Ovalbumin Inhalation Research Articles

A Novel Dithiol Amide CB3 Attenuates Allergic Airway Disease through Negative Regulation of p38 Mitogen-activated Protein Kinase

Cellular redox homeostasis altered by excessive production of reactive oxygen species (ROS) and weakening of the antioxidant defense leads to oxidative stress. Oxidative stress is characterized as a decrease in glutathione/glutathione disulfide (GSH/GSSG) and the triggering of a number of the redox-sensitive signaling cascades. Recent studies have demonstrated that ROS play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Here we characterized for the first time the protective properties of a new hydrophobic thiol compound, N-acetyl cysteine proline cysteine amide (CB3), in allergic airway diseases. We used ovalbumin (OVA)-inhaled mice to evaluate the role of CB3 as an antiinflammatory reagent and to determine its molecular signaling activity in allergic airways. The administration of CB3 (1-50 mg/kg) to OVA-inhaled mice restored the decreased GSH levels, enhanced IL-10 expression, and significantly reduced the increase of Th2 cytokines and OVA-specific IgE. CB3 decreased the number of inflammatory cells and airway hyperresponsiveness in the lungs. We also found that the administration of CB3 dramatically decreased the nuclear translocation of the nuclear factor-κB (NF-κB) and the phosphorylation of p38 mitogen-activated protein kinases (MAPKs) in lungs after OVA inhalation. In addition, allergen-induced airway inflammation and hyperresponsiveness were substantially reduced by the administration of inhibitors of NF-κB and p38 MAPK, BAY 11-7085, and SB 239063, respectively. These results suggest that CB3 attenuates allergic airway disease by up-regulation of GSH levels as well as inhibition of NF-κB and p38 MAPK activity.

Analysis of pulmonary allergic vasculitis with eosinophil infiltration in asthma model of mice

ABSTRACTHere the authors report pulmonary allergic vasculitis with eosinophil infiltration in an asthma model of mice and investigated its pathogenesis. C57BL/6 and BALB/c mice were sensitized with ovalbumin (OVA). After the inhalation of OVA, the authors measured the cell number and cytokine concentration in the blood and bronchoalveolar lavage fluid (BALF). The authors also examined the histological changes of the pulmonary. The number of eosinophils increased in the blood and BALF in both strains; however, the number in C57BL/6 in BALF was significantly higher than that in BALB/c. Histological analysis demonstrated severe vasculitis of the pulmonary arteries with derangement of the muscle layer and smooth muscle cell hyperplasia in C57BL/6. Semiquantitative analysis of the severity of vasculitis in the pulmonary arteries revealed that the internal vascular space was highly reduced by smooth muscle hyperplasia in C57BL/6 compared to BALB/c mice. The concentrations of interleukin (IL)-4, IL-5, and interferon (IFN)-γ in BALF of C57BL/6 were significantly high compared to those of BALB/c. C57BL/6 mice exhibited severe allergic vasculitis in the pulmonary arteries compared to BALB/c mice. The high concentrations of IL-4, IL-5, and IFN-γ in the lung may play a critical role in the pathogenesis of allergic vasculitis in C57BL/6 mice.

Phosphoinositide 3-kinase inhibitor suppresses interleukin-17 expression in a murine asthma model

Phosphoinositide 3-kinases (PI3Ks) contribute to the pathogenesis of asthma by regulating the activation of inflammatory mediators, inflammatory cell recruitment and immune cell function. Recent findings have indicated that PI3Ks also regulate the expression of interleukin (IL)-17, which has been recognised as an important cytokine involved in airway inflammation. In the present study, we investigated a role of PI3Kδ in the regulation of IL-17 expression in allergic airway disease using a murine model of asthma. After ovalbumin inhalation, administration of a selective p110δ inhibitor, IC87114, significantly attenuated airway infiltration of total cells, lymphocytes, neutrophils and eosinophils, as well as airway hyperresponsiveness, and attenuated the increase in IL-17 protein and mRNA expression. Moreover, IC87114 reduced levels of IL-4, -5 and -13, expression of keratinocyte chemoattractant protein and mRNA, and nuclear factor (NF)-κB activity. In addition, a NF-κB inhibitor, BAY 11-7085 substantially reduced the increase in IL-17 protein levels. Our results also showed that inhibition of IL-17 activity with an anti-IL-17 antibody remarkably reduced airway inflammation and hyperresponsiveness. These findings suggest that inhibition of the p110δ signalling pathway suppresses IL-17 expression through regulation of NF-κB activity and, thus, has therapeutic potential in asthma.

