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1.  Inflammatory cell distribution in guinea pig airways and its relationship to airway reactivity. 
Mediators of Inflammation  2001;10(3):143-154.
Although airway inflammation and airway hyperreactivity are observed after allergen inhalation both in allergic humans and animals, little is known about the mechanisms by which inflammatory cells can contribute to allergen-induced airway hyperreactivity. To understand how inflammatory cell infiltration can contribute to airway hyperreactivity, the location of these cells within the airways may be crucial Using a guinea pig model of acute allergic asthma, we investigated the inflammatory cell infiltration in different airway compartments at 6 and 24 h (i.e. after the early and the late asthmatic reaction, respectively) after allergen or saline challenge in relation to changes in airway reactivity (AR) to histamine. At 6 h after allergen challenge, a threefold (p < 0.01) increase in the AR to histamine was observed. At 24 h after challenge, the AR to histamine was lower, but still significantly enhanced (1.6-fold, p < 0.05). Adventitial eosinophil and neutrophil numbers in both bronchi and bronchioli were significantly increased at 6 h post-allergen provocation as compared with saline (p < 0.01 for all), while there was a strong tendency to enhanced eosinophils in the bronchial submucosa at this time point (p = 0.08). At 24h after allergen challenge, the eosinophilic and neutrophilic cell infiltration was reduced. CD3+ T lymphocytes were increased in the adventitial compartment of the large airways (p < 0.05) and in the parenchyma (p < 0.05) at 24h post-allergen, while numbers of CD8+ cells did not differ from saline treatment at any time point post-provocation. The results indicate that, after allergen provocation, inflammatory cell numbers in the airways are mainly elevated in the adventitial compartment. The adventitial inflammation could be important for the development of allergen-induced airway hyperreactivity.
PMCID: PMC1781701  PMID: 11545251
2.  Inhalation of the Rho-kinase inhibitor Y-27632 reverses allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction 
Respiratory Research  2006;7(1):121.
Background
In guinea pigs, we have previously demonstrated that the contribution of Rho-kinase to airway responsiveness in vivo and ex vivo is enhanced after active sensitization with ovalbumin (OA). Using conscious, unrestrained OA-sensitized guina pigs, we now investigated the role of Rho-kinase in the development of airway hyperresponsiveness (AHR) after the allergen-induced early (EAR) and late asthmatic reaction (LAR) in vivo.
Methods
Histamine and PGF2α PC100-values (provocation concentrations causing 100% increase in pleural pressure) were assessed before OA-challenge (basal airway responsiveness) and after the OA-induced EAR (5 h after challenge) and LAR (23 h after challenge). Thirty minutes later, saline or the specific Rho-kinase inhibitor Y-27632 (5 mM, nebulizer concentration) were nebulized, after which PC100-values were reassessed.
Results
In contrast to saline, Y-27632 inhalation significantly decreased the basal responsiveness toward histamine and PGF2α before OA-challenge, as indicated by increased PC100 -values. Both after the allergen-induced EAR and LAR, AHR to histamine and PGF2α was present, which was reversed by Y-27632 inhalation. Moreover, there was an increased effectiveness of Y-27632 to reduce airway responsiveness to histamine and PGF2α after the EAR and LAR as compared to pre-challenge conditions. Saline inhalations did not affect histamine or PGF2α PC100-values at all. Interestingly, under all conditions Y-27632 was significantly more effective in reducing airway responsiveness to PGF2α as compared to histamine. Also, there was a clear tendency (P = 0.08) to a more pronounced degree of AHR after the EAR for PGF2α than for histamine.
Conclusion
The results indicate that inhalation of the Rho-kinase inhibitor Y-27632 causes a considerable bronchoprotection to both histamine and PGF2α. Moreover, the results are indicative of a differential involvement of Rho-kinase in the agonist-induced airway obstruction in vivo. Increased Rho-kinase activity contributes to the allergen-induced AHR to histamine and PGF2α after both the EAR and the LAR, which is effectively reversed by inhalation of Y-27632. Therefore, Rho-kinase can be considered as a potential pharmacotherapeutical target in allergic asthma.
doi:10.1186/1465-9921-7-121
PMCID: PMC1586199  PMID: 17002806
3.  BCG infection during pre-sensitization or even post-sensitization inhibits airway sensitivity in an animal model of allergic asthma. 
Journal of Korean Medical Science  2000;15(3):265-272.
The objective of this study is to investigate whether BCG infection before, during or after sensitization suppresses allergen-induced airway hyperresponsiveness and eosinophilic inflammation in allergic asthma rats, and to determine the required dose of BCG to induce such an inhibition. Eighty-seven Sprague-Dawley (SD) rats were sensitized and provoked with ovalbumin (OA). A pretreatment of 6 x 10(4) or 6 x 10(5) colony forming units (CFUs) of BCG or saline was done at four different times: 3 days before sensitization, at sensitization, 3 days before provocation, or at provocation. The assessment of tracheal smooth muscle (TSM) responsiveness to electrical field stimulation or acetylcholine (ACh) and bronchoalveolar lavage (BAL) were performed 1 day after OA provocation. Doses of 6 x 10(4) CFUs inhibited TSM sensitivity of rats infected 3 days before sensitization or at sensitization, but not 3 days before provocation or at provocation. However, doses of 6 x 10(5) CFUs significantly inhibited not only the airway eosinophilia of rats infected 3 days before sensitization or at sensitization, but also the TSM sensitivity of rats infected 3 days before provocation or at provocation. In conclusion, BCG infection suppresses the development of sensitivity of airway smooth muscle and airway eosinophilic inflammation in allergic asthma rats. Furthermore, a relatively high dose of BCG infection inhibits airway sensitivity, even after allergen sensitization.
