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Increased insulin is associated with obesity-related airway hyperreactivity and asthma. We tested whether the use of metformin, an antidiabetic drug used to reduce insulin resistance, can reduce circulating insulin, thereby preventing airway hyperreactivity in rats with dietary obesity. Male and female rats were fed a high- or low-fat diet for 5 wk. Some male rats were simultaneously treated with metformin (100 mg/kg orally). In separate experiments, after 5 wk of a high-fat diet, some rats were switched to a low-fat diet, whereas others continued a high-fat diet for an additional 5 wk. Bronchoconstriction and bradycardia in response to bilateral electrical vagus nerve stimulation or to inhaled methacholine were measured in anesthetized and vagotomized rats. Body weight, body fat, caloric intake, fasting glucose, and insulin were measured. Vagally induced bronchoconstriction was potentiated only in male rats on a high-fat diet. Males gained more body weight, body fat, and had increased levels of fasting insulin compared with females. Metformin prevented development of vagally induced airway hyperreactivity in male rats on high-fat diet, in addition to inhibiting weight gain, fat gain, and increased insulin. In contrast, switching rats to a low-fat diet for 5 wk reduced body weight and body fat, but it did not reverse fasting glucose, fasting insulin, or potentiation of vagally induced airway hyperreactivity. These data suggest that medications that target insulin may be effective treatment for obesity-related asthma.

Dendritic cell (DC) trafficking from lung to the draining mediastinal lymph nodes (MLNs) is a key step for initiation of T cell responses in allergic asthma. In the present study, we investigate the role of DC-mediated airway inflammation after inhibition of p21-activated kinase-1 (PAK1), an effector of Rac and Cdc42 small GTPases, in the allergen-induced mouse models of asthma. Systemic administration of PAK1 specific inhibitor IPA-3 significantly attenuates not only the airway inflammation but also the airway hyperresponsiveness in a mouse model of ovalbumin-induced asthma. Specifically, intratracheal administration of low dosage of IPA-3 consistently decreases not only the airway inflammation but also the DC trafficking from lung to the MLNs. Importantly, intratracheal instillation of IPA-3-treated and ovalbumin-pulsed DCs behaves largely the same as that of either Rac inhibitor-treated and ovalbumin-pulsed DCs or Cdc42 inhibitor-treated and ovalbumin-pulsed DCs in attenuation of the airway inflammation in ovalbumin-challenged mice. Mechanistically, PAK1 is not involved in the maturation, apoptosis, antigen uptake, and T cell activation of cultured DCs, but PAK1 dose lie on the downstream of Rac and Cdc42 to regulate the DC migration toward the chemokine C-C motif chemokine ligand 19. Taken together, this study demonstrates that inhibition of PAK1 attenuates the cardinal features of asthma through suppressing the DC trafficking from lung to the MLN, and that interfere with DC trafficking by a PAK1 inhibitor thus holds great promise for the therapeutic intervention of allergic diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO synthase and because NO modulates bronchial tone and inflammation. Our objective was to investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Arginase 1 expression in the lung was ablated by crossing Arg1(fl/fl) with Tie2Cre(tg/-) mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with quantitative PCR, ELISA, and immunohistochemistry. Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but it improved peripheral lung function (tissue elastance and resistance) and attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL-13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA; lung content of IL-4, IL-5, IL-10, TNF-α, and IFN-γ protein; and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism but had little effect on airway inflammation.

Nebulized lidocaine may be a corticosteroid-sparing drug in human asthmatics, reducing airway resistance and peripheral blood eosinophilia. We hypothesized that inhaled lidocaine would be safe in healthy and experimentally asthmatic cats, diminishing airflow limitation and eosinophilic airway inflammation in the latter population. Healthy (n = 5) and experimentally asthmatic (n = 9) research cats were administered 2 weeks of nebulized lidocaine (2 mg/kg q8h) or placebo (saline) followed by a 2-week washout and crossover to the alternate treatment. Cats were anesthetized to measure the response to inhaled methacholine (MCh) after each treatment. Placebo and doubling doses of methacholine (0.0625-32.0000 mg/ml) were delivered and results were expressed as the concentration of MCh increasing baseline airway resistance by 200% (EC200Raw). Bronchoalveolar lavage was performed after each treatment and eosinophil numbers quantified. Bronchoalveolar lavage fluid (BALF) % eosinophils and EC200Raw within groups after each treatment were compared using a paired t-test (P <0.05 significant). No adverse effects were noted. In healthy cats, lidocaine did not significantly alter BALF eosinophilia or the EC200Raw. There was no difference in %BALF eosinophils in asthmatic cats treated with lidocaine (36±10%) or placebo (33 ± 6%). However, lidocaine increased the EC200Raw compared with placebo 10 ± 2 versus 5 ± 1 mg/ml; P = 0.043). Chronic nebulized lidocaine was well-tolerated in all cats, and lidocaine did not induce airway inflammation or airway hyper-responsiveness in healthy cats. Lidocaine decreased airway response to MCh in asthmatic cats without reducing airway eosinophilia, making it unsuitable for monotherapy. However, lidocaine may serve as a novel adjunctive therapy in feline asthmatics with beneficial effects on airflow obstruction.