The role of phosphorylation of c-Jun NH2-terminal kinase in airway remodeling of asthmatic rats and the effect of glucocorticoids

To study the role of phosphorylation of c-Jun NH2-terminal kinase (JNK) in asthmatic airway remodeling and to explore the effect of glucocorticoids on IL-1beta, JNK and airway remodeling. Forty-eight 4 - 6 week old male SD rats were randomly divided into 4 groups with 12 rats in each group: the control group, the asthma group, the budesonide (BUD) group, and the dexamethasone (DXM) group. The asthma airway remodeling models were made by intra-peritoneal injection of ovalbumin (OVA) on days 1 and 8 and inhalation of OVA every other day for 12 weeks since day 15. The BUD group underwent inhalation of BUD 30 min before every inhalation; the DXM group received intra-peritoneal injection of DXM 30 min before every inhalation; while the control group received normal saline instead of OVA. The histopathology and ultrastructural changes of pulmonary tissues were observed by light microscope and transmission electron microscope (TEM). The total bronchial wall thickness (Wat) and the airway smooth muscle thickness (Wam) were measured by image analysis system. The concentrations of IL-1beta in serum and BALF were tested by sandwich ELISA. The protein expressions of P-JNK and P-c-Jun were detected by immunohistochemistry technique. Lung tissue extracts were analyzed for phosphorylation of JNK by Western blot. Linear correlation analysis was used to evaluate correlations between Wat and P-JNK protein (mA), Wam and P-JNK protein (mA), P-JNK protein (mA) and levels of IL-1beta in serum, P-JNK protein (mA) and levels of IL-1beta in BALF. In the asthma group, HE-staining showed inflammatory cell infiltration around bronchi and mucous gland hyperplasia. TEM examination showed airway smooth muscle and collagen fiber proliferation, and widening of intercellular distance. The Wat and Wam of the asthma group were significantly higher than those of the control group, while the thickness of airway wall in the glucocorticoid intervention groups became significantly decreased. The concentrations of IL-1beta in serum and BALF of the asthma group [(81 +/- 4) ng/L, (331 +/- 15) ng/L] were significantly higher than those of the control group [(53 +/- 6) ng/L, (130 +/- 9) ng/L] (t = -8.62 and t = -24.10, both P < 0.01). Mean absorbance values (by immunohistochemistry) of P-JNK and P-c-Jun in the asthma group were significantly higher than those of the control group, and the mean absorbance values of P-JNK and P-c-Jun in the BUD and DXM groups were significantly lower than those of the asthma group, but higher than those of the control group (F = 223.59 and F = 76.53, both P < 0.01). Absorbance (by Western blot) of P-JNK in the control, asthma, BUD, and DXM groups were (1.00 +/- 0.00), (1.66 +/- 0.16), (1.18 +/- 0.12), and (1.29 +/- 0.14), respectively; that of the asthma group was significantly higher than that of the control group, while absorbance of P-JNK in the BUD and the DXM groups were significantly lower than that of the asthma group, but higher than that of the control group (F = 17.84, P < 0.05). Strong positive correlations were found between Wat or Wam and P-JNK (mA) (r = 0.700 and r = 0.769, P < 0.01, respectively, n = 48). Strong positive correlations were also found between P-JNK (mA) and concentration of IL-1beta in serum or BALF (r = 0.689 and r = 0.805, P < 0.01, respectively, n = 48). Phosphorylation of JNK is closely related to asthma airway remodeling. Glucocorticoids can inhibit phosphorylation of JNK, one mechanism of which may be down-regulation of IL-beta expression.

