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We investigated whether CTLA4-Ig can reverse established asthma manifestations in a novel murine model of ongoing disease. In BALB/c mice, sensitized to ovalbumin (OVA) without adjuvant, airway inflammation was induced by a first series of OVA aerosol challenges. Murine CTLA4-IgG was then administered, followed by a second series of OVA inhalations. In control-treated mice, two series of OVA challenges induced upregulation of OVA-specific IgE in serum, eosinophils in the bronchoalveolar lavage fluid (BALF), and IL-5 production by lung lymphocytes upon OVA restimulation in vitro , compared with saline-challenged mice. CTLA4-IgG significantly inhibited all of these parameters in OVA-challenged mice. Importantly, mCTLA4-IgG performed better than the gold-standard dexamethasone because this corticosteroid did not inhibit the upregulation of OVA-specific IgE in serum. In a more “severe” ongoing model, induced by sensitization to OVA emulsified in aluminum hydroxide, resulting in airway hyperresponsiveness to methacholine and stronger inflammatory responses, mCTLA4IgG was less effective in that only the number of eosinophils in the BALF was reduced ( P � 0.053), whereas dexamethasone inhibited both BALF eosinophilia and cytokine production by lung lymphocytes. Thus, CTLA4-Ig might be an effective alternative therapy in established allergic asthma, especially in situations of mild disease.

The inflammatory response as seen in human allergic asthma is thought to be regulated by Th2 cells. It has been shown that interferon-gamma (IFN-gamma) can downregulate the proliferation of Th2 cells and therefore might be of therapeutic use. In the present study we have investigated the in vivo role of endogenous and exogenous IFN-gamma in a murine model with features reminiscent of human allergic asthma. IFN-gamma gene knockout (GKO) and wild-type mice were sensitized with ovalbumin and exposed to repeated ovalbumin aerosol challenges. In addition, wild-type mice were treated with intraperitoneal or nebulized recombinant murine IFN-gamma during the challenge period. Sensitized wild-type mice exhibited upregulated ovalbumin-specific IgE in serum, and airway hyperresponsiveness and infiltration of eosinophils and mononuclear cells in the bronchoalveolar lavage fluid (BALF) after ovalbumin challenge. In contrast, in GKO mice only reduced eosinophilic infiltration in the BALF was observed after ovalbumin challenge. In wild-type mice, parenteral IFN-gamma treatment downregulated ovalbumin-specific IgE levels in serum, and airway hyperresponsiveness and cellular infiltration in the BALF, whereas aerosolized IFN-gamma treatment only suppressed airway hyperresponsiveness. In vitro experiments showed that these effects of IFN-gamma appear not to be mediated via a direct effect on the cytokine production of antigen-specific Th2 cells. These data indicate that airway hyperresponsiveness can be downregulated by IFN-gamma locally in the airways, whereas for downregulation of IgE and cellular infiltration systemic IFN-gamma is needed. The present study shows that exogenous IFN-gamma can downregulate the allergic response via an antigen-specific T-cell independent mechanism, but at the same time endogenous IFN-gamma plays a role in an optimal response.

Experiments were designed to investigate the role of IL-16 in a mouse model of allergic asthma. OVA-sensitized mice were repeatedly exposed to OVA or saline aerosols. Bronchoalveolar lavage fluid (BALF) was collected after the last aerosol, and the presence of IL-16 was evaluated using a migration assay with human lymphocytes. Migration of lymphocytes was significantly increased in the presence of cell-free BALF from OVA-challenged mice compared with BALF from saline-challenged controls. This response was significantly inhibited after addition of antibodies to IL-16, demonstrating the presence of IL-16 in BALF of OVA-challenged animals. Immunohistochemistry was performed and revealed IL-16 immunoreactivity particularly in airway epithelial cells but also in cellular infiltrates in OVA-challenged mice. IL-16 immunoreactivity was absent in nonsensitized animals; however, some reactivity was detected in epithelial cells of sensitized but saline-challenged mice, suggesting that sensitization induced IL-16 expression in airway epithelium. Treatment of mice with antibodies to IL-16 during the challenge period significantly suppressed up-regulation of OVA-specific IgE in OVA-challenged animals. Furthermore, antibodies to IL-16 significantly inhibited the development of airway hyper-responsiveness after repeated OVA inhalations, whereas the number of eosinophils in bronchoalveolar lavage or airway tissue was not affected. In conclusion, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Furthermore, IL-16 plays an important role in airway hyper-responsiveness and up-regulation of IgE but is not important for eosinophil accumulation in a mouse model of allergic asthma.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.

