OVA-sensitized Animals Research Articles

Sex differences in plasma metabolites in a guinea pig model of allergic asthma.

Sex seems to be a contributing factor in the pathogenesis of bronchial asthma. This study aimed to find sex-related differences in metabolome measured by hydrogen-1 nuclear magnetic resonance ((1)H NMR) spectroscopy in healthy and ovalbumin (OVA)-sensitized guinea pigs. Adult male and female animals were divided into controls and OVA-sensitized groups. OVA-sensitization was performed by OVA systemic and inhalational administration within 14 days; on day 15, animals were killed by anesthetic overdose followed by exsanguination. Blood was taken and differential white blood cell count was measured. Left lung was saline-lavaged and differential cell count in the bronchoalveolar lavage fluid (BALF) was measured. After blood centrifugation, plasma was processed for (1)H NMR analysis. Metabolomic data was evaluated by principal component analysis (PCA). Eosinophil counts elevated in the BALF confirming eosinophil-mediated inflammation in OVA-sensitized animals of both sexes. Sex differences for lactate, glucose, and citrate were found in controls, where these parameters were lower in males than in females. In OVA-sensitized males higher glucose and lower pyruvate were found compared to controls. OVA-sensitized females showed lower lactate, glucose, alanine, 3-hydroxy-butyrate, creatine, pyruvate, and succinate concentrations compared to controls. In OVA-sensitized animals, lactate concentration was lower in males. Data from females (healthy and OVA-sensitized) were generally more heterogeneous. Significant sex differences in plasma concentrations of metabolites were found in both healthy and OVA-sensitized animals suggesting that sex may influence the metabolism and may thereby contribute to different clinical picture of asthma in males and females.

Experimental evaluation of the nonselective and selective TMEM16 family calcium-activated chloride channel blockers in the airways

Abstract Background The family of calcium-activated chloride channels, TMEM16, plays a significant role in contributing to the pathogenesis of airway inflammatory diseases. Targeting these ion channels and aiming to modulate them may provide an interesting new approach to the therapy of these potentially fatal diseases. Methods We tested this hypothesis in both healthy and ovalbumin (OVA)-sensitized male Dunkin-Hartley guinea pigs. The ion channel activity was modulated by TMEM16A-nonselective (benzbromarone) and TMEM16A-selective blockers (CaCCinh-A01). In vivo airway reactivity, induced by histamine and methacholine, was expressed as specific airway resistance (sRaw) values. The number of citric acid-induced coughs was counted using a double-chambered body plethysmograph, and the frequency of ciliary beating (CBF) was assessed in vitro by brushing method. For comparison, salbutamol and codeine were tested under the same conditions. Results The results showed significant differences in the responses of unsensitized and sensitized airways to both TMEM16 blockers administered. CaCCinh-A01 and benzbromarone significantly reduced the number of cough efforts in the group of OVA-sensitized guinea pigs. Significant improvement in sRaw values could be observed in OVA-sensitized TMEM16 blocker–treated animals compared to salbutamol when challenged with inhalational histamine, and the outcome was similar to salbutamol when challenged with methacholine. CBF was significantly inhibited in animals sensitized to OVA treated with selective inhibition of TMEM16A. Conclusions The results demonstrated that treatment with blockers of TMEM16 can reduce both cough effort and sRaw, and the difference between TMEM16A-selective and TMEM16A-nonselective blocking is only negligibly in favor of CaCCinh-A01. It is also worthwhile to note the impairment of CBF in OVA-sensitized animals treated with CaCCinh-A01.

Inhibition of Rho-kinase improves response to deep inspiration in ovalbumin-sensitized guinea pigs.