Content and localization of leukotriene C4 in lung tissue during biphasic allergic bronchoconstriction in the guinea pig

Leukotriene C4 (LTC4) is a very potent bronchoconstrictor, and is believed to be an important mediator during immediate (IAR) and late asthmatic response (LAR). No direct measurement of LTC4 in lung tissue during IAR and LAR has been reported, however, and its localized site in the lung remains unclear. In an effort to clarify these issues, the content of LTC4 in lung tissue was evaluated at the onset of antigen-induced IAR and LAR, and the distribution of LTC4 was examined by immunohistochemistry. IAR and LAR were induced by inhalation of aerosolized ovalbumin (OA) in conscious guinea pigs. The development of IAR and LAR following inhaled OA exposure was determined in sensitized guinea pigs by measurement of respiratory resistance using the oscillation method. LTC4 in lung tissue was measured by radioimmunoassay at six different stages (S1: non-treated group; S2: group before OA-challenge following sensitization; S3: IAR group; S4: group two hours after IAR; S5: LAR-positive group; S6: LAR-negative group). For immunohistochemistry, non-treated guinea pigs and the sensitized animals at the onset of LAR were used. Lung sections were stained with anti-LTC4 antibody by avidin biotin peroxidase complex method (ABC method). About 70% of the guinea pigs displayed IAR, followed by LAR that peaked at 6-20 hours after OA challenge. The LTC4 content in lung tissue was significantly increased (p less than 0.01) at the onset of IAR, but was significantly decreased (p less than 0.01) after two hours, and significantly increased (p less than 0.01) at the onset of LAR.(ABSTRACT TRUNCATED AT 250 WORDS)

A standardized aqueous extract of Anoectochilus formosanus modulated airway hyperresponsiveness in an OVA-inhaled murine model

Anoectochilus formosanus HAYATA, a Chinese herb, is a valued folk medicine for fever, pain, and diseases of the lung and liver. Allergic asthma is characterized by increased serum IgE level and inflammation of the airways with high levels of interleukin (IL)-4 and IL-5 in bronchoalveolar lavage fluids (BALF). Constriction of airway smooth muscle and development of airway hyperresponsiveness (AHR) are the most important symptoms of allergic asthma. In our previous study, a standardized aqueous extract of A. formosanus (SAEAF) was used to modulate innate immunity of normal mice. In this study, airway inflammatory infiltrations, including T cell differentiation, cytokine modulation, allergic antibodies estimation, pulmonary pathology, and enhanced pause (Penh) of AHR were used to evaluate SAEAF treatment of an ovalbumin (OVA)-inhaled airway allergic murine model. The resulting cytokine profiles demonstrated that SAEAF can significantly reduce Th2 polarization after administration of SAEAF in OVA inhalation. These results also suggest that SAEAF modulates cytokine secretion in allergic asthma. Modulated natural T regulatory cells (CD25+/CD4+, Treg) were also shown to increase immuno-suppression in the allergic lung inflammation and further down-regulate airway inflammatory infiltration in eosinophils and macrophages. Finally, decreased airway anti-OVA IgE secretion and reduced AHR were observed. Our results indicate that the administration of SAEAF can modulate cytokines and T cell subpopulation by regulating inflammatory cell infiltration and modulating the allergic response.

Amphiregulin Is Not Essential for Ovalbumin-Induced Acute Airway Inflammation in Mice

The number of amphiregulin (AR)-positive mast cells in the bronchial mucosa and the levels of AR in sputum from asthmatic patients have been reported to be increased. In addition, AR can promote mucin gene expression in human epithelial cells, suggesting that AR contributes to the pathogenesis of allergic asthma. To elucidate the role of AR in the pathogenesis of asthma, we immunized AR-deficient mice with ovalbumin (OVA) and then induced airway inflammation in them after OVA inhalation. The OVA-induced airway inflammation was assessed on the basis of the lung histology, number of leukocytes in the bronchoalveolar lavage (BAL) fluid, Th2 cytokine levels in the BAL fluid and OVA-specific IgG1 and IgE levels in the serum and compared between AR-sufficient and -deficient mice. The OVA-induced airway inflammation was comparable in the AR-sufficient and -deficient mice. Amphiregulin is not essential for induction of acute airway inflammation by OVA in mice.

