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Background: Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis.Methods: Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting.Results: Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (RL) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge.Conclusion: Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis. [ABSTRACT FROM AUTHOR]

Background: Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma., Objective: We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming., Methods: Wild-type (WT) and B cell-deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation., Results: Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell-deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and antigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell-deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3 + regulatory T cells after in vitro coincubation with OVA/IgG-containing AF., Conclusion: Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Bleomycin-induced lung injury leads to surfactant dysfunction and permanent loss of alveoli due to a remodeling process called collapse induration. Collapse induration also occurs in acute interstitial lung disease and idiopathic pulmonary fibrosis in humans. We hypothesized that surfactant dysfunction aggravates lung injury and early remodeling resulting in collapse induration within 7 days after lung injury. Rats received bleomycin to induce lung injury and either repetitive surfactant replacement therapy (SRT: 100 mg Curosurf/kg BW = surf group) or saline (0.9% NaCl = saline group). After 3 (D3) or 7 (D7) days, invasive pulmonary function tests were performed to determine tissue elastance (H) and static compliance (Cst). Bronchoalveolar lavage (BAL) was taken for surfactant function, inflammatory markers, and protein measurements. Lungs were fixed by vascular perfusion for design-based stereology and electron microscopic analyses. SRT significantly improved minimum surface tension of alveolar surfactant as well as H and Cst at D3 and D7. At D3 decreased inflammatory markers including neutrophilic granulocytes, IL-1β, and IL-6 correlated with reduced BAL-protein levels after SRT. Numbers of open alveoli were significantly increased at D3 and D7 in SRT groups whereas at D7 there was also a significant reduction in septal wall thickness and parenchymal tissue volume. Septal wall thickness and numbers of open alveoli highly correlated with improved lung mechanics after SRT. In conclusion, reduction in surface tension was effective to stabilize alveoli linked with an attenuation of parameters of acute lung injury at D3 and collapse induration at D7. Hence, SRT modifies disease progression to collapse induration., (Copyright © 2017 the American Physiological Society.)

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly., Objectives: To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice., Methods: Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis., Measurements and Main Results: We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice., Conclusions: Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The surfactant-associated proteins SP-A and D are pattern recognition molecules with collectin structure. A single nucleotide polymorphism (SNP) exchanging a methionine (Met) for a threonine (Thr) in the amino-terminal SP-D domain influences the oligomeric structure and function of the protein. In this study, we investigated the susceptibility of mice transgenic for the human SP-D Met(11)Thr SNP to allergic airway inflammation and consequences for microRNA (miRNA, miR) expression. Mice expressing either human Met or Thr SP-D were sensitized and challenged with ovalbumin (OVA) in an acute model of allergic asthma. The influence of the SP-D polymorphism on the allergic airway inflammation was evaluated by lung function measurement, pulmonary inflammation parameters, morphological analysis and miRNA expression. Airway hyperresponsiveness, allergic inflammation, and mucus metaplasia were not significantly different between mice expressing one or the other allelic variant of SP-D. OVA sensitization and challenge led to significant airway hyperresponsiveness in wildtype mice and significantly lower eosinophil numbers and interleukin 5 levels in Thr SP-D mice. OVA challenge induced an upregulation of miR-21 and 155 in Thr SP-D mice and a downregulation of miR-21 in Met SP-D mice. Our results show that murine expression of human polymorphic SP-D variants does not significantly influence the severity of allergic airway inflammation. MiR-21 and 155 are differentially regulated in transgenic mice in response to allergic inflammation. Further studies are required to elucidate the impact of this SNP on inflammatory conditions of the lung.

Animal models with a high predictive value for human trials are needed to develop novel human-specific therapeutics for respiratory diseases. The aim of the present study was to examine lung-function parameters in marmoset monkeys (Callithrix jacchus) that can be used to detect pharmacologically or provocation-induced AHR (airway hyper-responsiveness). Therefore a custom-made lung-function device that allows application of defined aerosol doses during measurement was developed. It was hypothesized that LPS (lipopolysaccharide)-challenged marmosets show AHR compared with non-challenged healthy subjects. Invasive plethysmography was performed in 12 anaesthetized orotracheally intubated and spontaneously breathing marmosets. Pulmonary data of R(L) (lung resistance), C(dyn) (dynamic compliance), EF50 (mid-expiratory flow), P(oes) (oesophageal pressure), MV (minute volume), respiratory frequency (f) and V(T) (tidal volume) were collected. Measurements were conducted under baseline conditions and under MCh (methacholine)-induced bronchoconstriction. The measurement was repeated with the same group of animals after induction of an acute lung inflammation by intratracheal application of LPS. PDs (provocative doses) of MCh to achieve a certain increase in RL were significantly lower after LPS administration. AHR was demonstrated in the LPS treated compared with the naïve animals. The recorded lung-function data provide ground for pre-clinical efficacy and safety testing of anti-inflammatory substances in the common marmoset, a new translational NHP (non-human primate) model for LPS-induced lung inflammation.