Purpose: To determine the effect of pre-exercise exposure to diesel exhaust (DE) on 20-km cycling performance, pulmonary function, and cardio-respiratory variables during exercise., Methods: Eight endurance-trained males participated in the study. Test days consisted of a 60-min exposure to either filtered air (FA) or DE, followed by a 20 km cycling time trial. Exposures to DE were at a concentration of 300 µg/m³ of PM(2.5). Forced expiratory volume in 1 s (FEV₁) and forced vital capacity (FVC) were measured before and after exposure, and after exercise. Oxygen consumption (VO₂) and carbon dioxide production (VCO₂), minute ventilation (V(E)), tidal volume (V(T)), breathing frequency (F(B)), heart rate and oxyhemoglobin saturation (SpO₂), were collected during the time trials. The effect of condition on time trial duration, an order effect, and mean cardio-respiratory variables were each analysed using paired T-tests. Repeated-measures ANOVA were used to assess the effect of DE exposure on pulmonary function., Results: There was a main effect of condition (FA vs. DE) on the change in FEV₁ from baseline, and in exercise heart rate. Post hoc tests revealed that exercise-induced bronchodilation was significantly attenuated following DE compared to FA. There were no main effects of condition on 20 km cycling performance, or VO₂, VCO₂, V(E), V(T), F(B) and SpO₂ during a 20 km time trial., Conclusion: A 60-min exposure to DE prior to exercise significantly attenuated exercise-induced bronchodilation and significantly increased heart rate during exercise. Pre-exercise exposure to diesel exhaust did not significantly impair 20 km cycling time trial performance.

Background: Airway smooth muscle contraction causes bronchial constriction and is the main cause of bronchospasm in response to stimulants in asthma patients. In this pilot study, we tested the possibility of using a commercially available neurotoxin-botulinum toxin A (BTX-A)-to reduce bronchial hyperreactivity in dogs., Methods: Two bronchoscopic sessions were conducted in 6 healthy mongrel dogs. In the first session, BTX-A (concentration 10 U/mL) was injected in small aliquots submucosally in 1 caudal lobe and its subsegments, leaving the other side as control. During the second bronchoscopy conducted 2 weeks later, the airway calibers of the treated and untreated sides were measured in each animal before and after instillation of methacholine in the airways to induce bronchial hyperreactivity (concentration 25 mg/mL)., Results: The mean pretreatment diameter was 3.356 (± 1.294) mm and 2.765 (± 0.603) mm in the treated and untreated airways, respectively. After provocation with methacholine, the diameter of the treated airways was 1.985 (± 0.888) mm versus 0.873 (± 0.833) mm in the untreated airways (P=0.000). Local injection of BTX-A in the airway resulted in reduction of bronchial hyperreactivity by 58.6% (P=0.001). There were no complications resulting from the submucosal injection of BTX-A in the airways., Conclusions: Endobronchial injection of BTX-A reduces bronchial hyperreactivity in the airways of healthy dogs.

Background: Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role., Materials and Methods: To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days., Results: When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation., Conclusion: In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.

Penh is a dimensionless index normally used to evaluate changes in the shape of the airflow pattern entering and leaving a whole-body flow plethysmograph as an animal breathes. The index is sensitive to changes in the distribution of area under the waveform during exhalation and increases in a nonlinear fashion as the normalized area increases near the beginning of the curve. Enhanced pause (Penh) has been used to evaluate changes in pulmonary function and as a method to evaluate airway reactivity. However, the use of Penh to assess pulmonary function has been challenged (Bates et al., 2004; Lundblad et al., 2002; Mitzner et al., 2003; Mitzner & Tankersley, 1998; Petak et al., 2001; Sly et al., 2005). The objective of this study was to show how Penh of the thorax and plethysmograph flow patterns are related. That relationship is used to describe the conditions under which whole-body plethysmograph Penh measurements can be used to detect changes in sRaw.