Objective(s):The modulatory effect of deep inspiration (DI) on airway constriction is impaired in asthma. However, mechanisms underlying this impairment are not clear. Since there is evidence indicating that Rho-kinase activation mediates force maintenance under oscillatory strain, we investigated the impact of Rho-kinase inhibition on the bronchodilatory effect of DI in ovalbumin (OVA) sensitized guinea pigs.Materials and Methods:forty-eight male Dunkin Hartley guinea pigs were divided into 8 groups including saline/ constant, saline/DI, OVA/constant, OVA/DI, Rho-I/OVA/constant, Rho-I/OVA/DI, OVA-Rho-I/MCh/constant, and OVA-Rho-I/MCh/DI. Animals were subjected to 12 inhalations of OVA or saline aerosol. Guinea pigs in Rho-I/OVA/constant or DI groups were treated with the Rho-kinase inhibitor (Rho-I) (Y-27632, 1 mM aerosols) prior to the last 8 allergen inhalations and OVA-Rho-I/MCh/constant or DI groups received Y-27632 at the end of allergen sensitization protocol before methacholine challenge. The bronchodilatory effect of DI in guinea pigs that were exposed to methacholine was assessed by using an animal ventilator. The bronchodilatory effect was assessed using several parameters: the airway pressure maintenance, airway pressure recovery, and decline of airway pressure.Results:Results indicated that application of Y-27632 prior to methacholine challenge reduces the airway smooth muscle ability to maintain pressure and also causes further decline in airway pressure in OVA-sensitized animals undergone DI. However, the inhibition of Rho-kinase before OVA inhalations had minimal effect.Conclusion:We propose that alteration of Rho-kinase signaling pathway may be one of the mechanisms underlying the impairment of DI-induced bronchodilation in OVA-sensitized guinea pigs.

Dehydrodieugenol improved lung inflammation in an asthma model by inhibiting the STAT3/SOCS3 and MAPK pathways

BackgroundEugenol, a common phenylpropanoid derivative found in different plant species, has well-described anti-inflammatory effects associated with the development of occupational hypersensitive asthma. Dehydrodieugenol, a dimeric eugenol derivative, exhibits anti-inflammatory and antioxidant activities and can be found in the Brazilian plant species Nectandra leucantha (Lauraceae). The biological effects of dehydrodieugenol on lung inflammation remain unclear. PurposeThis study aimed to investigate the effects of eugenol and dehydrodieugenol isolated from N. leucantha in an experimental model of asthma. MethodsIn the present work, the toxic effects of eugenol and dehydrodieugenol on RAW 264.7 cells and their oxidant and inflammatory effects before lipopolysaccharide (LPS) exposure were tested. Then, male BALB/c mice were sensitized with ovalbumin through a 29-day protocol and treated with vehicle, eugenol, dehydrodieugenol or dexamethasone for eight days beginning on the 22nd day until the end of the protocol. Lung function; the inflammatory profile; and the protein expression of ERK1/2, JNK, p38, VAChT, STAT3, and SOCS3 in the lung were evaluated by immunoblotting. ResultsEugenol and dehydrodieugenol were nontoxic to cells. Both compounds inhibited NO release and the gene expression of IL-1β and IL-6 in LPS-stimulated RAW 264.7 cells. In OVA-sensitized animals, dehydrodieugenol reduced lung inflammatory cell numbers and the lung concentrations of IL-4, IL-13, IL-17, and IL-10. These anti-inflammatory effects were associated with inhibition of the JNK, p38 and ERK1/2, VAChT and STAT3/SOCS3 pathways. Moreover, treatment with dehydrodieugenol effectively attenuated airway hyperresponsiveness. Conclusion: The obtained data demonstrate, for the first time, that dehydrodieugenol was more effective than eugenol in counteracting allergic airway inflammation in mice, especially its inhibition of the JNK, p38 and ERK1/2, components of MAPK pathway. Therefore, dehydrodieugenol can be considered a prototype for the development of new and effective agents for the treatment of asthmatic patients.

Treadmill exercise restores memory and hippocampal synaptic plasticity impairments in ovalbumin-sensitized juvenile rats: Involvement of brain-derived neurotrophic factor (BDNF)

Studies demonstrate that asthma, especially during childhood, affects the functions of the brain including learning and memory. Exercise is well known for its neuroprotective functions and for its beneficial effects on asthma. We aimed to assess the effects of exercise on cognitive function, synaptic plasticity, and hippocampal brain-derived neurotrophic factor (BDNF) levels in ovalbumin (OVA) sensitized juvenile rats. Rats were sensitized by intraperitoneal administration and inhaled OVA. Animals were subjected to treadmill running exercise during the OVA-challenged period. T-helper type 2 (Th2) cytokine [interleukin (IL)-4], Th1 cytokine (INF-γ) levels, and INF-γ/IL-4 (Th1/Th2) ratio in bronchoalveolar lavage fluid (BALF), and tracheal response to methacholine and OVA were measured. Further, memory behaviors and BDNF levels were measured in the hippocampus as well as long-term potentiation (LTP) was assessed by recording field excitatory postsynaptic potentials (fEPSPs) in the hippocampus. The levels of IL-4 and TGF-β were decreased but INF-γ level and INF-γ/IL-4 ratio increased in the BALF due to exercise in the OVA-sensitized animals. In addition, exercise improved OVA-sensitization induced cognitive impairments, increased BDNF levels, and enhanced hippocampal LTP in OVA-sensitized rats. Exercise is not only effective in the alleviation of airway inflammation by restoring Th1/Th2 cytokines balance, but also is a candidate for improvement of memory and synaptic plasticity deficits partially through increasing the levels of hippocampal BDNF in OVA-sensitized rats.