Involvement of sirtuin 1 in airway inflammation and hyperresponsiveness of allergic airway disease

Bronchial asthma is a chronic inflammatory disorder of the airways characterized by increased expression of multiple inflammatory genes. Acetylation of histones by histone acetyltransferases is associated with increased gene transcription, whereas hypoacetylation induced by histone deacetylases is associated with suppression of gene expression. Sirtuin 1 (SIRT1) is a member of the silent information regulator 2 family that belongs to class III histone deacetylase. This study aimed to investigate the role of SIRT1 and the related molecular mechanisms in the pathogenesis of allergic airway disease. By using a murine model of ovalbumin (OVA)-induced allergic airway disease and murine tracheal epithelial cells, this study investigated the involvement of SIRT1 and its signaling networks in allergic airway inflammation and hyperresponsiveness. In this study with mice after inhalation of OVA, the increased levels of SIRT1, hypoxia-inducible factor 1alpha (HIF-1alpha), and vascular endothelial growth factor protein in the lungs after OVA inhalation were decreased substantially by the administration of a SIRT1 inhibitor, sirtinol. We also showed that the administration of sirtinol reduced significantly the increased numbers of inflammatory cells of the airways; airway hyperresponsiveness; increased levels of IL-4, IL-5, and IL-13; and increased vascular permeability in the lungs after OVA inhalation. In addition, we have found that inhibition of SIRT1 reduced OVA-induced upregulation of HIF-1alpha in airway epithelial cells. These results indicate that inhibition of SIRT1 might attenuate antigen-induced airway inflammation and hyperresponsiveness through the modulation of vascular endothelial growth factor expression mediated by HIF-1alpha in mice.

Induction of long-lived allergen-specific plasma cells by mucosal allergen challenge

Allergen-specific IgE antibodies are responsible for the pathogenesis of type I hypersensitivity. In patients with allergy, IgE titers can persist in the apparent absence of allergen for years. Seasonal allergen exposure triggers clinical symptoms and enhances allergen-specific IgE. Whether allergen-specific plasma cells originating from seasonal allergen exposures can survive and become long-lived is so far unclear. We analyzed the localization and lifetimes of allergen-specific IgE-secreting, IgA-secreting, and IgG(1)-secreting plasma cells after allergen inhalation in an ovalbumin-induced murine model of allergic asthma. Ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in lungs, spleen, and bone marrow were isolated and tested for antibody secretion by the ELISpot technique. Longevity of ovalbumin-specific plasma cells was determined by cyclophosphamide treatment, which depletes proliferating plasmablasts but leaves plasma cells untouched. Ovalbumin aerosol-induced infiltrates in lungs were localized by confocal microscopy. Long-lived ovalbumin-specific plasma cells were generated by systemic sensitization and survived in bone marrow and spleen, maintaining systemic ovalbumin-specific titers of IgG, IgA, and IgE. On inhalation of ovalbumin-containing aerosol, sensitized mice developed airway inflammation and more ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in the lungs and in secondary lymphoid organs. These plasma cells joined the pool of ovalbumin-specific plasma cells in the bone marrow and became long-lived-that is, they are resistant to cyclophosphamide. Termination of ovalbumin inhalation depleted ovalbumin-specific plasma cells from the lungs, but they persisted in spleen and bone marrow. Our results show that inhalation of aerosolized allergen generates long-lived, allergen-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting plasma cells that survive cytostatic treatment.

Oral Treatment with Probiotics Reduces Allergic Symptoms in Ovalbumin-Sensitized Mice: A Bacterial Strain Comparative Study

Background/Aim: Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma. Methods: OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-γ. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8. Results: Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA. Conclusion: B. breve M-16V was identified as the most potent antiallergic strain.

Effects of sulfur dioxide on expressions of p53, bax and bcl-2 in lungs of asthmatic rats

Inhibition of cell apoptosis is an increasingly important factor in modulating airway inflammation in asthma, which is related to environmental pollutants. To investigate the effects of sulfur dioxide (SO2) on the mRNA and protein expressions of apoptosis-related genes in lungs from asthmatic rats, male Wistar rats were challenged by ovalbumin (OVA) or SO2 (2 ppm) inhalation alone or together. Examinations were performed 24 h after the last treatment. The mRNA and protein levels of p53, bax, and bcl-2 were analyzed in lungs using real-time reverse transcription-polymerase chain reaction (RT-PCR) assay and Western blot analysis, respectively. The results indicated that increases of bcl-2 or decreases of p53 and bax mRNA and protein levels were not significant in lungs of rats exposed to SO2 alone, compared with controls, but elevated or reduced levels of these genes appeared in lungs of asthmatic rats exposed to SO2 plus OVA, compared with controls, suggesting that SO2 exposure could result in OVA-induced increases or decreases of transcription and translation levels of these apoptosis-related genes in rat lungs, and may have relations to airway inflammation in asthma. The regulation mechanism of apoptosis in asthma disease exposure to SO2 needs further study.

