed in asthma. The objective of this study was to provide a simple functional tool able to detect tidal airway closure during bronchoconstriction in asthma. Methods. In 20 subjects with mild persistent asthma, we sequentially performed the measurement of functional residual capacity (FRC) by body plethysmography (pleth) and multibreath helium dilutional technique (He) and then computed residual volume (RV) and total lung capacity (TLC) at baseline, at the end of methacholine (MCh) challenge and after bronchodilator (albuterol). Measurements and main results. Despite substantial bronchoconstriction (fall in FEV1 = 35 ± 7%), TLC,pleth did not change following MCh challenge, but FRC,pleth because of dynamic pulmonary hyperinflation (+0.68 ± 0.54 L) and RV,pleth because of air trapping (+0.65 ± 0.37 L), invariably increased (on average by 22% and 46%, respectively). In contrast, FRC,He (and RV,He and TLC,He) could either increase, as seen in 13 subjects (Group I), or decrease, as seen in 7 subjects (Group II). Hence, the difference between FRC,pleth and FRC,He (Diff. FRC,pleth – FRC,He) was much greater in Group II (1.03 ± 0.41 L) than in Group I (0.22 ± 0.20 L) (p < .01). No functional differences were found between the two groups, including baseline PD20FEV1 and absolute and percent change in forced vital capacity (FVC) at the end of the MCh challenge. Conclusions. Comparison between FRC,pleth and FRC,He is useful to identify asthmatics prone to tidal airway closure during MCh-induced bronchoconstriction and Diff. FRC,pleth – FRC,He can be used to measure the overall unventilated lung volume upstream of the airways closed at end-expiratory lung volume (EELV).

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma

BackgroundExposure to chlorine (Cl2) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl2-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury.MethodsBalb/C mice were exposed to Cl2 gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl2, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl2 exposure.ResultsMice exposed to Cl2 had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl2 exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl2-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl2 prevented lipid peroxidation in the lung. Following Cl2 exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl2 exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU.ConclusionOur data show that the anti-oxidant DMTU is effective in attenuating Cl2 induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.

No role for neutrophil elastase in influenza-induced cellular recruitment, cytokine production or airway hyperresponsiveness in mice

Previous studies have suggested that in vitro modulation of neutrophil chemokines and inflammatory cytokines by neutrophil elastase (NE) does not translate to the in vivo setting. We aimed to test the role of NE in the recruitment of neutrophils, cytokine production and lung function responses to respiratory viral infection. To address this, we inoculated neutrophil elastase (NE(-/-)) deficient and wild-type (WT) 129Sv mice with 50μL of 10(4.5)pfu Influenza A/Mem71 (H3N1) or a control preparation. Mice were subjected to methacholine (MCh) challenge at 3-4 days post-infection during the peak of cellular inflammation. Inflammation, protein content and cytokines (TNF-α and MIP-2) were assessed in bronchoalveolar lavage fluid. Influenza-infected mice had a heightened responsiveness to MCh, increased cellular inflammation, increased protein leak and altered cytokine production, none of which were influenced by the absence of NE. These data demonstrate that NE does not modulate neutrophil recruitment, cytokine production, epithelial permeability or responsiveness to bronchoconstricting agents during acute influenza infection in mice.

Establishment of airway eosinophilic bronchitis mouse model without hyperresponsiveness by ovalbumin

Eosinophilic bronchitis (EB) is a useful tool for studying airway hyperresponsiveness (AHR), but an exact EB animal model is lacking. Our objective was to establish an EB mouse model using ovalbumin (OVA). Mice were divided into asthma, normal saline (NS) control and three model groups. Asthma mice were challenged intranasally with 200 μg OVA. Model groups were challenged with one of the three OVA doses (10, 20 or 100 μg), and NS control mice received normal saline. Changes in lung resistance (R(L)), serum OVA-specific immunoglobulin-E (IgE) and differential inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were determined after exposure to increasing doses of methacholine (MCh). Lung histological sections were examined for inflammatory infiltration. R(L) in the 10-μg OVA-challenged model group was not significantly different compared with the NS group at any MCh concentration but was significantly different compared with the asthma group (P < 0.01). R(L) in the other two model groups was intermediate between the asthma and NS groups. Serum OVA-specific IgE and eosinophils in BALF were increased significantly in all model and asthma groups compared with the NS group, but no significant differences were observed between model and asthma groups. Inflammatory cells were seen around bronchioles and capillaries in model and asthma groups but not the NS group. A mouse model of EB without AHR can be established by 10 μg OVA challenge and provides a useful tool for studying AHR mechanisms.