Allergic airway sensitization impairs antibacterial IgG antibody responses during bacterial respiratory tract infections

Mycoplasma pneumoniae, an atypical human pathogen, has been associated with asthma initiation and exacerbation. Asthmatic patients have been reported to have higher carriage rates of M pneumoniae compared with nonasthmatic subjects and are at greater risk for invasive respiratory infections. We sought to study whether prior allergen sensitization affects the host response to chronic bacterial infection. BALB/cJ and IL-4 receptor α-/- mice were sensitized with ovalbumin (OVA) and then infected with M pneumoniae or Streptococcus pneumoniae. Immune parameters were analyzed at 30days postinfection and included cellular profiles in bronchoalveolar lavage fluid (BALF) and serum IgG and IgE antibody levels to whole bacterial lysate, recombinant P1 adhesin, and OVA. Total lung RNA was examined for transcript levels, and BALF was examined for cytokine protein profiles. Anti-M pneumoniae antibody responses were decreased in allergen-sensitized, M pneumoniae-infected animals compared with control animals, but OVA-specific IgG responses were unaffected. Similar decreases in anti-S pneumoniae antibody levels were found in OVA-sensitized animals. However, M pneumoniae, but not S pneumoniae, infection augmented anti-OVA IgE antibody responses. Loss of IL-4 receptor signaling partially restored anti-M pneumoniae antibody responses in IgG2a and IgG2b subclasses. Inflammatory cytokine levels in BALF from OVA-sensitized, M pneumoniae-infected or S pneumoniae-infected animals were reduced compared with those in uninfected OVA-sensitized control animals. Unexpectedly, airway hyperreactivity to methacholine was essentially ablated in M pneumoniae-infected, OVA-sensitized animals. An established type 2-biased host immune response impairs the host immune response to respiratory bacterial infection in a largely pathogen-independent manner. Some pathogens, such as M pneumoniae, can augment ongoing allergic responses and inhibit pulmonary type 2 cytokine responses and allergic airway hyperreactivity.

Aqueous extracts from Uncaria tomentosa (Willd. ex Schult.) DC. reduce bronchial hyperresponsiveness and inflammation in a murine model of asthma

Ethnopharmacological relevanceUncaria tomentosa (Willd. Ex Schult) DC is used by indigenous tribes in the Amazonian region of Central and South America to treat inflammation, allergies and asthma. The therapeutic properties of U. tomentosa have been attributed to the presence of tetracyclic and pentacyclic oxindole alkaloids and to phenolic acids. Aims of the studyTo characterize aqueous bark extracts (ABE) and aqueous leaf extracts (ALE) of U. tomentosa and to compare their anti-inflammatory effects. Materials and methodsConstituents of the extracts were identified by ultra performance liquid chromatography-mass spectrometry. Anti-inflammatory activities were assessed in vitro by exposing lipopolysaccharide-stimulated macrophage cells (RAW264.7-Luc) to ABE, ALE and standard mitraphylline. In vivo assays were performed using a murine model of ovalbumin (OVA)-induced asthma. OVA-sensitized animals were treated with ABE or ALE while controls received dexamethasone or saline solution. Bronchial hyperresponsiveness, production of Th1 and Th2 cytokines, total and differential counts of inflammatory cells in the bronchoalveolar lavage (BAL) and lung tissue were determined. ResultsMitraphylline, isomitraphylline, chlorogenic acid and quinic acid were detected in both extracts, while isorhyncophylline and rutin were detected only in ALE. ABE, ALE and mitraphylline inhibited the transcription of nuclear factor kappa-B in cell cultures, ALE and mitraphylline reduced the production of interleukin (IL)−6, and mitraphylline reduced production of tumor necrosis factor-alpha. Treatment with ABE and ALE at 50 and 200 mg kg−1, respectively, reduced respiratory elastance and tissue damping and elastance. ABE and ALE reduced the number of eosinophils in BAL, while ALE at 200 mg kg−1 reduced the levels of IL-4 and IL-5 in the lung homogenate. Peribronchial inflammation was significantly reduced by treatment with ABE and ALE at 50 and 100 mg kg−1 respectively. ConclusionThe results clarify for the first time the anti-inflammatory activity of U. tomentosa in a murine model of asthma. Although ABE and ALE exhibited distinct chemical compositions, both extracts inhibited the production of pro-inflammatory cytokines in vitro. In vivo assays revealed that ABE was more effective in treating asthmatic inflammation while ALE was more successful in controlling respiratory mechanics. Both extracts may have promising applications in the phytotherapy of allergic asthma.