In the guinea pig, interleukin-5 (IL-5) has been shown to induce airway hyperresponsiveness as well as eosinophilia, which are important symptoms in asthma. IL-5 seems to be a critical cytokine since it selectively affects eosinophil functions. The mechanism of action by which IL-5 leads to airway hyperresponsiveness may be important for our understanding of the pathogenesis of asthma. Neurogenic inflammation, which is mediated by nonadrenergic noncholinergic nerves (NANC), may play a role in the IL-5-induced effects in guinea pig airways. In this study, the role of neuropeptides in the IL-5-induced airway hyperresponsiveness and eosinophilia in the guinea pig was examined using selective neurokinin receptor antagonists. Intra-airway application of IL-5 (1 microgram, twice) induces a selective eosinophil migration (control: 12 [8-22] x 10(5) cells and IL-5: 90 [67-187] x 10(5) cells, p0.05) and activation (control: 6.3 +/- 0.9 ng eosinophil peroxidase [EPO]/ml bronchoalveolar lavage [BAL] fluid and IL-5: 29.3 +/- 4.9 ng EPO/ml BAL fluid, p0.05) and a pronounced airway hyperresponsiveness in vivo. The maximal responses to histamine are increased by 160 +/- 16% (p0.05) after IL-5. Treatment of guinea pigs with either the nonselective neurokinin (NK)-receptor antagonist, FK224, or the selective NK2-receptor antagonist, SR48968, results in a complete inhibition of the in vivo hyperresponsiveness found after application of IL-5. Vice versa, intra-airway administration of substance P (10 micrograms, twice) results in an airway hyperresponsiveness (increased maximal response after substance P: 166 +/- 15% [p0.05]) without inducing migration or activation of eosinophils. All examined NK-receptor antagonists do not influence the IL-5-induced eosinophil accumulation. In addition, no effect of the NK-receptor antagonists is observed on the IL-5-induced eosinophil activation, as determined by BAL fluid EPO levels. The release of NK2-receptor active tachykinins plays an important role in the development of IL-5-induced airway hyperresponsiveness. This feature appears to be a step following eosinophil infiltration and activation since there are no effects on eosinophil function by pretreatment of the used NK-receptor antagonists.

There is need for an accurate diagnostic test in mastocytosis patients with wasp venom allergy (WVA) and monitoring of these patients during immunotherapy (IT). In this study, we aimed to evaluate sensitivity and specificity of the Basophil Activation Test (BAT) as a diagnostic and monitoring test in patients with mastocytosis and WVA.Seventeen patients with mastocytosis and WVA and six mastocytosis patients without WVA were included. BAT was performed before the start of IT (first visit) and at 6 weeks (second visit) and 1 year (third visit), after reaching the maintenance dose. Of 17 patients included, 11 completed the third visit. In mastocytosis patients with WVA, dose-dependent wasp-venom induced upregulation of CD63 and CD203c expression on basophils was observed compared with mastocytosis patients without WVA. Serum specific IgE, IgG4, and tryptase levels were measured in all patients.BAT had a sensitivity of 87% and specificity of 100% in diagnosing WVA in mastocytosis patients. Basophil allergen threshold sensitivity with respect to CD63 and CD203c was significantly decreased in the second visit compared with the first visit and increased significantly in the third visit compared with the second visit. Specific IgE levels increased significantly in the second visit compared with first and decreased significantly in the third visit compared with the second. Specific IgG4 levels rose significantly in the second visit compared with the first and on the third visit compared with the second. Tryptase levels did not change significantly during the study.BAT represents a diagnostic test with 100% specificity in allergic patients with mastocytosis and these patients are better to be monitored for a longer period during IT.