T-bet gene modified dendritic cells abrogate and reverse the airway inflammation in allergic asthma: experiment with mice

To explore the feasibility of the therapeutic strategy to use T-bet gene modified dendritic cells (DCs) to reverse the course of asthma. (1) Mature DCs were derived from mononuclear cells obtained from femur of BALB/c mouse and divided into 3 groups, T-bet group transfected with recombinant adenovirus Ad-T-bet containing T-bet gene, LacZ group transfected with recombinant adenovirus Ad-LacZ containing LacZ gene, and control group. Seven days later ELISA was used to detect the interferon (IFN)-gamma level in the culture fluid. (2) Airway inflammation abrogating trial. Twenty-four BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) (on day 1 and 15) to establish asthma models, and then randomly divided into 3 equal groups: T-bet group injected intravenously with T-bet-modified DCs on day 27, LacZ group injected with LacZ-modified DCs, and model control group without intravenous injection. Two days later the model mice began to undergo challenge by inhalation of OVA twice (on day 29 - 31). Eight mice were used as control group treated with PBS. On day 37 all mice were killed, ELISA was used to detect the blood interleukin (IL)-4 and IFN-gamma levels, and microscopy was conducted to observe the airway inflammation. (3) Airway inflammation reversing trial. Another 24 model mice were divided into 3 equal groups as well: re-challenged T-bet group injected intravenously with T-bet-modified DCs on day 27 and 42, re-challenged LacZ group injected intravenously with LacZ-modified DCs on day 27 and 42, and model control group. Since the day 45 OVA inhalation was given once a day for successive 3 days. On day 49 these mice were all killed to undergo the tests as mentioned above. The IFN-gamma level in the culture fluid of the T-bet gene modified DCs was (15.24 +/- 4.75) ng/ml, significantly higher than that of the LacZ gene modified DCs and control DCs [(3.08 +/- 0.61) and (2.35 +/- 0.41) ng/ml respectively, both P < 0.01]. The IFN-gamma in mice blood plasma of T-bet groups in abrogating and reversing trial were (130.2 +/- 10.5) and (145.7 +/- 16.7) pg/ml respectively, both significantly higher than those of the abrogating and reversing trial normal control groups [(25.0 +/- 6.5) and (24.6 +/- 5.9) pg/ml respectively], asthmatic model control groups [(20.7 +/- 4.5) and (16.5 +/- 7.0) pg/ml respectively] and LacZ groups [(17.6 +/- 7.0) and (24.2 +/- 9.0) pg/ml respectively] (all P < 0.01). However, the IL-4 levels in mice blood plasma of T-bet groups were both significantly lower than those of asthmatic model control groups and LacZ groups (all P < 0.01). The airway inflammation of T-bet groups were remarkable milder than those of the model control groups and LacZ groups. The asthma management strategy based on T-bet gene modified DCs is feasible with the plausible mechanism that the T-bet gene modified DCs regulate the T cells differentiation and polarization on the antigen presenting level.

Inhibitory effect of Platycodi Radix on ovalbumin-induced airway inflammation in a murine model of asthma

Asthma is a chronic inflammatory disease of the airways characterized by an associated increase in airway responsiveness. In this study, we investigated the inhibitory effect of an aqueous extract from the root of Platycodi Radix (Changkil: CK) on airway inflammation in a murine model of asthma. Mice were sensitized and challenged by ovalbumin (OVA) inhalation to induce chronic airway inflammation and airway remodeling. CK markedly decreased the number of infiltrated inflammatory cells and the levels of Th1 and Th2 cytokines and chemokines compared with those in the OVA-induced group. In addition, CK reduced OVA-specific IgE levels in bronchoalveolar lavage (BAL) fluid. Based on lung histopathological studies, inflammatory cell infiltration and mucus hypersecretion were inhibited by CK administration compared to that in the OVA-induced group. Lung weight was reduced after CK administration. Also, increased generation of ROS in BAL fluid, as well as NF-κB nuclear translocation, by inhalation of OVA was diminished by CK. Moreover, CK reduced the OVA-induced upregulation of matrix metalloproteases activity. These findings indicate that oxidative stress may play a crucial role in the pathogenesis of bronchial asthma induced by OVA and that CK may be useful as an adjuvant therapy for the treatment of bronchial asthma.