Rock Inhibitor Fasudil Reduces Leukocyte-Endothelium Interactions in the Microvasculature of a Sickle Cell Mouse Model of Allergic Inflammation

Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. DisclosuresConran:Bayer AG: Research Funding. Fertrin:Alexion Pharmaceuticals: Consultancy.

Effect of α-Hederin on IL-2 and IL-17 mRNA and miRNA-133a Levels in Lungs of Ovalbumin-Sensitized Male Rats.

α-hederin, a saponin that is a major constituent of English Ivy (Hedera helix) is effective in the treatment of asthma. In the present study, the effect of α-hederin on lung tissue pathology and the levels of the inflammatory mediators; IL-2 mRNA, IL-17 mRNA, and MicroRNAs (miRNA)-133a was evaluated in a rat ovalbumin (OVA)-sensitized model of asthma. Rats were divided randomly into control (C), OVA-sensitized (S), OVA-sensitized pretreated with the antioxidant, thymoquinone (3 mg/kg, S + TQ) or OVA-sensitized pretreated with α-hederin (0.02 mg/kg, S + AH) groups. Levels of IL-2 and IL-17 mRNA were higher in the OVA-sensitized group than controls while the level of miRNA-133a gene expression was lower. IL-2 mRNA and miRNA-133a gene expression in the S + TQ group was higher than in the control and OVA-sensitized groups while the level of IL-17 mRNA in the S + TQ group was lower than in the OVA-sensitized group. Pretreatment with α-hederin decreased IL-17 mRNA levels and increased miRNA-133a gene expression compared with OVA-sensitized animals. All pathological changes in pretreated groups were lower than the OVA-sensitized group. These results showed a beneficial effect of α-hederin in OVA-sensitized rats, suggesting that α-hederin affects the IL-2 and IL-17 secretion pathways, altering miRNA-133a expression.

Characteristics of IL-25 and allergen-induced airway fibrosis in a murine model of asthma.

Interleukin (IL)-25 has been implicated in the pathogenesis of human asthma by inducing a Th2 cytokine response, but its possible role in the development of airway remodelling is less clear. We developed a murine surrogate of chronic airway inflammation induced by intranasal application of IL-25 alone. Comparison was with the 'classical' surrogate of ovalbumin (OVA) intranasal instillation into previously sensitized animals. Airway fibrotic biomarkers were analysed by immunohistochemistry and enzyme-linked immunosorbent assay. Additionally, proliferation assay and real-time polymerase chain reaction analysis were performed to assess IL-25's effects on primary human bronchial fibroblasts in vitro. In Balb/c mice, intranasal instillation of IL-25 alone induced florid airway fibrosis, including increased lay down of extracellular matrix proteins such as collagen I, III, V and fibronectin, increased numbers of fibroblasts/myofibroblasts, a profibrotic imbalance in matrix metalloproteinase/tissue inhibitor of metalloproteinase production and increased expression of profibrotic mediators including connective tissue growth factor and transforming growth factor-β1. These changes broadly reproduced those seen with classical intranasal OVA challenge in OVA-sensitized animals. Furthermore, IL-25 induced proliferation and expression of collagen I and III and smooth muscle α-actin in primary human lung fibroblasts. We conclude that chronic exposure of the airways to IL-25 alone is sufficient to cause functionally relevant airway remodelling, with the corollary that targeting of IL-25 may attenuate bronchial remodelling and fibrosis in human asthmatics.