Asthma is a chronic inflammatory disease of the airways characterized by variable airway obstruction and airway hyperresponsiveness (AHR). The T regulatory (Treg) cell subset is critically important for the regulation of immune responses. Adoptive transfer of Treg cells has been shown to be sufficient for the suppression of airway inflammation in experimental allergic asthma. Intervention strategies aimed at expanding the Treg cell population locally in the airways of sensitized individuals are therefore of high interest as a potential therapeutic treatment for allergic airway disease. Here, we aim to test whether long-term suppression of asthma manifestations can be achieved by locally expanding the Treg cell subset via intranasal administration of a TLR-2 agonist. To model therapeutic intervention aimed at expanding the endogenous Treg population in a sensitized host, we challenged OVA-sensitized mice by OVA inhalation with concomitant intranasal instillation of the TLR-2 agonist Pam3Cys, followed by an additional series of OVA challenges. Pam3Cys treatment induced an acute but transient aggravation of asthma manifestations, followed by a reduction or loss of AHR to methacholine, depending on the time between Pam3Cys treatment and OVA challenges. In addition, Pam3Cys-treatment induced significant reductions of eosinophils and increased numbers of Treg cells in the lung infiltrates. Our data show that, despite having adverse acute effects, TLR2 agonist treatment as a therapeutic intervention induces an expansion of the Treg cell population in the lungs and results in long-term protection against manifestation of allergic asthma upon subsequent allergen provocation. Our data indicate that local expansion of Tregs in allergic airway disease is an interesting therapeutic approach that warrants further investigation.

Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin-proteasome pathway. In the present study, we aimed to find out whether exposure of alveolar epithelial cells to CS induces an endoplasmic reticulum (ER) stress response by accumulation of damaged proteins that are inefficiently degraded by the proteasomes. The hypothesis was tested in a human alveolar epithelial cell line (A549) exposed to gas-phase CS. Exposure to gas-phase CS for 5 min caused an increase in the amount of ubiquitin-protein conjugates within 4 h. Cigarette smoke exposure also induced the ER stress response marker eIF2α, followed by a significant reduction of nascent protein synthesis and increase in the level of free intracellular amino acids. Moreover, CS exposure significantly reduced all three proteasomal activities (caspase-, trypsin- and chymotrypsin-like activity) within 4 h, which was still present after 24 h. It can be concluded that gas-phase CS induces ER stress in A549 alveolar epithelial cells, leading to inadequate protein turnover caused by an accumulation of damaged proteins, reduction in nascent protein synthesis and inhibition of the proteasome. We suggest that prolonged ER stress may lead to excessive cell death with disruption of the epithelial barrier, contributing to development of chronic obstructive pulmonary disease.

Mechanisms of the Development of Allergy (MeDALL), a Seventh Framework Program European Union project, aims to generate novel knowledge on the mechanisms of initiation of allergy. Precise phenotypes of IgE-mediated allergic diseases will be defined in MeDALL. As part of MeDALL, a scientific seminar was held on January 24, 2011, to review current knowledge on the IgE-related phenotypes and to explore how a multidisciplinary effort could result in a new integrative translational approach. This article provides a summary of the meeting. It develops challenges in IgE-related phenotypes and new clinical and epidemiologic approaches to the investigation of allergic phenotypes, including cluster analysis, scale-free models, candidate biomarkers, and IgE microarrays; the particular case of severe asthma was reviewed. Then novel approaches to the IgE-associated phenotypes are reviewed from the individual mechanisms to the systems, including epigenetics, human in vitro immunology, systems biology, and animal models. The last chapter deals with the understanding of the population-based IgE-associated phenotypes in children and adolescents, including age effect in terms of maturation, observed effects of early-life exposures and shift of focus from early life to pregnancy, gene-environment interactions, cohort effects, and time trends in patients with allergic diseases. This review helps to define phenotypes of allergic diseases in MeDALL. (J Allergy Clin Immunol 2012;129:943-54.)

Background Cigarette smoke, the major risk factor for COPD, is known to activate matrix metalloproteinases in airway epithelium. We investigated whether metalloproteinases, particularly A Disintegrin and Metalloproteinase (ADAM)17, contribute to increased pro-inflammatory epithelial responses with respect to the release of IL-8 and TGF-α, cytokines implicated in COPD pathogenesis. Methods We studied the effects of cigarette smoke extract (CSE) and metalloproteinase inhibitors on TGF-α and IL-8 release in primary bronchial epithelial cells (PBECs) from COPD patients, healthy smokers and non-smokers. Results We observed that TGF-α was mainly shed by ADAM17 in PBECs from all groups. Interestingly, IL-8 production occurred independently from ADAM17 and TGF-α shedding, but was significantly inhibited by broad-spectrum metalloproteinase inhibitor TAPI-2. CSE did not induce ADAM17-dependent TGF-α shedding, while it slightly augmented the production of IL-8. This was accompanied by reduced endogenous inhibitor of metalloproteinase (TIMP)-3 levels, suggesting that CSE does not directly but rather indirectly alter activity of ADAM17 through the regulation of its endogenous inhibitor. Furthermore, whereas baseline TGF-α shedding was lower in COPD PBECs, the early release of IL-8 (likely due to its shedding) was higher in PBECs from COPD than healthy smokers. Importantly, this was accompanied by lower TIMP-2 levels in COPD PBECs, while baseline TIMP-3 levels were similar between groups. Conclusions Our data indicate that IL-8 secretion is regulated independently from ADAM17 activity and TGF-α shedding and that particularly its early release is differentially regulated in PBECs from COPD and healthy smokers. Since TIMP-2-sensitive metalloproteinases could potentially contribute to IL-8 release, these may be interesting targets to further investigate novel therapeutic strategies in COPD.