PMCID: PMC3054645  PMID: 10895966
4.  Opposite Effects of Mepyramine on JNJ 7777120-Induced Amelioration of Experimentally Induced Asthma in Mice in Sensitization and Provocation 
PLoS ONE  2012;7(1):e30285.
Background
Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, JNJ 7777120, an antagonist at the histamine H4 receptor, reduces asthmatic symptoms, while the histamine H1 receptor-selective antagonist mepyramine is virtually without effect. In the present study, we analyzed the effect of combined antagonism at the histamine H1 and H4 receptors in a murine asthma model in relation to the timing of their application, i.e. sensitization or provocation.
Methodology/Principal Findings
Asthma was induced in mice by sensitization and provocation with ovalbumin. JNJ 7777120 and/or mepyramine were injected subcutaneously either during sensitization or during provocation, and typical asthma parameters were analyzed. JNJ 7777120, but not mepyramine, reduced serum concentrations of anti-OVA IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar-lavage (BAL)-fluids independently of the timing of application. Upon application of JNJ 7777120 plus mepyramine in combination during provocation, mepyramine inhibited the effects of JNJ 7777120. In contrast, when applied during sensitization, mepyramine enhanced the disease-ameliorating effects of JNJ 7777120.
Conclusions/Significance
Our study indicates that both histamine H1 and H4 receptors play important roles in the course of murine experimental asthma. Unexpectedly, the contribution of these receptors to the pathogenesis differs between the two phases, sensitization or provocation. Since in human asthma, repeated contact to the allergen is not only provocation but also a boost of sensitization, a combined pharmacological targeting of histamine H1 and H4 receptors could be taken into consideration as an option for the prevention of asthma and maybe other allergic diseases.
doi:10.1371/journal.pone.0030285
PMCID: PMC3260279  PMID: 22272324
5.  Repeated allergen exposure reduce early phase airway response and leukotriene release despite upregulation of 5-lipoxygenase pathways 
Background
Allergen induced early phase airway response and airway plasma exudation are predominantly mediated by inflammatory mast cell mediators including histamine, cysteinyl leukotrienes (cysLTs) and thromboxane A2 (TXA2). The aim of the present study was to evaluate whether repeated allergen exposure affects early phase airway response to allergen challenge.
Methods
A trimellitic anhydride (TMA) sensitized guinea pig model was used to investigate the effects of low dose repeated allergen exposure on cholinergic airway responsiveness, early phase airway response and plasma exudation, as well as local airway production of mast cell derived cysteinyl leukotrienes and thromboxane B2 (TXB2) after allergen challenge.
Results
Repeated low dose allergen exposure increased cholinergic airway responsiveness. In contrast, early phase airway response and plasma exudation in response to a high-dose allergen challenge were strongly attenuated after repeated low dose allergen exposure. Inhibition of the airway response was unspecific to exposed allergen and independent of histamine receptor blocking. Furthermore, a significant reduction of cysteinyl leukotrienes and TXB2 was found in the airways of animals repeatedly exposed to a low dose allergen. However, in vitro stimulation of airway tissue from animals repeatedly exposed to a low dose allergen with arachidonic acid and calcium ionophore (A23187) induced production of cysteinyl leukotrienes and TXB2, suggesting enhanced activity of 5-lipoxygenase and cyclooxygenase pathways.
Conclusions
The inhibition of the early phase airway response, cysteinyl leukotriene and TXB2 production after repeated allergen exposure may result from unresponsive effector cells.
doi:10.1186/2045-7022-2-7
PMCID: PMC3348058  PMID: 22439792
Guinea pig; Allergen exposure; Cholinergic responsiveness; Early phase airway response; Plasma exudation; 5-lipoxygenase; Cyclooxygenase
6.  Glutathione S-transferase P1 Ile105Val polymorphism modulates allergen-induced airway inflammation in human atopic asthmatics in vivo 
Background
Glutathione S-transferase P1 is a Phase II cytoprotective and detoxifying enzyme that is widely expressed in human airways. The glutathione S-transferase P1 Ile105Val polymorphism has been linked with atopic disorders and asthma. Yet, little is known about the regulation of allergic inflammation by glutathione S-transferase P1 in human asthmatics.
Objective
To establish the effect of the glutathione S-transferase P1 Ile105Val polymorphism on allergen-induced airway inflammation and oxidant stress, and nonspecific bronchial hyperresponsiveness to methacholine and reactivity to specific allergen in mild human atopic asthmatics in vivo.
Methods
Five Val105/Val105, twelve Val105/Ile105, and twenty Ile105/Ile105 mild atopic asthmatics underwent methacholine challenge, inhaled allergen challenge, and endobronchial allergen provocation through a bronchoscope. A panel of inflammatory cytokines and chemokines, F2-isoprostanes and isofuranes, markers of oxidative stress, thromboxane B2, and immunoglobulin E were measured in bronchoalveolar lavage fluid at baseline and 24 hrs after allergen instillation.