Intraluminal acid activates esophageal nodose C fibers after mast cell activation

Acid reflux in the esophagus can induce esophageal painful sensations such as heartburn and noncardiac chest pain. The mechanisms underlying acid-induced esophageal nociception are not clearly understood. In our previous studies, we characterized esophageal vagal nociceptive afferents and defined their responses to noxious mechanical and chemical stimulation. In the present study, we aim to determine their responses to intraluminal acid infusion. Extracellular single-unit recordings were performed in nodose ganglion neurons with intact nerve endings in the esophagus using ex vivo esophageal-vagal preparations. Action potentials evoked by esophageal intraluminal acid perfusion were compared in naive and ovalbumin (OVA)-challenged animals, followed by measurements of transepithelial electrical resistance (TEER) and the expression of tight junction proteins (zona occludens-1 and occludin). In naive guinea pigs, intraluminal infusion with either acid (pH = 2-3) or capsaicin did not evoke an action potential discharge in esophageal nodose C fibers. In OVA-sensitized animals, following esophageal mast cell activation by in vivo OVA inhalation, intraluminal acid infusion for about 20 min started to evoke action potential discharges. This effect is further confirmed by selective mast cell activation using in vitro tissue OVA challenge in esophageal-vagal preparations. OVA inhalation leads to decreased TEER and zona occludens-1 expression, suggesting an impaired esophageal epithelial barrier function after mast cell activation. These data for the first time provide direct evidence of intraluminal acid-induced activation of esophageal nociceptive C fibers and suggest that mast cell activation may make esophageal epithelium more permeable to acid, which subsequently may increase esophageal vagal nociceptive C fiber activation.

Prostaglandin D2 receptord-type prostanoid receptor 2 mediates eosinophil trafficking into the esophagus

Eosinophilic esophagitis is characterized by eosinophil-predominant inflammation in the esophagus. How eosinophils migrate and infiltrate into the esophagus, however, is less clear. Our previous study demonstrated that mast cell activation led to eosinophil infiltration in the esophagus. Prostaglandin D2 (PGD2) is an important mediator released from activated mast cells. The present study aims to determine whether PGD2 induces eosinophil infiltration into the esophagus via a d-type prostanoid receptor 2 (DP2) receptor-dependent mechanism. Using an in vivo guinea pig model, PGD2, d-type prostanoid receptor 1 (DP1) agonist, or DP2 agonist were injected into the esophagus. Esophageal tissues were removed 2 hours after injections and proceeded to either hematoxylin-eosin (HE) staining or immunofluorescent staining of eosinophil major basic protein (MBP) to compare each treatment-induced eosinophil infiltration in the esophagus. In a separate study, ovalbumin (OVA)-sensitized guinea pigs were pretreated with either DP2 or DP1 antagonists, followed by inhalation of OVA to induce mast cell activation. Esophageal tissues were then processed for immunofluorescent staining of MBP. PGD2 injection in the esophagus led to an increase of eosinophil infiltration in esophageal epithelium at the injection site as revealed by HE staining. Increased infiltration of eosinophils was further confirmed by the increased presence of MBP-labeled immunopositive (MBP-LI) cells in esophageal epithelium. Injection with DP2 agonist 15(R)-PGD2, but not DP1 agonist BW 245C, mimicked the PGD2-induced response. In OVA-sensitized animals, antigen inhalation increased MBP-LI cells in esophageal epithelium. Pretreatment with DP2 antagonist BAY-u3405, but not DP1 antagonist BW 868C, inhibited the antigen inhalation-induced increase of MBP-LI cells in esophageal epithelium. These data support the hypothesis that PGD2 induces eosinophil trafficking into the esophageal epithelium via a DP2-mediated pathway, suggesting a role of DP2 antagonist in the prevention of eosinophilic esophagitis.