Background Regulatory T-cells (Treg) hamper immune responses elicited by cancer vaccines. Therefore, depletion of Treg is being used to improve the outcome of vaccinations. Methods We studied whether an alphavirus vector-based immunotherapeutic vaccine changes the number and/or activity of Treg and if Treg depletion improves the efficacy of this vaccine against tumours. The vaccine is based on a Semliki Forest virus (SFV). The recombinant SFV replicon particles encode a fusion protein of E6 and E7 from human papillomavirus (HPV) type 16 (SFVeE6,7). Results We demonstrated that SFVeE6,7 immunization did not change Treg levels and their suppressive activity. Depletion of Treg in mice, using the novel anti-folate receptor 4 antibody, did not enhance the immune response induced by SFVeE6,7 immunization. Both the priming and the proliferation phases of the HPV-specific response elicited with SFVeE6,7 were not affected by the immune-suppressive activity of Treg. Moreover, Treg depletion did not improve the therapeutic antitumour response of SFVeE6,7 in a murine tumour model. Conclusions The efficacy of the SFVeE6,7 vaccine was not hampered by Treg. Therefore, SFVeE6,7 seems a very promising candidate for the treatment of HPV-induced disease, as it may not require additional immune interventions to modulate Treg activity.

Allergy is defined as a disease following a response by the immune system to an otherwise innocuous antigen. Allergic diseases include allergic rhinitis, atopic dermatitis, systemic Anaphylaxis, food allergy, allergic asthma and acute urticaria and are mediated by unwanted type-I hypersensitivity reactions (see chapter A9) to extrinsic allergens like pollen, house dust, animal dander, drugs and insect venom.

Allergen-specific immunotherapy (IT) uniquely renders long-term relief from allergic symptoms and is associated with elevated serum levels of allergen-specific IgG and IgA. The allergen-specific IgG response induced by IT treatment was shown to be critical for suppression of the immediate phase of the allergic response in mice, and this suppression was partially dependent on signaling through Fc gamma RIIB. To investigate the relevance of the allergen-specific IgG responses for suppression of the Th2-driven late-phase allergic response, we performed IT in a mouse model of allergic asthma in the absence of FcgRIIB or Fc gamma RI/Fc gamma RIII signaling. We found that suppression of Th2 cell activity, allergic inflammation, and allergen-specific IgE responses is independent of Fc gamma RIIB and Fc gamma RI/Fc gamma RIII signaling. Moreover, we show that the IT-induced allergen-specific systemic IgG or IgA responses and B cell function are dispensable for suppression of the late-phase allergic response by IT treatment. Finally, we found that the secretory mucosal IgA response also is not required for suppression of the Th2-driven allergic inflammation by IT. These data are in contrast to the suppression of the immediate phase of the allergic response, which is critically dependent on the induced allergen-specific serum IgG response. Hence, IT-induced suppression of the immediate and late phases of the allergic response is governed by divergent and independent mechanisms. Our data show that the IT-induced suppression of the Th2 cell-dependent late-phase allergic response is independent of the allergen-specific IgG and IgA responses that are associated with IT treatment. The Journal of Immunology, 2010, 185: 3857-3865.