Oxidative stress is a hallmark of asthma, and increased levels of oxidants are considered markers of the inflammatory process. Most studies to date addressing the role of oxidants in the etiology of asthma were based on the therapeutic administration of low m.w. antioxidants or antioxidant mimetic compounds. To directly address the function of endogenous hydrogen peroxide in the pathophysiology of allergic airway disease, we comparatively evaluated mice systemically overexpressing catalase, a major antioxidant enzyme that detoxifies hydrogen peroxide, and C57BL/6 strain matched controls in the OVA model of allergic airways disease. Catalase transgenic mice had 8-fold increases in catalase activity in lung tissue, and had lowered DCF oxidation in tracheal epithelial cells, compared with C57BL/6 controls. Despite these differences, both strains showed similar increases in OVA-specific IgE, IgG1, and IgG2a levels, comparable airway and tissue inflammation, and identical increases in procollagen 1 mRNA expression, following sensitization and challenge with OVA. Unexpectedly, mRNA expression of MUC5AC and CLCA3 genes were enhanced in catalase transgenic mice, compared with C57BL/6 mice subjected to Ag. Furthermore, when compared with control mice, catalase overexpression increased airway hyperresponsiveness to methacholine both in naive mice as well as in response to Ag. In contrast to the prevailing notion that hydrogen peroxide is positively associated with the etiology of allergic airways disease, the current findings suggest that endogenous hydrogen peroxide serves a role in suppressing both mucus production and airway hyperresponsiveness.

Inhalation challenges with occupational agents are used to confirm the aetiology of occupational asthma. It has been proposed that using closed-circuit equipment rather than the realistic challenge method would improve the methodology of these tests. Changes in forced expiratory volume in one second (FEV1) were examined in 496 subjects with "positive specific inhalation challenges", i.e. changes in FEVI of > or = 20% after exposure to an occupational agent, including 357 subjects exposed by the realistic method, 108 using the closed-circuit method and 31 by both methods. For immediate reactions, 18 of 95 (19%) showed changes in FEV1 of > or = 30% with the closed-circuit method, whereas a significantly larger proportion, i.e. 77 of 200 (38.5%), showed such changes using the realistic method. As regards nonimmediate reactions, changes in FEV1 of > or = 30% occurred in 16 of 43 (37%) cases with the closed-circuit method as compared to a larger proportion, i.e. 87 of 180 (48%) cases, using the realistic method. This favourable effect was significantly more pronounced in workers with higher levels of bronchial hyperresponsiveness to methacholine. It is concluded that, for agents that can be generated using the closed-circuit method, use of such apparatus results in a smaller proportion of exaggerated bronchoconstriction than does the realistic method, this being particularly true for low-molecular weight agents.

Trimellitic anhydride (TMA) is a low-molecular-weight chemical known to cause occupational asthma. The dose-response study was designed to determine whether respiratory responses during a single inhalation challenge with TMA (25-30 mg/m3 for 30 min, 3 weeks after the initial induction), the ensuing non-specific airway hyperresponsiveness (AH) to methacholine (MCh) aerosol, and infiltration of eosinophilic granulocytes into the lungs of sensitized Brown Norway (BN) rats are associated and dependent on the concentration of TMA used for topical induction. The initial topical exposure concentrations were 1, 5, and 25% TMA in acetone:olive oil (AOO) followed by a booster induction 1 week later. In the time course study BN rats received AOO alone or were sensitized to the minimal sensitizing topical concentration of TMA (5%) and were the subsequently challenged with TMA on Days 17, 24, 41, 47, 55, and 66, followed by a MCh challenge 1 day later. One additional group of rats was sensitized to 5% TMA but were repeatedly challenged with MCh without prior TMA challenge. In the dose-response study the rats sensitized topically to TMA (5 and 25% in AOO) displayed unequivocal changes in breathing patterns upon challenge with TMA, including an increased responsiveness to MCh aerosol. These findings were associated with a sustained pulmonary eosinophilic inflammation. All endpoints demonstrated consistently that 5% TMA in AOO constitutes the minimal sensitizing concentration. When rats were topically sensitized with this concentration and repeatedly challenged with TMA over a time period of 7 weeks, it became apparent that challenge exposures in BN rats may be false negative when performed at time periods less than 3 weeks after the initial induction. Despite the time-related increased responsiveness elicited by the repeated TMA challenge exposures, the MCh challenge revealed increased non-specific airway hyperreactivity exclusively on Day 17. After the sixth TMA-challenge, the respiratory response and lung weights of rats sensitized topically were essentially similar to those observed in the repetitively re-challenged control group (induction: vehicle only; repeated booster challenge exposures with TMA). Thus, it appears, that in this animal model the effective concentration for successful topical sensitization must be at least approximately 5%. The repeated low-dose re-challenge with TMA in topically sensitized rats resulted in similar or slightly aggravated time-related responses over a period of 7 weeks. An over-proportionally increased susceptibility of rats receiving a topical priming dose prior to repeated inhalation challenge exposures was not observed. In summary, this study shows that the analysis of functional changes in breathing patterns is suitable to identify respiratory allergy. Repeated short-term inhalation exposures to mildly irritant concentrations (but low doses) of chemical asthmagens may be of higher concern than topical exposures.