Inhalation of urokinase-type plasminogen activator reduces airway remodeling in a murine asthma model

The airway remodeling that occurs in asthma is characterized by an excess of extracellular matrix deposition in the submucosa, hyperplasia/hypertrophy of smooth muscle, goblet cell metaplasia, and accumulation of fibroblasts/myofibroblasts. The urokinase-type plasminogen activator (uPA)/plasmin system participates in pericellular proteolysis and is capable of directly degrading matrix components, activating latent proteinases, and activating growth factors. In a mouse ovalbumin (OVA) asthma model, we increased plasminogen activator activity in the lung by administering exogenous uPA or by using mice genetically deficient in the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1) to assess the role of this system in asthma pathogenesis. After intraperitoneal OVA sensitization, mice inhaled OVA plus uPA (500 IU/mouse) or saline by ultrasonic nebulization for 3 wk. When studied 24 h after the final exposure, the groups with upregulated plasmin activity had significantly reduced subepithelial fibrosis within the airway walls and had decreased airway hyperresponsiveness (AHR) to methacholine. Morphometric analysis showed that subepithelial wall thickening of the bronchi (subepithelial area ratio) was also reduced, as were collagen and alpha-smooth muscle actin. Upregulation of plasmin activity also increased the level of hepatocyte growth factor activity in bronchoalveolar lavage fluid, whereas the release of transforming growth factor-beta was decreased. The administration of uPA 1 wk after the last OVA inhalation also significantly reduced lung hydroxyproline content and AHR. These results show that enhancing uPA/plasmin activity lessens the airway remodeling in a murine asthma model.

SH2‐Bβ expression in alveolar macrophages in BAL fluid of asthmatic guinea pigs and its role in NGF–TrkA‐mediated asthma

Nerve growth factor (NGF)/tyrosine kinase receptor A (TrkA) signalling may play an important role in the pathogenesis of asthma, and SH2-B beta, a TrkA-binding protein, modulates the NGF signalling pathway. In this study, SH2-B beta expression in alveolar macrophages (AM) in guinea pig BAL fluid and its role in asthma pathogenesis through the NGF-TrkA signalling pathway were investigated. Guinea pigs were randomized into five groups: control, a model of asthma, anti-SH2-B beta antibody treatment, anti-NGF antibody treatment and anti-TrkA antibody treatment. The asthmatic model was established in guinea pigs by inhalation of ovalbumin. Specific anti-SH2-B beta, anti-NGF and anti-TrkA antibodies were administered and AM were isolated from BAL fluid to assess SH2-B beta expression using an immunofluorescence assay. SH2-B beta and TrkA protein expression were determined by western blotting, IL-1 beta and IL-4 levels in the BAL fluid supernatants were determined by ELISA, and pathological changes in the bronchi and lung tissues were examined by HE staining. Lymphocyte, eosinophil and total inflammatory cell numbers in BAL fluid were significantly higher in the asthma model group than in the other groups (P < 0.01). NGF expression in the asthma model group was significantly higher than that in the PBS control group (P < 0.01). SH2-B beta was expressed in AM of control animals and expression was significantly higher in the asthma model than in the other groups (P < 0.01). TrkA protein expression was significantly higher in the asthma model group than in the PBS group (P < 0.01), and treatment with anti-NGF antibody resulted in significant reduction of TrkA expression (P < 0.01). SH2-B beta is expressed in AM of normal guinea pigs, and SH2-B beta may participate in asthma pathogenesis through the NGF-TrkA signalling pathway.