Background: Cigarette smoking causes oxidative stress and severe damage of proteins in the lungs. One of the main systems to protect cells from damaged proteins is the ubiquitin-proteasome pathway. In this work, we follow up on an earlier observation that cigarette smoke (CS)-treated airway epithelial cells contain increased levels of free intracellular amino acids (AA). Objective: We hypothesize that i) CS-induced increased levels of free intracellular AA are due to activation of the proteasome, ii) CS increases the amount of ubiquitin-protein conjugates, iii) CS interferes with proteasomal degradation and de novo protein synthesis. Materials and methods: Airway epithelial cells (A549) were exposed to CS for 1 or 5 min and cultured for the next 4 or 24 h. Ubiquitin-protein conjugates were analyzed by Western blot. Proteasomal subunits were analyzed using the fluorescent activity-based probe MV151. Proteasomal activities were determined using fluorogenic substrates. Free amino acids in the lysates of CS-treated cells were quantified by Liquid Chromatography - Mass Spectrometry using stable isotope labeling. De novo protein synthesis was assayed using the AA analog L-azidohomoalanine. Results: Exposure of airway epithelial cells to CS increased the amount of ubiquitin-protein conjugates. Degradation of ubiquitin-protein conjugates by the proteasome was accompanied by an increase in free intracellular AA and inhibition of nascent protein synthesis. Activity-based labeling of the proteasomal active sites was not affected, except after 5-min exposure to CS. Caspase- and chymotrypsin-like proteasomal activities decreased, while trypsin-like activity increased. Conclusions: Our data imply that CS induces damage to proteins which are subsequently ubiquitinated and directed for proteasomal degradation. Proteasomal activities and protein synthesis are disturbed depending on the exposure time to CS. Inadequate protein turnover due to the effect of CS may thus be related to the disruption of normal structure and functions of the airway epithelium as observed in smokers.

Background Allergic asthma is characterized by airway hyperresponsiveness (AHR) and allergic inflammation of the airways, driven by allergen-specific Th2 cells. The asthma phenotypes and especially AHR are sensitive to the presence and activity of regulatory T (Treg) cells in the lung. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is known to have a co-stimulatory function on effector CD4+ T cells, rendering these cells insensitive to Treg suppression. However, the effects of GITR signaling on polarized Th1 and Th2 cell effector functions are not well-established. We sought to evaluate the effect of GITR signaling on fully differentiated Th1 and Th2 cells and to determine the effects of GITR activation at the time of allergen provocation on AHR and airway inflammation in a Th2-driven mouse model of asthma. Methods CD4+CD25- cells were polarized in vitro into Th1 and Th2 effector cells, and re-stimulated in the presence of GITR agonistic antibodies to assess the effect on IFNγ and IL-4 production. To evaluate the effects of GITR stimulation on AHR and allergic inflammation in a mouse asthma model, BALB/c mice were sensitized to OVA followed by airway challenges in the presence or absence of GITR agonist antibodies. Results GITR engagement potentiated cytokine release from CD3/CD28-stimulated Th2 but not Th1 cells in vitro. In the mouse asthma model, GITR triggering at the time of challenge induced enhanced airway hyperresponsiveness, serum IgE and ex vivo Th2 cytokine release, but did not increase BAL eosinophilia. Conclusion GITR exerts a differential effect on cytokine release of fully differentiated Th1 and Th2 cells in vitro, potentiating Th2 but not Th1 cytokine production. This effect on Th2 effector functions was also observed in vivo in our mouse model of asthma, resulting in enhanced AHR, serum IgE responses and Th2 cytokine production. This is the first report showing the effects of GITR activation on cytokine production by polarized primary Th1 and Th2 populations and the relevance of this pathway for AHR in mouse models for asthma. Our data provides crucial information on the mode of action of the GITR signaling, a pathway which is currently being considered for therapeutic intervention.

Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of (13)C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of (13)C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C(18) reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.

Systemic corticosteroids and additional short-acting beta 2-agonists are commonly used in exacerbations of chronic obstructive pulmonary disease (COPD). In this double-blind study, the combination of a high-dose inhaled corticosteroid with a rapid-onset long-acting beta 2-agonist was evaluated in the treatment of out-patient COPD exacerbations. The primary aim was to compare 14-day treatment effects of budesonide/formoterol to placebo on sputum eosinophils and, secondarily, on other indices of inflammation, forced expiratory flow in one second (FEV(1)), symptoms, health status, and adverse events. Forty-five patients not using steroids (37 male, 21/24 currentlex smoker, median packyears 38, age 65 years, FEV(1) 61% predicted), experiencing a COPD exacerbation, were treated at home with budesonide/formoterol (320/9 mu g 4 times daily), prednisolone (30 mg daily), or placebo for 14 days. Sputum eosinophils were significantly reduced by budesonide/formoterol (-57%) compared to placebo (+24%) (p = 0.01). Budesonide/formoterol reduced total symptom scores significantly (p = 0.01) compared to placebo. The increase in FEV(1) by 2 weeks of treatment with budesonide/formoterol (125 ml) was not significantly different from that of placebo (43 ml) (p = 0.07). Budesonide/formoterol treatment did not suppress morning serum cortisol compared to placebo (-16 %; p = 0.50). In conclusion, budesonide/formoterol reduces sputum eosinophils and improves symptoms in the treatment of out-patient COPD exacerbations.