A rat bioassay has been developed to provide an objective approach for the identification and classification of respiratory allergy using trimellitic anhydride (TMA), which is a known respiratory tract irritant and asthmagen. Particular emphasis was placed on the study of route-of-induction-dependent effects and their progression upon inhalation challenge with TMA (approximately 23 mg m(-3) for a duration of 30 min), which included analysis of specific and non-specific airway hyperreactivity and pulmonary inflammation initiated and sustained by immunological processes. Refinement of the bioassay focused on procedures to probe changes occurring upon challenge with TMA or methacholine aerosols using physiological, biochemical and immunological procedures. Following challenge with TMA, the rats sensitized to TMA showed marked changes in peak inspiratory and expiratory air flows and respiratory minute volume. In these animals, a sustained pulmonary inflammation occurred, characterized by specific endpoints determined in bronchoalveolar lavage (lactate dehydrogenase, protein, nitrite, eosinophil peroxidase, myeloperoxidase). When compared with the naive controls, lung weights were increased significantly, as were the weights of lung-associated lymph nodes following inhalation induction and auricular lymph nodes following topical induction. The extent of changes observed was equal or more pronounced in animals sensitized epicutaneously (day 0:150 microl vehicle/50% TMA on each flank, day 7; booster administration to the skin of the dorsum of both ears using half the concentration and volume used on day 0) when compared with rats sensitized by 5 x 3 h day(-1) inhalation exposures (low dose: 25 mg TMA m(-3), high dose: 120 mg TMA m(-3)). In summary, the findings support the conclusion that the Brown Norway rat model is suitable for identifying TMA as an agent that causes both an immediate-type change of breathing patterns and a delayed-type sustained pulmonary inflammatory response. However, it remains unresolved whether the marked effects observed in the topically sensitized rats are more related to a route-of-induction or dose-dependent phenomenon., (Copyright 2002 John Wiley & Sons, Ltd.)

Proadrenomedullin NH2-terminal 20 peptide (PAMP), a newly identified hypotensive peptide, may play physiological roles in airway and cardiovascular controls. This study was designed to determine the mechanism responsible for the bronchoprotective effects of PAMP on capsaicin-induced bron-choconstriction in anesthetized guinea pigs. PAMP (10(-8)-10(-6) M) significantly inhibited capsaicin-induced bronchoconstriction in a dose-dependent manner. The bronchoprotective effect of PAMP (10(-6) M) was as large as that of isoproterenol (10(-7) M) and lasted > 10 min. The concentration of immunoreactive substance P (SP) in bronchoalveolar lavage fluid after administration of capsaicin (4 x 10(-6) M) was 120 +/- 10 fmol/ml. PAMP significantly inhibited the release of immunoreactive SP in a dose-dependent manner (60 +/- 6 fmol/ml for (10(-6) M PAMP, P < 0.01; 84 +/- 6 fmol/ml for 10(-7) M PAMP, P < 0.01; and 95 +/- 6 fmol/ml for 10(-8) M PAMP, P < 0.05). PAMP (10(-6) M) did not significantly affect exogenous neurokinin A (NKA) or NKA + SP-induced bronchoconstriction, whereas isoproterenol (10(-7) M) significantly inhibited exogenous tachykinin-induced bronchoconstriction. These findings suggest that the bronchoprotective effects of PAMP are mainly due to inhibition of the release of tachykinins at airway C-fiber endings.