Functional and morphological alterations induced by uranyl nitrate in rat submandibular gland

Exposure to uranium (U) is an occupational hazard to workers who continually handle it and an environmental risk to the population at large. Adverse effects of U on different tissues, particularly kidneys, have been reported. The aim of the present study was to investigate whether U might produce damage to the rat submandibular gland (SMG). Uranyl nitrate (UN) was used to evaluate the secretory responses to norepinephrine (NE) or to the parasympathomimetic agent, methacholine (MC), along with some morphological and histological parameters. In addition, the presence of U in saliva was determined by atomic absorption spectrometric techniques. Results indicate that intraperitoneal (i.p.) injection of a single dose of UN (2 or 4 mg kg−1) induced a functional decrease in the induced secretory responses in the rat SMG, demonstrating for the first time that U interferes with salivary secretion. Moreover, salivary responses to MC as well as to NE were decreased after UN administration, in time- and dose-dependent manner, displaying a higher diminution after 7 days post injection. In agreement with the functional studies, the injection of UN produced morphological alterations of SMG, consisting in a reduction of mean acinar area and a marked vacuolization. Data indicate that UN produced an adverse effect in a dose- and time-dependent manner on SMG function and morphology. Furthermore, it was shown that U was incorporated in saliva and therefore, these findings may contribute to create a useful, noninvasive method to detect the exposure to U.

Acute cigarette smoke inhalation blunts lung responsiveness to methacholine and allergen in rabbit: differentiation of central and peripheral effects

Despite the prevalence of active smoking in asthmatics, data on the short-term effect of acute mainstream tobacco smoke exposure on airway responsiveness are very scarce. The aim of this study was to assess the immediate effect of acute exposure to mainstream cigarette smoke on airway reactivity to subsequent nonspecific and allergenic challenges in healthy control (n = 5) and ovalbumin-sensitized rabbits (n = 6). We combined low-frequency forced oscillations and synchrotron radiation CT imaging to differentiate central airway and peripheral airway and lung parenchymal components of the response to airway provocation. Acute exposure to smoke generated by four successive cigarettes (CS) strongly inhibited the central airway response to subsequent IV methacholine (MCh) challenge. In the sensitized animals, although the response to ovalbumin was also inhibited in the central airways, mainstream CS did not blunt the peripheral airway response in this group. In additional groups of experiments, exposure to HEPA-filtered CS (n = 6) similarly inhibited the MCh response, whereas CO (10,000 ppm for 4 min, n = 6) or nitric oxide inhalation instead of CS (240 ppm, 4 x 7 min, n = 5) failed to blunt nonspecific airway responsiveness. Pretreatment with alpha-chymotrypsin to inhibit endogenous VIP before CS exposure had no effect (n = 4). Based on these observations, the gas phase of mainstream cigarette smoke may contain one or more short-term inhibitory components acting primarily on central airways and inhibiting the response to both specific and nonspecific airway provocation, but not on the lung periphery where both lung mechanical parameters, and synchrotron-imaging derived parameters, showed large changes in response to allergen challenge in sensitized animals.

Deficiency in the Serum-Derived Hyaluronan-Associated Protein-Hyaluronan Complex Enhances Airway Hyperresponsiveness in a Murine Model of Asthma

Background: Serum-derived hyaluronan (HA)-associated proteins (SHAPs), the heavy chains of inter-α-trypsin inhibitor, covalently bind to HA to form the SHAP-HA complex. The SHAP-HA complex is involved in the pathophysiology of inflammatory diseases, including rheumatoid arthritis. We investigated whether this complex is also involved in airway allergy. Methods: SHAP-HA-deficient (bikunin knockout, KO) mice and wild-type (WT) mice were immunized twice by intraperitoneal injection of ovalbumin (OVA) and exposed to aerosol OVA for 30 min each day for 2 weeks. Twenty-four hours after the final OVA challenge, airway responsiveness to inhaled methacholine (MCh) was measured, and analysis of bronchoalveolar lavage fluid (BALF) and lung histological studies were done. Results: Compared to WT mice, KO mice showed higher airway hyperresponsiveness to inhaled MCh and higher late-phase responses to OVA whereas the early-phase responses were similar. Cell differentials of BALF showed an increased number of macrophages and neutrophils in KO mice. Furthermore, decreased concentrations of soluble tumor necrosis factor receptor-1 (sTNFR1) were found in BALF from KO mice whereas the levels of Th1 and Th2 cytokines were not different from WT mice. Immunochemical study of the lung tissues revealed stronger staining of sTNFR1 in KO than in WT mice. Conclusions: Our results suggest that in this murine asthma model, the SHAP-HA complex has an inhibitory role in the development of airway hyperresponsiveness and allergic airway inflammation which may be attributed, at least in part, to negative feedback mechanisms exerted by sTNFR1, the shedding of which from the cell surface might also be promoted by the SHAP-HA complex.