Airway epithelial cells are well-known producers of thymus- and activation-regulated chemokine (TARC), a Th2 cell-attracting chemokine that may play an important role in the development of allergic airway inflammation. However, the mechanism responsible for up-regulation of TARC in allergy is still unknown. In the asthmatic airways, loss of expression of the cell-cell contact molecule E-cadherin and reduced epithelial barrier function has been observed, which may be the result of an inadequate repair response. Because E-cadherin also suppressed multiple signaling pathways, we studied whether disruption of E-cadherin-mediated cell contact may contribute to increased proallergic activity of epithelial cells, e.g., production of the chemokine TARC. We down-regulated E-cadherin in bronchial epithelial cells by small interference RNA and studied effects on electrical resistance, signaling pathways, and TARC expression (by electric cell-substrate impedance sensing, immunodetection, immunofluorescent staining, and real-time PCR). Small interference RNA silencing of E-cadherin resulted in loss of E-cadherin-mediated junctions, enhanced phosphorylation of epidermal growth factor receptor (EGFR), and the downstream targets MEK/ERK-1/2 and p38 MAPK, finally resulting in up-regulation of TARC as well as thymic stromal lymphopoietin expression. The use of specific inhibitors revealed that the effect on TARC is mediated by EGFR-dependent activation of the MAPK pathways. In contrast to TARC, expression of the Th1/Treg cell-attracting chemokine RANTES was unaffected by E-cadherin down-regulation. In summary, we show that loss of E-cadherin-mediated epithelial cell-cell contact by damaging stimuli, e.g., allergens, may result in reduced suppression of EGFR-dependent signaling pathways and subsequent induction of Th2 cell-attracting molecule TARC. Thus, disruption of intercellular epithelial contacts may specifically promote Th2 cell recruitment in allergic asthma.

Sputum induction (SI) is considered to be a safe tool for assessing airway inflammation in stable patients with COPD, but little is known about its safety during exacerbations. We therefore assessed the safety of SI during COPD exacerbations. SI data from 44 COPD patients were assessed both in the stable phase and during exacerbation. The median FEV, for the stable phase and exacerbation were 61% predicted (interquartile range [IQR], 49 to 74% predicted) and 51% predicted (IQR, 45 to 60% predicted), respectively. The median decrease in FEV, with SI during an exacerbation was 0.27 L (IQR, 0.17 to 0.40 L) vs 0.28 L (IQR, 0.22 to 0.44 L) during the stable phase (p = 0.03). The patients experienced the associated dyspnea well; no other adverse events occurred. All FEV, values returned to within 90% of their initial value within 30 min. A larger decrease in FEV, due to SI during an exacerbation was associated with the following parameters in the stable phase of disease: lower total sputum cell count (r = -0.37; p = 0.01); higher percentage of eosinophils (r = 0.33; p = 0.04); and a larger decrease in FEV1 after SI (r = 0.39; p = 0.03). In a multivariate analysis, the only independent association was with the larger decrease in FEV, in the stable phase. We concluded that SI can be safely carried out in patients with mild-to-moderate COPD who experience an exacerbation, and this occurs with no greater risk than in stable patients with COPD.

The type 2 T-helper (Th2) lymphocyte can be regarded as an important target cell for the treatment of allergic asthma as it plays a crucial role in the initiation, progression and persistence of disease. Several strategies to target Th2 cells can be envisioned. Drugs that prevent Th2-cells from migrating into the lung tissue, such as antibodies to the chemokine receptor CCR4 and inhibitors of the adhesion molecule VLA-4, are promising for the treatment of asthma. To inhibit Th2-cell activation, novel asthma drugs that act on Th2-selective transciption factors such as GATA3 are being developed. Although initial strategies aimed to block the action of Th2-derived cytokines, the generation of counter-regulatory Th1 lymphocytes and regulatory T cells is currently being explored.