Results
Asthmatics with glutathione S-transferase P1 Val105/Val105 compared to asthmatics with the glutathione S-transferase P1 Val105/Ile105 and Ile105/Ile105 had greater generation of acute phase cytokines (TNF-α, IL-6, CXCL8), IL-12, CCL11, thromboxane B2, and immunoglobulin E at 24 hrs after local allergen challenge. The GSTP1 genotype had no effect on airway hyperresponsiveness to methacholine and the reactivity to specific allergen.
Conclusion
The glutathione S-transferase P1 Ile105Val polymorphism markedly modifies allergen-provoked airway inflammation in atopic asthmatics in vivo. Modulation of the biochemical milieu in response to allergen provides a mechanistic explanation for regulatory effects of glutathione S-transferase P1 polymorphism on airway pathophysiology, and may guide improvement of future therapeutic methods in human atopic asthmatics. These findings must me confirmed in a larger study population of asthmatics with various ethnicities.
doi:10.1111/cea.12086
PMCID: PMC3633143  PMID: 23600543
Glutathione S-transferase P1; Ile105Val polymorphism; atopic asthma; allergen challenge; methacholine challenge; allergic inflammation; oxidant stress
7.  Airway responses and inflammation in subjects with asthma after four days of repeated high-single-dose allergen challenge 
Respiratory Research  2012;13(1):78.
Background
Both standard and low-dose allergen provocations are an established tool in asthma research to improve our understanding of the pathophysiological mechanism of allergic asthma. However, clinical symptoms are less likely to be induced. Therefore, we designed a protocol for repetitive high-dose bronchial allergen challenges to generate clinical symptoms and airway inflammation.
Methods
A total of 27 patients aged 18 to 40 years with positive skin-prick tests and mild asthma underwent repetitive high-dose allergen challenges with household dust mites for four consecutive days. Pulmonary function and exhaled NO were measured at every visit. Induced sputum was analysed before and after the allergen challenges for cell counts, ECP, IL-5, INF-γ, IL-8, and the transcription factor Foxp3.
Results
We found a significant decrease in pulmonary function, an increased use of salbutamol and the development of a late asthmatic response and bronchial hyperresponsiveness, as well as a significant induction of eNO, eosinophils, and Th-2 cytokines. Repeated provocation was feasible in the majority of patients. Two subjects had severe adverse events requiring prednisolone to cope with nocturnal asthma symptoms.
Conclusions
Repeated high-dose bronchial allergen challenges resulted in severe asthma symptoms and marked Th-2-mediated allergic airway inflammation. The high-dose challenge model is suitable only in an attenuated form in diseased volunteers for proof-of-concept studies and in clinical settings to reduce the risk of severe asthma exacerbations.
Trial registration
ClinicalTrials.govNCT00677209
doi:10.1186/1465-9921-13-78
PMCID: PMC3445853  PMID: 22989372
Bronchial allergen challenge; Bronchial hyperresponsiveness; Exhaled NO; Eosinophils; IL-5; IL-8; IFN-γ; Foxp3
8.  Can AMP induce sputum eosinophils, even in subjects with complete asthma remission? 
Respiratory Research  2010;11(1):106.
Background
The definition of "clinical asthma remission" is based on absence of symptoms and use of medication. However, in the majority of these subjects airway inflammation is still present when measured. In the present study we investigated whether "complete asthma remission", additionally defined by the absence of bronchial hyperresponsiveness (BHR) and the presence of a normal lung function, is associated with the absence of airway inflammation.
Methods
Patients with a former diagnosis of asthma and a positive histamine provocation test were re-examined to identify subjects with complete asthma remission (no asthma symptoms or medication, PC20 histamine > 32 mg/ml, FEV1 > 90% predicted). Patients with PC20 histamine ≤ 32 mg/ml were defined as current asthmatics and were divided in two groups, i.e. asthmatics with and without BHR to adenosine 5'monophoshate (AMP). Sputum induction was performed 1 week before and 1 hour after AMP provocation. Sputum induction and AMP provocation were previously shown to be sensitive markers of airway inflammation.
Results
Seven patients met criteria for complete asthma remission. Twenty-three were current asthmatics, including twelve without hyperresponsiveness to AMP. Subjects with complete asthma remission showed no AMP-induced sputum eosinophilia (median (range) 0.2 (0 - 4.6)% at baseline and 0.2 (0 - 2.6)% after AMP). After AMP, current asthmatics had a significant increase in sputum eosinophils (0.5 (0 - 26.0)% at baseline and 2.6 (0 - 32.0) % after AMP), as had the subgroup of current asthmatics without hyperresponsiveness to AMP (0.2 (0 - 1.8)% at baseline and 1.3 (0 - 6.3)% after AMP).
Conclusions
Subjects with complete asthma remission, in contrast to subjects with current asthma, do not respond with eosinophilic inflammation in sputum after AMP provocations. These data lend support to the usefulness of the definition of complete asthma remission.
doi:10.1186/1465-9921-11-106
PMCID: PMC2923115  PMID: 20678209
9.  Effects of Respiratory Mycoplasma pneumoniae Infection on Allergen-Induced Bronchial Hyperresponsiveness and Lung Inflammation in Mice  
Infection and Immunity  2003;71(3):1520-1526.