The relationship between antibronchospasmic, tachycardiac, or hypokalaemic effects and plasma concentration of L-isoproterenol (ISP) hydrochloride was investigated in guinea-pigs in vivo. ISP was infused at the rate of 10, 30, 50, 100, and 300 ng kg-1 min-1. The antibronchospasmic effect was expressed as the attenuation of methacholine-induced bronchospasm. The EC50 values of ISP for antibronchospasmic and tachycardiac effects were 5.12 nM and 3.95 nM, respectively. Although they were comparable to the values reported in vitro (7.23-0.358 nM, 1.77 nM), the concentration response relationship of ISP for antibronchospasmic effect was quite steep with a slope factor of more than six. Moreover, a decrease in plasma potassium level was not clearly detected. The experimental procedure in our present study was useful for evaluating antibronchospasmic and tachycardiac effects of beta-agonists.

This study was designed to investigate if animals exposed to urban levels of air pollution develop pulmonary hyperresponsiveness and to test if this change was reversed after moving the animals to a nonpolluted environment. One hundred twenty male Wistar rats were kept in (a) São Paulo (polluted environment) for 3 months (SP3); (b) Atibaia (clean region), for 3 months (A3); (c) São Paulo for 3 months and then Atibaia for a further 3 months (SPA6); (d) Atibaia for 6 months (A6). After the exposure period, the rats were submitted to dose-response curves to inhaled methacholine. Older animals (SPA6 and A6) had lower responses to methacholine in terms of respiratory system resistance when compared to the animals studied after 3 months of experiment (SP3 and A3). However, the response in terms of respiratory system elastance of the SP3 group was significantly (P = 0.0004) greater than those of the other three groups. Our results suggest that the environmental conditions of the large urban centers can induce pulmonary hyperresponsiveness in rats that can be reversed when the animals are removed to a nonpolluted area.

The effects of histamine and methacholine aerosols and of a fixed inspiratory resistance on tidal breathing flow-volume loops (TBFVL) were investigated using 18 unsedated, standing, healthy thoroughbred horses. The data were first analysed using traditional flow-volume loop indices and then reduced using standardized factor scoring coefficients obtained in a previous study in this laboratory using similar experimental techniques. On the basis of resting TBFVL analysis, the degree of pulmonary dysfunction caused by inhalation of histamine and methacholine aerosols with concentrations of 10 and 2 mg/ml, respectively, was similar. The fixed resistance also caused significant changes in the resting spirogram and TBFVL indices, suggesting that this model may prove valuable for further studies involving upper respiratory tract (URT) conditions. Administration of histamine and methacholine aerosols resulted in significant changes in all factor scores, although most of the observed changes were due to the effects of these aerosols on the respiratory rate. These findings re-emphasize the importance of the effects of respiratory rate on pulmonary mechanics. Application of the resistance resulted in significant changes in factor score 3, the 'inspiratory' factor, which lends support to the validity of this model for URT conditions. The close agreement between the factor scores obtained under controlled conditions in this study and in a previous study in this laboratory confirms that the factor analysis used for both of these studies provides an adequate means of reducing TBFVL data obtained from thoroughbred horses. The large intra- and inter-individual variation observed both with the indices of TBFVL and with the factor scores limits the potential of these variables for detecting individual animals with obstructive airway disease. Re-evaluation of these indices under the stress of exercise may reduce the variability observed in these data and may increase the magnitude of differences between different animals, providing a means of detecting individual animals with subclinical obstructive airway conditions.

We studied the effect of intravenous administration of leukotriene (LT) C4 on bronchial responsiveness to histamine and airway wall thickening in guinea-pigs. Guinea-pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded sections of the lungs were stained and the airways that were cut in transverse sections were measured by tracing enlarged images using a digitizer. Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after the intravenous administration of LTC4. Intravenous administration of 3 microg/kg LTC4 for 1 hr induced an increase of the relative thickness of the airway wall in peripheral bronchi by the histological examination. In analysis of airway function, intravenous administration of 3 microg/kg LTC4 for 1 hr induced airway hyperresponsiveness to histamine with airway wall thickening. Thromboxane A2 receptor antagonists ONO-NT-126 and ONO-8809 inhibited the LTC4-induced airway hyperresponsiveness to histamine in a dose-dependent manner, but not the airway wall thickening induced by LTC4, suggesting that the effect of LTC4 on bronchial hyperresponsiveness is likely to be mediated through TXA2.