MAG‐EPA resolves lung inflammation in an allergic model of asthma

Asthma is a chronic disease characterized by airways hyperresponsiveness, inflammation and airways remodelling involving reversible bronchial obstruction. Omega-3 fatty acids and their derivatives are known to reduce inflammation in several tissues including lung. The effects of eicosapentaenoic acid monoacylglyceride (MAG-EPA), a newly synthesized EPA derivative, were determined on the resolution of lung inflammation and airway hyperresponsiveness in an in vivo model of allergic asthma. Ovalbumin (OVA)-sensitized guinea-pigs were treated or not with MAG-EPA administered per os. Isometric tension measurements, histological analyses, homogenate preparation for Western blot experiments or total RNA extraction for RT-PCR were performed to assess the effect of MAG-EPA treatments. Mechanical tension measurements revealed that oral MAG-EPA treatments reduced methacholine (MCh)-induced bronchial hyperresponsiveness in OVA-sensitized guinea-pigs. Moreover, MAG-EPA treatments also decreased Ca(2+) hypersensitivity of bronchial smooth muscle. Histological analyses and leucocyte counts in bronchoalveolar lavages revealed that oral MAG-EPA treatments led to less inflammatory cell recruitment in the lung of OVA-sensitized guinea-pigs when compared with lungs from control animals. Results also revealed a reduction in mucin production and MUC5AC expression level in OVA-sensitized animals treated with MAG-EPA. Following MAG-EPA treatments, the transcript levels of pro-inflammatory markers such as IL-5, eotaxin, IL-13 and IL-4 were markedly reduced. Moreover, per os MAG-EPA administrations reduced COX2 over-expression in OVA-sensitized animals. We demonstrate that MAG-EPA reduces airway hyperresponsiveness and lung inflammation in OVA-sensitized animals, a finding consistent with a decrease in IL-4, IL-5, IL-13, COX-2 and MUC5AC expression levels in the lung. The present data suggest that MAG-EPA represents a new potential therapeutic strategy for resolving inflammation in allergic asthma.

Effect of the circadian cycle in Late Asthmatic Response (LAR). Comparison between two allergic asthma models in rats and mice

The ovalbumin (OVA) model of allergic inflammation, both in rat and mouse is characterised by a late bronchoconstrictive response (LAR) after allergen challenge that can be measured using whole-body plethysmography (WBP) in conscious unrestrained animals. Circadian oscillations of lung mechanical properties have been reported in conscious undisturbed rodents. Potentially, these fluctuations could alter the pulmonary response induced by allergen provocation and the response of pharmacological treatments. This circadian influence has been showed in asthma patients [1], where an allergen inhalation challenge given in the evening produced a more frequent and severe LAR compared to that one given in the morning. The aim of this study was to assess whether LAR in OVA-sensitized rats and mice was affected differently when animals were exposed to allergen in different times of day. Firstly, to confirm the circadian oscillations, changes in several respiratory parameters were monitored during 24h in naive Brown Norway rats or C57/Black6 mice. Afterwards, OVA-sensitized animals were exposed to the allergen in different periods of the day and the pulmonary function parameters were monitored, including the LAR. In rats, LAR evoked in three different times of day (morning, afternoon or evening) showed no differences in severity, but was more frequent during the evening. In contrast, the LAR triggered in mice in the morning was more severe and more frequent than that evoked during the evening. These results suggest that species and day period of time to induce LAR in allergic animal models are critical and should be taken into account when evaluating the effects of new compounds on this read-out.

Allergic reactions compared between BN and Wistar rats after oral exposure to ovalbumin

There is currently no validated animal model for evaluating the potential allergenicity of food proteins. This study aimed to compare the allergic reactions between BN and Wistar rats after oral exposure to ovalbumin (OVA) by studying immune responses and clinical manifestations. Female BN and Wistar rats were orally exposed to OVA on days 1 and 14, and thereafter daily from day 15 to day 42. Sera and plasma were screened for OVA-specific antibodies and histamine. On day 49, all the OVA-sensitized animals were orally challenged with OVA before blood pressure was measured. One day later (on day 50), histopathology and differential cell counts were performed. The results indicate that oral exposure of BN rats to OVA yielded IgE, IgG, and IgG2a antibody responses that were generally of higher levels than those observed in Wistar rats (p < 0.05). However, the Wistar rats presented with more serious clinical manifestations and histopathologic changes that could have serious implications for any OVA-induced anaphylaxis. The studies here proved that OVA-sensitized BN and Wistar rats evinced different immune responses and clinical manifestations; these outcomes suggested that the two rat strains might differ in their immunologic mechanisms of allergy and that there was no correlation between immune responses and the severity of clinical symptoms. To be clear, the data from these studies should be viewed as ‘preliminary’, as only a single protein allergen was examined. Accordingly, further studies are needed to compare the allergic reactions between BN and Wistar rats by using purified strong-, weak-, and non-allergenic proteins based on the experiments reported here.