Airway mycoplasma infection may be associated with asthma pathophysiology. However, the direct effects of mycoplasma infection on asthma remain unknown. Using a murine allergic-asthma model, we evaluated the effects of different timing of airway Mycoplasma pneumoniae infection on bronchial hyperresponsiveness (BHR), lung inflammation, and the protein levels of Th1 (gamma interferon [IFN-γ]) and Th2 (interleukin 4 [IL-4]) cytokines in bronchoalveolar lavage fluid. When mycoplasma infection occurred 3 days before allergen (ovalbumin) sensitization and challenge, the infection reduced the BHR and inflammatory-cell influx into the lung. This was accompanied by a significant induction of Th1 responses (increased IFN-γ and decreased IL-4 production). Conversely, when mycoplasma infection occurred 2 days after allergen sensitization and challenge, the infection initially caused a temporary reduction of BHR and then increased BHR, lung inflammation, and IL-4 levels. Our data suggest that mycoplasma infection could modulate both physiological and immunological responses in the murine asthma model. Our animal models may also provide a new means to understand the role of infection in asthma pathogenesis and give evidence for the asthma hygiene hypothesis.
doi:10.1128/IAI.71.3.1520-1526.2003
PMCID: PMC148884  PMID: 12595471
10.  Changes in bronchial responsiveness to histamine at intervals after allergen challenge. 
Thorax  1987;42(4):302-308.
Bronchial responsiveness to inhaled histamine was measured two, seven, and 30 hours after allergen inhalation challenge in 19 atopic subjects. The provocative histamine concentrations causing a 20% fall in FEV1 (PC20) at these three times were compared with the baseline value, with values obtained two and seven hours after diluent inhalation, and with those obtained five to seven days after allergen challenge in the 12 late responders. Seven subjects had allergen induced isolated early asthmatic responses (delta FEV1 22.6% (SD 6.6%)) with less than a 5% late fall in FEV1. There was no change in the six histamine PC20 values measured in these seven subjects; the geometric mean PC20 was 1.0-1.3 mg/ml on all six occasions. Twelve subjects had an allergen induced early asthmatic response (delta FEV1 26.3% (9.8%)) followed by a definite (greater than 15% delta FEV1, n = 7) or equivocal (5-15% delta FEV1, n = 5) late asthmatic response. The geometric mean histamine PC20 was not significantly different two hours after allergen inhalation either from baseline (0.67 v 0.78 mg/ml) or from that seen two hours after diluent (0.67 v 0.95). It was significantly reduced at seven (0.24 mg/ml) and at 30 hours (0.44 mg/ml) but had returned to baseline when repeated five to seven days later (0.74 mg/ml). In 10 subjects with a dual response who had a repeat antigen challenge the mean early and late response and delta PC20 at seven and 30 hours were similar. These data show that bronchial responsiveness to a non-allergic stimulus has not increased two hours after allergen inhalation following spontaneous recovery of the early asthmatic response but before the start of the late asthmatic response.
PMCID: PMC460713  PMID: 3616989
11.  An Intratracheal Challenge Murine Model of Asthma: Can Bronchial Inflammation Affect the Nose? 
Purpose
Extensive data support the influence of the upper airway on lower airway inflammation and pathophysiology in allergic disease. However, few studies have focused on allergic inflammation in the nose after an isolated lower airway allergen challenge, a situation that can exist clinically when human subjects breathe primarily through the mouth, as occurs when nasally congested. This study used a mouse model to investigate whether upper airway inflammation and hyperresponsiveness were induced by an isolated lower airway allergen challenge.
Methods
BALB/c mice were sensitized by systemic intraperitoneal injection of ovalbumin/saline and challenged with intratracheal ovalbumin/saline. Inflammation in the nose and lungs was assessed by cytology and histology of nasal tissues and bronchoalveolar lavage fluid (BALF), while nasal airway resistance and response were measured over 3 days post-challenge.
Results
Intratracheal application of an allergen in anaesthetized mice resulted in exclusive deposition in the lower airway. Compared to control animals, ovalbumin-sensitized mice after challenge showed bronchial hyperreactivity and increased IL-5 in the serum BALF, as well as eosinophil infiltration in the lungs. However, nasal histology of the ovalbumin-sensitized mice showed no increase in eosinophil infiltration. The nasal lavage fluid revealed no increase in eosinophils or IL-5, and the nasal airway resistance did not increase after challenge either.
Conclusions
In a mouse allergy model, exclusive allergen challenge of the lower airway can elicit a pulmonary and systemic allergic response, but does not induce upper airway inflammatory or physiological responses.
doi:10.4168/aair.2015.7.1.76
PMCID: PMC4274473  PMID: 25553266
Asthma; rhinitis; mice; inflammation
12.  Effect of a platelet activating factor antagonist, WEB 2086, on allergen induced asthmatic responses. 
Thorax  1993;48(6):594-598.