Allergy is defined as a disease following a response by the IMMUNE SYSTEM to an otherwise innocuous agent. This chapter describes the actions of antiallergic drugs and therapeutics on diseases such as allergic rhinitis, atopic dermatitis, allergic conjunctivitis, systemic anaphylaxis, food ALLERGY, allergic asthma and acute urticaria. The putative MECHANISMS OF ACTION of disodium cromoglycate and nedocromil sodium (chromones) are discussed in detail as well as the biochemical and pharmacological effects, clinical applications and unwanted effects of these drugs. The characterisation of histamine receptors is detailed and the MECHANISMS OF ACTION as well as the observed biological effects of H1 and the relatively new H4 receptors are explained. Further, the biochemical and pharmacological effects of anti-IgE therapy are described and recent clinical trials with these drugs are briefly reviewed.

In asthmatics an immediate asthmatic response occurs after antigen provocation. Furthermore, asthmatic patients display airway hyperresponsiveness, accompanied by airway eosinophilia. In some patients late asthmatic responses can be detected. Many controversies still exist about the relations between the different airway responses and inflammatory cell infiltration, we therefore used a murine model to investigate associations between these phenomena. In this study we show the presence of antigen-induced early bronchoconstrictive responses, accompanied by increased serum mucosal mast cell protease-1 (MMCP-1) levels. However, we were unable to demonstrate late bronchoconstrictive responses either at the time when eosinophils start to infiltrate the lungs or when both airway hyperresponsiveness and eosinophilia are established. With sequential exposures to antigen, an association exists between development of airway hyperresponsiveness and eosinophilia. In contrast, resolution of this hyperreactivity appears to be dissociated from eosinophilia after stopping the antigen challenges. Based on these data, we conclude that mast cell degranulation is a plausible cause of early bronchoconstrictive responses. Furthermore, late bronchoconstrictive responses are not related to the infiltration of eosinophils or development of airway hyperresponsiveness in this murine model. Finally, we conclude that airway hyperresponsiveness and eosinophilia are only associated with each other during the induction phase and not after the final antigen challenge.

Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323–339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323–339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with pulmonary and extra-pulmonary manifestations. Although COPD is a complex disease, diagnosis and staging are still based on simple spirometry measurements. Different COPD phenotypes exist based on clinical, physiological, immunological and radiological observations. Cigarette smoking is the most important risk factor for COPD, but only 15-20% of smokers develop the disease, suggesting a genetic predisposition. Unfortunately, little is known about the pathogenesis of COPD, and even less on the very first steps that are associated with an aberrant response to smoke exposure. This study aims to investigate the underlying local and systemic inflammation of different clinical COPD phenotypes, and acute effects of cigarette smoke exposure in individuals susceptible and non-susceptible for the development of COPD. Furthermore, we will investigate mechanisms associated with corticosteroid insensitivity. Our study will provide valuable information regarding the pathogenetic mechanisms underlying the natural course of COPD.METHODS AND ANALYSIS: This cross-sectional study will include young and old individuals susceptible or non-susceptible to develop COPD. At a young age (18-40 years) 60 'party smokers' will be included who are called susceptible or non-susceptible based on COPD prevalence in smoking family members. In addition, 30 healthy smokers (age 40-75 years) and 110 COPD patients will be included. Measurements will include questionnaires, pulmonary function, low-dose CT scanning of the lung, body composition, 6 min walking distance and biomarkers in peripheral blood, sputum, urine, exhaled breath condensate, epithelial lining fluid, bronchial brushes and biopsies. Non-biased approaches such as proteomics will be performed in blood and epithelial lining fluid.ETHICS AND DISSEMINATION: This multicentre study was approved by the medical ethical committees of UMC Groningen and Utrecht, the Netherlands. The study findings will be presented at conferences and will be reported in peer-reviewed journals.TRIAL REGISTRATION: ClinicalTrials.gov, NCT00807469 (study 1) and NCT00850863 (study 2).