New Omega-3 Derivatives Reduce Airway Inflammation and Prevent Rho-Kinase Activation in an Allergic Model of Asthma

Asthma is a chronic inflammatory airways disease characterized by early and late asthmatic reactions that are associated with infiltration and activation of inflammatory cells in the airways. Bronchial hyperresponsiveness to a variety of stimuli is part of the symptomology which include neurotransmitters and inflammatory mediators. Omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to reduce inflammation in the lung. In the present study, we synthesized new omega-3 polyunsaturated fatty acid (n3-PUFA) derivatives, namely DHA monoglyceride (MAG-DHA) and EPA monoglyceride (MAG-EPA), and investigated their effects on lung inflammation and RhoA activation in an in vivo guinea pig model of allergic asthma. Histological analyses and leukocyte count in bronchoalveolar lavages revealed that oral MAG-DHA and MAG-EPA treatments led to less inflammatory cell recruitment in the lung of ovalbumine (OVA)-sensitized guinea pigs when compared to lung from control animals. Results also revealed a reduction in mucus production in OVA-sensitized animals treated with either MAG-DHA or MAG-EPA. Moreover, per os n-3 PUFA administrations prevented NF k B activation and COX2 over-expression in OVA-sensitized animals. Mechanical tension measurements revealed that oral MAG-DHA and MAG-EPA treatments prevented U-46619-induced bronchial hyperresponsiveness in OVA-sensitized guinea pigs. MAG-DHA and MAG-EPA also prevented U-46619-induced Ca 2+ hypersensitivity of bronchial smooth muscle through a decrease in RhoA activation. Together, these findings provide key evidences regarding the mode of action of MAG-DHA and MAG-EPA in the lung and point to new potential therapeutic strategies for modulating inflammation and airway hyperresponsiveness in asthma.

Local administration of uridine suppresses the cardinal features of asthmatic airway inflammation

The immuno-modulatory properties of nucleotides such as adenosine or inosine, have been described extensively. Recently, the nucleoside uridine and its analogue 4-thiouridine have gained attention for their protective role in acute lung inflammation. In this study, we investigated the influence of uridine on asthmatic airway inflammation. We used the classical ovalbumin (OVA)-alum model, as well as a model of house dust mite-(HDM)-induced airway inflammation. The degree of inflammation was determined by bronchoalveolar lavage (BAL), histology, and measurement of bronchial hyperresponsiveness. Intratracheal treatment of OVA-sensitized animals with uridine before allergen challenge resulted in a reduction in total BAL cells and BAL eosinophils. This was accompanied by reduced tissue infiltration and diminished production of T helper type 2-cytokines by mediastinal lymph node cells. Additionally, mice treated with uridine developed less bronchial hyperresponsiveness. Uridine was also effective in reducing airway inflammation in HDM-induced asthma. The protective effects of uridine were independent of myeloid dendritic cell (mDC) function, because in vitro pre-treatment of allergen-pulsed DCs with uridine did not alter the degree of inflammation. However, uridine inhibited the release of pro-inflammatory mediators in vivo and by cultured lung epithelial cells, suggesting an effect on lung structural cells. In summary, we were able to show that uridine inhibits the classical features of asthmatic airway inflammation. As uridine supplementation is well tolerated in humans, it might be a new therapeutic approach for the treatment of bronchial asthma.

Modulation of NF-κB and Hypoxia-Inducible Factor−1 byS-Nitrosoglutathione Does Not Alter Allergic Airway Inflammation in Mice