BACKGROUND--Platelet activating factor (PAF) has been implicated in the pathogenesis of airway hyperresponsiveness in asthma. The purpose of this study was to evaluate the effects of a selective PAF antagonist (WEB 2086), given in doses known to antagonise the effects of inhaled PAF in human subjects, on allergen induced early and late asthmatic responses and on airway hyperresponsiveness. METHODS--Eight atopic, mildly asthmatic subjects were studied during a screening period and two treatment periods. During the screening period subjects inhaled an allergen to which they were known to be sensitised and the response was measured as the fall in the forced expired volume in one second (FEV1) to show the presence of early (0-1 h) and late (3-7 h) asthmatic responses. On another day the subjects inhaled allergen diluent. During the treatment periods subjects inhaled allergen after one week's pretreatment with WEB 2086 (100 mg three times a day) or placebo administered in a randomised, double blind, crossover fashion. Histamine airway responsiveness was measured 24 hours before and 24 hours after allergen and the results were expressed as the provocative concentration causing a 20% fall in FEV1 (PC20). RESULTS--The maximal early asthmatic response after allergen with placebo treatment was 18.4% (SE 4.4%) and with WEB 2086 18.9% (4.4%). The maximal late response with placebo treatment was 21.7% (5.3%) and with WEB 2086 21.2% (3.0%). The log difference (before and after allergen) in histamine PC20 was 0.35 (0.06) after placebo treatment and 0.30 (0.1) after WEB 2086. CONCLUSIONS--These results indicate that one week of treatment with an orally administered PAF antagonist (WEB 2086) does not attenuate allergen induced early or late responses or airway hyperresponsiveness.
PMCID: PMC464572  PMID: 8346486
13.  Combined Sensitization of Mice to Extracts of Dust Mites, Ragweed and Aspergillus Breaks through Tolerance and Establishes Chronic Features of Asthma in Mice 
Background
Existing asthma models develop tolerance when chronically exposed to the same allergen.
Objective
To establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure.
Methods
We immunized and exposed mice to a combination of single, double or triple allergens (dust-mite, ragweed, and Aspergillus) intranasally for 8 weeks. Airway hyperreactivity and morphological features of asthma were studied 3 weeks after the allergen exposure. Signaling effects of the allergens were studied on dendritic cells.
Results
Sensitization and repeated exposure to a single allergen induced tolerance. Sensitization to double, and especially triple allergens broke through tolerance and established AHR, eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucus production and airway remodeling that persisted at least 3 weeks after allergen exposure. Mucosal exposure to triple allergens in the absence of an adjuvant was sufficient to induce chronic airway inflammation. Anti-IL5 and -IL13 antibodies inhibited inflammation and AHR in the acute asthma model but not in the chronic triple allergen model. Multiple allergens produce a synergy in p38 MAPK signaling and maturation of dendritic cells, which provides a heightened T cell co-stimulation at a level that cannot be achieved with a single allergen.
Conclusions
Sensitivity to multiple allergens leads to chronic asthma in mice. Multiple allergens synergize in dendritic cell signaling and T cell stimulation that allows escape from the single allergen-associated tolerance development.
Clinical Implications
We have developed a model of chronic asthma that allows for the study and treatment of long-lasting features of asthma obviating the need for acute de novo allergen challenges.
doi:10.1016/j.jaci.2009.02.009
PMCID: PMC2683988  PMID: 19348928
chronic asthma; mouse; inflammation; airway hyperreactivity; tolerance; dendritic cells
14.  In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs. 
Journal of Clinical Investigation  1996;97(4):1117-1121.
This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig.
PMCID: PMC507160  PMID: 8613536
15.  Evaluation of Effect of Taxus baccata Leaves Extract on Bronchoconstriction and Bronchial Hyperreactivity in Experimental Animals 
The present investigation was undertaken to evaluate the bronchodilating effect and bronchial hyperreactivity of alcoholic extract of Taxus baccata Linn. (AET) leaves in experimental animals. Bronchodilator activity of AET was studied on the histamine and acetylcholine aerosol induced bronchospasm in guinea pigs and bronchial hyperreactivity was studied on bronchoalveolar lavage fluid (BALF) in the egg albumin sensitized guinea pigs and by histopathological studies. In vitro mast cell stabilizing activity was studied using compound 48/80 as a degranulating agent. Treatment with AET (200 and 400 mg/kg, p.o., for 7 days) showed significant protection against histamine and acetylcholine aerosol induced bronchospasm in guinea pigs. Significant decrease in the total leukocyte and differential leukocyte count in the BALF of the egg albumin sensitized guinea pigs was observed by administration of AET (200 and 400 mg/kg, p.o., for 15 days). AET dose dependently protected the mast cell disruption induced by compound 48/80. These results suggest that AET not only has bronchodilating activity but also decreases bronchial hyperreactivity by decreasing the infiltration of inflammatory cells in the airway and inhibiting the release of histamine like mediators from the mast cell by stabilizing it.
doi:10.4103/0975-1483.76418
PMCID: PMC3094559  PMID: 21607053
Anti-asthmatic activity; alcoholic extract; leaves; Taxus baccata
16.  Early Life Co-Administration of Cockroach Allergen and Endotoxin Augments Pulmonary and Systemic Responses 
Background
Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease.
Objective
We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice.
Methods
C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue.
Results
Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen specific IgE, IgG1, and IgG2a production, and alveolar remodeling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG1, lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared to exposures with individual substances, the responses to co-exposures were more than additive.
Conclusions
Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodeling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.
doi:10.1111/j.1365-2222.2009.03254.x
PMCID: PMC3408223  PMID: 19438589
endotoxin; cockroach allergen; lung inflammation; allergy; enzyme hypothesis; synergistic effect
17.  Prevention of Diabetes in NOD Mice by Repeated Exposures to a Contact Allergen Inducing a Sub-Clinical Dermatitis 
PLoS ONE  2010;5(5):e10591.