Oral tolerance attenuates airway inflammation and remodeling in a model of chronic pulmonary allergic inflammation

We investigated the effects of oral tolerance (OT) in controlling inflammatory response, hyperresponsiveness and airway remodeling in guinea pigs (GP) with chronic allergic inflammation. Animals received seven inhalations of ovalbumin (1-5mg/mL-OVA group) or normal saline (NS group). OT was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st ovalbumin inhalation (OT1 group) or after the 4th (OT2 group). The induction of OT in sensitized animals decreased the elastance of respiratory system (Ers) response after both antigen and methacholine challenges, peribronchial edema formation, eosinophilic airway infiltration, eosinophilopoiesis, and airways collagen and elastic fiber content compared to OVA group (P<0.05). The number of mononuclear cells and resistance of respiratory system (Rrs) responses after antigen and methacholine challenges were decreased only in OT2 group compared to OVA group (P<0.05). Concluding, our results show that inducing OT attenuates airway remodeling as well as eosinophilic inflammation and respiratory system mechanics.

Mast Cells Can Mediate Vascular Permeability through Regulation of the PI3K–HIF-1α–VEGF Axis

Bronchial inflammation is usually accompanied by increased vascular permeability. Mast cells release a number of mediators that act directly on the vasculature, resulting in vasodilatation, increased permeability, and subsequent plasma protein extravasation. Vascular endothelial growth factor (VEGF) has been implicated to contribute to asthmatic tissue edema through its effect on vascular permeability. However, the effects of mast cells on VEGF-mediated signaling in allergic airway disease are not clearly understood. An aim of the present study was to investigate the role of mast cells on VEGF-mediated signal transduction in allergic airway disease. We used genetically mast cell-deficient WBB6F(1)-Kit(W)/Kit(W-v) (W/W(v)) mice and the congenic normal WBB6F(1)(+/+) mouse model for allergic airway disease to investigate the role of mast cells on VEGF-mediated signal transduction in allergic airway disease, more specifically in vascular permeability. Our present study, with ovalbumin (OVA)-sensitized without adjuvant and OVA-challenged mice, revealed the following typical pathophysiologic features of allergic airway diseases: increased inflammatory cells of the airways, airway hyperresponsiveness, increased vascular permeability, and increased levels of VEGF. However, levels of VEGF and plasma exudation in W/W(v) mice after OVA inhalation were significantly lower than levels in WBB6F(1)(+/+) mice. Moreover, mast cell-reconstituted W/W(v) mice restored vascular permeability and VEGF levels similar to those of the WBB6F(1)(+/+) mice. Our data also showed that VEGF expression was regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) activation through the phosphatidylinositol 3-kinase (PI3K)-HIF-1alpha pathway in allergic airway disease. These results suggest that mast cells modulate vascular permeability by the regulation of the PI3K-HIF-1alpha-VEGF axis.

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs

Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

Establishing the phenotype in novel acute and chronic murine models of allergic asthma

Allergic asthma is a chronic disease of the airways, with superimposed acute inflammatory episodes which correspond to exacerbations of asthma. Two novel models of allergic asthma have been developed in mice receiving the same allergen sensitisation, but with acute or chronic allergen exposures, the latter to mimic the human situation more closely. Ovalbumin-sensitised mice were challenged by ovalbumin inhalation twice on the same day for the acute model, and 18 times over a period of 6 weeks for the chronic model. Lung function was monitored in conscious, unrestrained mice immediately after the last challenge for up to 12 h. Airway responsiveness to inhaled methacholine and serum antibody levels were determined 24 h after challenge. Bronchoalveolar inflammatory cell recruitment was determined at 2 or 24 h.Acute and chronically treated mice had similar early and late asthmatic responses peaking at 2 h and 7---8 h, respectively. IgE and IgG antibody levels, compared with naïve mice, and eosinophil infiltration, compared with naïve and saline challenge, were elevated. Airway hyperresponsiveness to methacholine was observed 24 h after challenge in both models. The acute model had higher levels of eosinophilia, whereas the chronic model showed hyperresponsiveness to lower doses of methacholine and had higher levels of total IgE and ovalbumin-specific IgG antibodies.Both novel murine models of allergic asthma bear a close resemblance to human asthma, each offering particular advantages for studying the mechanisms underlying asthma and for evaluating existing and novel therapeutic agents.