Induction of nitric oxide synthase (NOS)-2 and production of nitric oxide (NO) are common features of allergic airway disease. Conditions of severe asthma are associated with deficiency of airway S-nitrosothiols, a biological product of NO that can suppress inflammation by S-nitrosylation of the proinflammatory transcription factor, NF-κB. Therefore, restoration of airway S-nitrosothiols might have therapeutic benefit, and this was tested in a mouse model of ovalbumin (OVA)-induced allergic inflammation. Naive or OVA-sensitized animals were administered S-nitrosoglutathione (GSNO; 50 μl, 10 mM) intratracheally before OVA challenge and analyzed 48 hours later. GSNO administration enhanced lung tissue S-nitrosothiol levels and reduced NF-κB activity in OVA-challenged animals compared with control animals, but did not lead to significant changes in total bronchoalveolar lavage cell counts, differentials, or mucus metaplasia markers. Administration of GSNO also altered the activation of hypoxia-inducible factor (HIF)-1, leading to HIF-1 activation in naive mice, but suppressed HIF-1 activation in OVA-challenged mice. We assessed the contribution of endogenous NOS2 in regulating NF-κB and/or HIF-1 activation and allergic airway inflammation using NOS2(-/-) mice. Although OVA-induced NF-κB activation was slightly increased in NOS2(-/-) mice, associated with small increases in bronchoalveolar lavage neutrophils, other markers of allergic inflammation and HIF-1 activation were similar in NOS2(-/-) and wild-type mice. Collectively, our studies indicate that instillation of GSNO can suppress NF-κB activation during allergic airway inflammation, but does not significantly affect overall markers of inflammation or mucus metaplasia, thus potentially limiting its therapeutic potential due to effects on additional signaling pathways, such as HIF-1.

Ambient ultrafine particles provide a strong adjuvant effect in the secondary immune response: implication for traffic-related asthma flares.

We have previously demonstrated that intranasal administration of ambient ultrafine particles (UFP) acts as an adjuvant for primary allergic sensitization to ovalbumin (OVA) in Balb/c mice. It is important to find out whether inhaled UFP exert the same effect on the secondary immune response as a way of explaining asthma flares in already-sensitized individuals due to traffic exposure near a freeway. The objective of this study is to determine whether inhalation exposure to ambient UFP near an urban freeway could enhance the secondary immune response to OVA in already-sensitized mice. Prior OVA-sensitized animals were exposed to concentrated ambient UFP at the time of secondary OVA challenge in our mobile animal laboratory in Los Angeles. OVA-specific antibody production, airway morphometry, allergic airway inflammation, cytokine gene expression, and oxidative stress marker were assessed. As few as five ambient UFP exposures were sufficient to promote the OVA recall immune response, including generating allergic airway inflammation in smaller and more distal airways compared with the adjuvant effect of intranasally instilled UFP on the primary immune response. The secondary immune response was characterized by the T helper 2 and IL-17 cytokine gene expression in the lung. In summary, our results demonstrated that inhalation of prooxidative ambient UFP could effectively boost the secondary immune response to an experimental allergen, indicating that vehicular traffic exposure could exacerbate allergic inflammation in already-sensitized subjects.

Bacterial strain‐specific induction of Foxp3+ T regulatory cells is protective in murine allergy models

The incidence of atopic disease has increased dramatically during recent decades and the potential immunoregulatory influence of the microbiota in these individuals is under investigation. The aim of our study was to identify a bacterial strain that is protective in murine allergy models and to determine if microbial induction of T regulatory cells was associated with protection from allergic inflammation. Three microbes (Bifidobacterium breve AH1205, B. longum AH1206 and Lactobacillus salivarius AH102) of human origin were fed to newborn, adult and germ-free animals. Induction of Foxp3(+) T regulatory cells was assessed by flow cytometry. Gene array analysis was performed on Peyer's patches. Strains were also examined for their protective effects in the ovalbumin (OVA) respiratory allergy model and the OVA-cholera toxin dietary allergy model. Bifidobacterium longum AH1206 consumption resulted in increased numbers of Foxp3(+) T regulatory cells in infant, adult and germ-free animals. B. breve AH1205 induced Foxp3(+) T regulatory cell expansion only in infant mice while L. salivarius AH102 did not alter T regulatory cell numbers in any animal model tested. B. longum AH1206 reduced the Peyer's patch gene expression associated with antigen presentation, TLR signalling and cytokine production while increasing the expression of genes associated with retinoic acid metabolism. B. longum AH1206 protected against airway inflammation in OVA-sensitized animals and B. longum AH1206 blocked the induction of IgE to orally administered OVA. Neither B. breve AH1205 nor L. salivarius AH102 had a protective effect in either model. Bacterial strain-specific induction of Foxp3(+) T regulatory cells in vivo is associated with protection from respiratory and oral allergy.