Background
Type 1 diabetes is an autoimmune disease, while allergic contact dermatitis although immune mediated, is considered an exposure driven disease that develops due to epicutanous contact with reactive low-molecular chemicals. The objective of the present study was to experimentally study the effect of contact allergens on the development of diabetes in NOD mice. As the link between contact allergy and diabetes is yet unexplained we also examined the effect of provocation with allergens on Natural Killer T (NKT) cells, since involvement of NKT cells could suggest an innate connection between the two diseases.
Method
NOD mice 4 weeks of age were exposed, on the ears, to two allergens, p-phenylenediamine and 2,4-dinitrochlorobenzene respectively, to investigate the diabetes development. The mice were followed for a maximum of 32 weeks, and they were either repeatedly exposed to the allergens or only sensitized a week after arrival. The stimulation of NKT cells by the two allergens were additionally studied in C57BL/6 mice. The mice were sensitized and two weeks later provocated with the allergens. The mice were subsequently euthanized at different time points after the provocation.
Results
It was found that repeated application of p-phenylenediamine reduced the incidence of diabetes compared to application with water (47% vs. 93%, P = 0.004). Moreover it was shown that in C57BL/6 mice both allergens resulted in a slight increment in the quantity of NKT cells in the liver. Application of the allergens at the same time resulted in an increased number of NKT cells in the draining auricular lymph node, and the increase appeared to be somewhat allergen specific as the accumulation was stronger for p-phenylenediamine.
Conclusion
The study showed that repeated topical application on the ears with a contact allergen could prevent the development of diabetes in NOD mice. The contact allergens gave a non-visible, sub-clinical dermatitis on the application site.
The preventive effect on diabetes may be due to stimulation of peripheral NKT cells, as shown for provocation with p-phenylenediamine in the C57BL/6 mouse. This epicutanous procedure may lead to new strategies in prevention of type 1 diabetes in humans.
doi:10.1371/journal.pone.0010591
PMCID: PMC2868032  PMID: 20485668
18.  Poly(ADP-ribose) polymerase inhibition with HYDAMTIQ reduces allergen-induced asthma-like reaction, bronchial hyper-reactivity and airway remodelling 
Activation of poly(ADP-ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper-reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma-like events to airway damage. Ovalbumin-sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma-like signs, bronchial hyper-reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1-10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen-induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ-treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8-hydroxy-2′-deoxyguanosine and the lung content of pro-inflammatory cytokines (TNF-α, interleukin (IL)-1β, IL-5, IL-6 and IL-18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ-treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.
doi:10.1111/jcmm.12197
PMCID: PMC3955153  PMID: 24444146
asthma-like reaction; poly (ADP-ribose) polymerase (PARP); airway inflammation and remodelling; hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ); histamine release
19.  Antigen Sensitization Influences Organophosphorus Pesticide–Induced Airway Hyperreactivity 
Environmental Health Perspectives  2008;116(3):381-388.
Background
Recent epidemiologic studies have identified organophosphorus pesticides (OPs) as environmental factors potentially contributing to the increase in asthma prevalence over the last 25 years. In support of this hypothesis, we have demonstrated that environmentally relevant concentrations of OPs induce airway hyperreactivity in guinea pigs.
Objectives
Sensitization to allergen is a significant contributing factor in asthma, and we have shown that sensitization changes virus-induced airway hyperreactivity from an eosinophil-independent mechanism to one mediated by eosinophils. Here, we determine whether sensitization similarly influences OP-induced airway hyperreactivity.
Methods
Nonsensitized and ovalbumin-sensitized guinea pigs were injected subcutaneously with the OP parathion (0.001–1.0 mg/kg). Twenty-four hours later, animals were anesthetized and ventilated, and bronchoconstriction was measured in response to either vagal stimulation or intravenous acetylcholine. Inflammatory cells and acetylcholinesterase activity were assessed in tissues collected immediately after physiologic measurements.
Results
Ovalbumin sensitization decreased the threshold dose for parathion-induced airway hyperreactivity and exacerbated parathion effects on vagally induced bronchoconstriction. Pretreatment with antibody to interleukin (IL)-5 prevented parathion-induced hyperreactivity in sensitized but not in nonsensitized guinea pigs. Parathion did not increase the number of eosinophils in airways or the number of eosinophils associated with airway nerves nor did it alter eosinophil activation as assessed by major basic protein deposition.
Conclusions
Antigen sensitization increases vulnerability to parathion-induced airway hyperreactivity and changes the mechanism to one that is dependent on IL-5. Because sensitization to allergens is characteristic of 50% of the general population and 80% of asthmatics (including children), these findings have significant implications for OP risk assessment, intervention, and treatment strategies.
doi:10.1289/ehp.10694
PMCID: PMC2265045  PMID: 18335107
airway hyperreactivity; asthma; atopy; eosinophils; organophosphorus pesticides; parathion; sensitization
20.  Social Stress Enhances Allergen-Induced Airway Inflammation in Mice and Inhibits Corticosteroid Responsiveness of Cytokine Production 
Chronic psychosocial stress exacerbates asthma but the underlying mechanisms remain poorly understood. We hypothesized that psychosocial stress aggravates allergic airway inflammation by altering innate immune cell function. The effects of stress on airway inflammation, lung function and glucocorticoid responsiveness were studied in a novel in vivo murine model of combined social disruption stress and allergic sensitization. The effects of corticosterone were assessed on cytokine profile and glucocorticoid receptor activation in LPS-stimulated spleen cell cultures in vitro.