Recently, we demonstrated that antibody to interleukin-5 (IL-5) prevents the infiltration of eosinophils in the respiratory airways and the development of bronchial hyperreactivity in an animal model of allergic asthma. In this study we investigated the influence of long-term intranasal administration of IL-5 on airway responsiveness in vitro, the infiltration of inflammatory leukocytes, and mucosal exudation. Mice (BALB/c) received 1 μg of recombinant human IL-5 in 30 μl of saline solution or vehicle alone twice a day for 1, 3, and 7 days. At 3 and 7 days after IL-5 administration, the number of bronchoalveolar lavage eosinophils increased approximately fourfold and sixfold, respectively. Blood eosinophil numbers showed a similar increase. In addition, 7 days after IL-5 treatment, total lung eosinophil peroxidase activity was significantly increased by 170% as compared with controls. The maximal responsiveness of the trachea in vitro to methacholine was significantly increased by 34%, as compared with controls, only at 7 days after IL-5 administration. Furthermore, mucosal exudation was also only increased significantly at 7 days after IL-5 administration. It can be concluded that the IL-5–induced eosinophil infiltration precedes the development of airway hyperreactivity and mucosal exudation. (J Allergy Clin Immunol 1995;96:104-12.)

Background: Regulatory T-cell dysfunction is associated with development of the complex genetic conditions atopy and asthma. Therefore, we hypothesized that single nucleotide polymorphisms in genes involved in the development and function of regulatory T cells are associated with atopy and asthma development.Objective: To evaluate main effects and gene-gene interactions of haplotype tagging single nucleotide polymorphisms of genes involved in regulatory T-cell function-IL6, IL6R, IL10, heme-oxygenase 1 (HMOX1), IL2, Toll-like receptor 2 (TLR2), TGFB1, TGF-beta receptor (TGFBR)-1, TGFBR2, IL2RA, and forkhead box protein 3 (FOXP3)-in relation to atopy and asthma.Methods: Single-locus and multilocus associations with total IgE (3rd vs 1st tertile); specific IgE to egg, milk, and indoor allergens; and asthma were evaluated by x 2 tests and the multifactor dimensionality-reduction method in 3 birth cohorts (Allergenic study).Results: Multiple statistically significant multilocus associations existed. IL2RA rs4749926 and TLR2 rs4696480 associated with IgE in both age groups tested (1-2 and 6-8 years). TGFBR2 polymorphisms associated with total and specific IgE in both age groups and with asthma. TGFBR2 rs9831477 associated with specific IgE for milk at age 1 to 2 years and indoor allergens at age 6 to 8 years. For milk-specific IgE, interaction between TGFBR2 and FOXP3 polymorphisms was confirmed by logistic regression and consistent in 2 birth cohorts and when stratified for sex, supplying internal replications.Conclusion: Genes involved in the development and function of regulatory T cells, specifically IL2RA, TLR2, TGFBR2, and FOXP3, associate with atopy and asthma by gene-gene interaction. Modeling of multiple gene-gene interactions is important to unravel further the genetic susceptibility to atopy and asthma. (J Allergy Clin Immunol 2010;126:338-46.)

Bacterial endotoxin (E. coli O111:B4) induces, 4 days after intraperitoneal injection, a 30% reduction of guinea pig lung beta-adrenoceptor number (Bmax). No change in affinity (Kd) for the receptors occurred. Bilateral electrolytic lesions centered in the anterior hypothalamic nucleus prevent this reduction in Bmax and even reverse the reduction into a small increase in beta-adrenoceptor number. Since it is known from the literature data that anterior hypothalamic lesions as well as beta-adrenoceptor stimulants have an inhibitory influence on the immune system, the mechanism by which these lesions inhibit the reduction of beta-adrenoceptor sites after bacterial endotoxin and influence immune functions, may be related.

Epithelium-derived Fas ligand is believed to modulate inflammation within various tissues. In this paper, we report findings that suggest a similar immunoregulatory role for Fas ligand in the lung. First, Fas ligand was localized to nonciliated, cuboidal airway epithelial cells (Clara cells) throughout the airways in the normal murine lung by employing nonisotopic in situ hybridization and immunohistochemistry. Second, gldmutant mice, which express a dysfunctional Fas ligand protein, were noted to develop prominent infiltration of inflammatory cells in submucosal and peribronchial regions of the upper and lower airways. Third, during allergic airway inflammation induced by ovalbumin in mice, cell-associated staining for Fas ligand mRNA and protein was markedly reduced in the airway epithelium. These data suggest that Clara cell-derived Fas ligand may control immune activity in the airway; thus alterations in this protective mechanism may be involved in the pathogenesis of certain inflammatory conditions of the airway, such as asthma.

Background Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. Methods BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. Results Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p Conclusion Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Background Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis. Methods We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls. Results We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1. Conclusion The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD. © 2013 Hoffmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.