Airway inflammation resolved 48 hours after a single allergen provocation in sensitized control mice but not in animals that were repeatedly exposed to stress prior to allergen challenge. The enhanced eosinophilic airway inflammation 48 hours after allergen challenge in these mice was associated with increased levels of IL-5, GM-CSF, IgG1, TARC, TNF-α and IL-6 in the airways and a diminished inhibition of these mediators by corticosterone in LPS-stimulated splenocyte cultures in vitro. Stress-induced reduction of the corticosteroid effects paralleled increased p65 expression and a decreased DNA binding capability of the glucocorticoid receptor in vitro. Further, glucocorticoid receptor mRNA and protein expression in the lungs of mice exposed to both stress and allergen was markedly reduced in comparison with that in either condition alone or in naïve mice. Thus, exposure to repeated social stress prior to allergen inhalation enhances and prolongs airway inflammation and alters corticosterone responsiveness. We speculate that these effects were mediated at least in part by impaired glucocorticoid receptor expression and function.
doi:10.4049/jimmunol.0800891
PMCID: PMC2767120  PMID: 19494313
Rodent; Allergy; Lung; Inflammation; Th1/Th2 Cells
21.  Plasma 9α,11ß-PGF2, a PGD2 metabolite, as a sensitive marker of mast cell activation by allergen in bronchial asthma 
Thorax  2004;59(6):459-464.
Background: Prostaglandin D2 (PGD2) is a major cyclooxygenase product generated by activated mast cells during an allergic response. Assessment of PGD2 and its metabolites in patients with asthma has mostly been performed in urine, bronchoalveolar lavage fluid and induced sputum, whereas human plasma determinations have been performed only sporadically.
Methods: In 32 patients with allergic asthma and 50 healthy non-allergic controls, baseline plasma and urinary levels of 9α,11ß-PGF2, a primary PGD2 metabolite, were assessed by gas chromatography/mass spectrometry. Serum tryptase levels were measured by fluoroenzyme immunoassay and urinary leukotriene E4 (LTE4) by ELISA. In a subgroup of 10 asthmatics (randomly selected from the 32 study patients) in whom bronchial allergen challenges with specific allergens (Dermatophagoides pteronyssinus, n = 4, mixed grass pollens, n = 6) were carried out, measurements were taken both before and after provocation.
Results: At baseline no significant differences between mean plasma and urinary levels of the PGD2 metabolite and serum tryptase levels were found in asthmatics or controls. Asthmatic patients had significantly higher urinary LTE4 levels. Allergen challenge resulted in a significant early increase in the mean plasma 9α,11ß-PGF2 level and only a borderline but significant increase in the urinary 9α,11ß-PGF2 level within 2 hours after provocation. The challenge did not produce statistically significant changes in serum tryptase levels. Urinary LTE4 levels remained significantly increased 4 hours after provocation.
Conclusions: PGD2 is actively involved in the early asthmatic response to allergens. Measurement of 9α,11ß-PGF2 release into plasma rather than urine following allergen challenge is a sensitive marker of enhanced PGD2 synthesis, most probably due to mast cell activation.
doi:10.1136/thx.2003.013573
PMCID: PMC1747024  PMID: 15170023
22.  Effects of nebulized ketamine on allergen-induced airway hyperresponsiveness and inflammation in actively sensitized Brown-Norway rats 
Since airway hyperresponsiveness (AHR) and allergic inflammatory changes are regarded as the primary manifestations of asthma, the main goals of asthma treatment are to decrease inflammation and maximize bronchodilation. These goals can be achieved with aerosol therapy. Intravenous administration of the anesthetic, ketamine, has been shown to trigger bronchial smooth muscle relaxation. Furthermore, increasing evidence suggests that the anti-inflammatory properties of ketamine may protect against lung injury. However, ketamine inhalation might yield the same or better results at higher airway and lower ketamine plasma concentrations for the treatment of asthma. Here, we studied the effect of ketamine inhalation on bronchial hyperresponsiveness and airway inflammation in a Brown-Norway rat model of ovalbumin(OVA)-induced allergic asthma. Animals were actively sensitized by subcutaneous injection of OVA and challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitizened rats received inhalation of aerosol of phosphate-buffered saline (PBS) or aerosol of ketamine or injection of ketamine respectivity. Airway reactivity to acetylcholine (Ach) was measured in vivo, and various inflammatory markers, including Th2 cytokines in bronchoalveolar lavage fluid (BALF), as well as induciable nitric oxide synthase (iNOS) and nitric oxide (NO) in lungs were examined. Our results revealed that delivery of aerosolized ketamine using an ultrasonic nebulizer markedly suppressed allergen-mediated airway hyperreactivity, airway inflammation and airway inflammatory cell infiltration into the BALF, and significantly decreased the levels of interleukin-4 (IL-4) in the BALF and expression of iNOS and the concentration of NO in the inflamed airways from OVA-treated rats. These findings collectively indicate that nebulized ketamine attenuated many of the central components of inflammatory changes and AHR in OVA-provoked experimental asthma, potentially providing a new therapeutic approach against asthma.
doi:10.1186/1476-9255-4-10
PMCID: PMC1876456  PMID: 17480224
23.  Segmental Allergen Challenge Alters Multimeric Structure and Function of Surfactant Protein D in Humans 
Rationale: Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells.
Objectives: We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation.
Methods: Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis.
Measurements and Main Results: The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate–polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol–SP-D compared with patients without cross-linked SP-D.
Conclusions: We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.
doi:10.1164/rccm.201004-0654OC
PMCID: PMC3086753  PMID: 21131470
human asthma; pulmonary collectins; nitric oxide; surfactant proteins; biomarker
24.  Airway hyperresponsiveness and bronchial mucosal inflammation in T cell peptide‐induced asthmatic reactions in atopic subjects 
Thorax  2007;62(9):750-757.
Background
Subjects with allergic asthma develop isolated late asthmatic reactions after inhalation of allergen‐derived T cell peptides. Animal experiments have shown that airway hyperresponsiveness (AHR) is CD4+ cell‐dependent. It is hypothesised that peptide inhalation produces increases in non‐specific AHR and a T cell‐dominant bronchial mucosal inflammatory response.
Methods
Bronchoscopy, with bronchial biopsies and bronchoalveolar lavage (BAL), was performed in 24 subjects with cat allergy 6 h after aerosol inhalation of short overlapping peptides derived from Fel d 1, the major cat allergen. Biopsy specimens and BAL fluid were studied using immunohistochemistry and ELISA.
Results
Twelve of the 24 subjects developed an isolated late asthmatic reaction without a preceding early (mast cell/histamine‐dependent) reaction characteristic of whole allergen inhalation. These responders had significant between‐group differences (responders vs non‐responders) in the changes (peptide vs diluent) in AHR (p = 0.007) and bronchial mucosal CD3+ (p = 0.005), CD4+ (p = 0.006) and thymus‐ and activation‐regulated chemokine (TARC)+ (p = 0.003) but not CD8+ or CD25+ cells or eosinophils, basophils, mast cells and macrophages. The between‐group difference for neutrophils was p = 0.05 but with a non‐significant within‐group value (peptide vs diluent, responders, p = 0.11). In BAL fluid there was a significant between‐group difference in TARC (p = 0.02) but not in histamine, tryptase, basogranulin, C3a or C5a, leukotrienes C4/D4/E4, prostaglandins D2 or F2α.
Conclusions
Direct activation of allergen‐specific airway T cells by peptide inhalation in patients with atopic asthma leads to increased AHR with local increases in CD3+ and CD4+ cells and TARC but no significant changes in eosinophils or basophil/mast cell products. These findings support previous animal experiments which showed a CD4+ dependence for AHR.
doi:10.1136/thx.2006.072041
PMCID: PMC2117301  PMID: 17389757
25.  Attenuation of early and late phase allergen-induced bronchoconstriction in asthmatic subjects by a 5-lipoxygenase activating protein antagonist, BAYx 1005 
Thorax  1997;52(4):348-354.
BACKGROUND: The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) have been implicated in the pathogenesis of allergen-induced airway responses. The effects of pretreatment with BAYx 1005, an inhibitor of leukotriene biosynthesis via antagonism of 5-lipoxygenase activating protein, on allergen-induced early and late asthmatic responses has been evaluated. METHODS: Eight atopic subjects with mild asthma participated in a two period, double blind, placebo controlled, cross-over trial. Subjects were selected on the basis of a forced expiratory volume in one second (FEV1) of > 70% predicted, a methacholine provocative concentration causing a 20% fall in FEV1 (PC20) of < 32 mg/ ml, a documented allergen- induced early response (EAR, > 15% fall in FEV1 0-1 hour after allergen inhalation) and late response (LAR, > 15% fall in FEV1 3-7 hours after allergen inhalation), and allergen-induced airway hyperresponsiveness (at least a doubling dose reduction in the methacholine PC20 30 hours after allergen inhalation). During the treatment periods subjects received BAYx 1005 (500 mg twice daily) or placebo for 3.5 days; treatment periods were separated by at least two weeks. On the third day of treatment, two hours after administration of medication, subjects performed an allergen inhalation challenge and FEV1 was measured for seven hours. RESULTS: Treatment with BAYx 1005 attenuated the magnitude of both the allergen-induced early and late asthmatic responses. The mean (SE) maximal fall in FEV1 during the EAR was 26.6 (3.3)% during placebo treatment and 11.4 (3.3)% during treatment with BAYx 1005 (mean difference 15.2 (95% confidence interval (CI) 9.4 to 21.00) with a mean protection afforded by BAYx 1005 of 57.1%. The mean (SE) maximal fall in FEV1 during the LAR was 19.8 (5.7)% during placebo treatment and 10.7 (4.4)% during BAYx 1005 treatment (mean difference 9.2 (95% CI 1.4 to 17.0) with a mean protection afforded by BAYx 1005 of 46.0%. The area under the time response curve (AUC0-3) was also reduced after treatment with BAYx 1005 compared with placebo by 86.5%.h (mean difference 26.3 (95% CI 17.1 to 38.5)) and the AUC3-7 by 59.6%.h (mean difference 26.9 (95% CI-3.8 to 57.6)). CONCLUSIONS: These results show that antagonism of 5-lipoxygenase activating protein can attenuate allergen-induced bronchoconstrictor responses and support an important role for the cysteinyl leukotrienes in mediating these asthmatic responses. 



PMCID: PMC1758536  PMID: 9196518

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