Severe asthma and viral-induced asthma exacerbations represent a high unmet medical need as no therapy is currently available for these patients. HRV (human rhinovirus) is prominently associated with asthma exacerbations in humans. The aim of the present study was to establish a mouse model of severe asthma with additional rhinovirus infection to investigate the interplay between chronic allergic airway inflammation and acute respiratory viral infection. Balb/c mice were sensitized with HDM (house dust mite) extract (25 μg in 50 μl of saline) by i.n. (intranasal) delivery to the lung over 7 weeks. HRV1B (HRV serotype 1B) inoculation was performed i.n. on the last 3 days. Therapeutic treatment with FP (fluticasone propionate) was performed to assess steroid efficacy. Lung resistance was measured invasively to assess AHR (airway hyper-responsiveness). BAL (bronchoalveolar lavage) differential cell count, cytokines, lung histology and the proliferative and cytokine response of MLN (mediastinal lymph node) cells upon in vitro restimulation were analysed. Chronic HDM application induced a strong Th2-skewed eosinophilic airway inflammation and AHR, which was not exacerbated by superimposed HRV1B infection. Therapeutic steroid intervention in the chronic HDM model reduced BAL eosinophil cell counts, cytokine levels and AHR, while neutrophil numbers were unaffected. Steroid efficacy against inflammatory readouts was maintained during additional HRV1B infection. Animals with chronic allergic airway inflammation exhibited a diminished immune response towards superimposed HRV1B infection compared with HRV1B alone, as induction of the anti-viral and pro-inflammatory cytokines IFN (interferon)-α, IFN-γ and IL (interleukin)-12 were suppressed. Although superimposed HRV1B infection did not provoke asthma exacerbation in this severe model, a deficient anti-viral immune response to HRV1B was present under chronic allergic airway inflammatory conditions. Thus, this model is able to reflect some aspects of the complex interplay of respiratory virus infection in chronic allergic asthma.

The CD26-associated enzymatic activity of dipeptidyl peptidase-4 (DPP4) as well as the recruitment of CD26(+) T cells increase under allergic airway inflammation. Furthermore, genetic deficiency of CD26/DPP4 exerts protective effects in experimental asthma. Therefore, CD26/DPP4 might represent a novel therapeutic target in asthma. To study the effects of pharmacological inhibition of DPP4 on allergic airway inflammation the DPP4-inhibitor isoleucine thiazolidide was tested using different doses at different time points (at sensitization, immediately before and simultaneously with the allergen challenge, as well as continuously via drinking water), and different routes (intraperitoneal, oral, and by inhalation). Allergic-like airway inflammation was induced in Fischer 344 rats (Charles River) sensitized against ovalbumin (OVA) using OVA aerosols. Intraperitoneal application of the DPP4 inhibitor showed effects neither at sensitization nor at challenge, whereas a continuous application via drinking water using high doses of the inhibitor led to an aggravation of the histomorphological signs of airway inflammation. In contrast, aerosolization of the DPP4 inhibitor simultaneously with the allergen significantly reduced airway hyperresponsiveness and ameliorated histopathological signs compared to controls. In addition, this treatment resulted in increased mRNA levels of surfactant proteins, suggesting an involvement of DPP4 inhibitors in surfactant metabolism in OVA-challenged rats. Continuous systemic inhibition of DPP4 via the oral route aggravates allergic airway inflammation. In contrast, topical inhibition of DPP4 exerts potential protective effects, and further research in humans is needed.

Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae-specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae/HDM allergen-treated group than in the C. pneumoniae/NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae/HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The ICH guideline S7A requires safety pharmacology tests including measurements of pulmonary function. In the first step - as part of the "core battery" - lung function tests in conscious animals are requested. If potential adverse effects raise concern for human safety, these should be explored in a second step as a "follow-up study." For these two stages of safety pharmacology testing, both non-invasive and invasive techniques are needed which should be as precise and reliable as possible. A short overview of typical in vivo measurement techniques is given, their advantages and disadvantages are discussed and out of these the non-invasive head-out body plethysmography and the invasive but repeatable body plethysmography in orotracheally intubated rodents are presented in detail. For validation purposes the changes in the respective parameters such as tidal midexpiratory flow (EF(50)) or lung resistance have been recorded in the same animals in typical bronchoconstriction models and compared. In addition, the technique of head-out body plethysmography has been shown to be useful to measure lung function in juvenile rats starting from day two of age. This allows safety pharmacology testing and toxicological studies in juvenile animals as a model for the young developing organism as requested by the regulatory authorities (e.g., EMEA Guideline 1/2008). It is concluded that both invasive and non-invasive pulmonary function tests are capable of detecting effects and alterations on the respiratory system with different selectivity and area of operation. The use of both techniques in a large number of studies in mice and rats in the last years have demonstrated that they provide useful and reliable information on pulmonary mechanics in safety pharmacology and toxicology testing, in investigations of respiratory disorders, and in pharmacological efficacy studies.

Background: Our laboratory has shown that inhalational sensitization to new antigens is facilitated through an ongoing T(H)2-polarized inflammation of the lung, a phenomenon we call "collateral priming.", Objective: We were interested to analyze whether a T(H)1-polarized pulmonary inflammation also facilitates priming toward new antigens and which cytokine or cytokines are involved., Methods: T(H)1-polarized T cells were generated in vitro and transferred into congenic mice. Mice were challenged initially with cognate antigen and an unrelated antigen; consecutively, they received cognate antigen or the secondary antigen. Airway inflammation, antigen-specific IgG2a levels, and airway hyperresponsiveness were assessed to determine the inflammatory phenotype, with antibody blocking studies used to determine cytokine requirements for T(H)1 collateral priming., Results: Our experiments revealed that ongoing inflammation of the lung induced by the transfer of T(H)1-polarized cells also facilitates priming toward new antigens, which results in lymphocytic inflammation of the lung. Interestingly, blocking studies identified IL-17A as a major contributor to this pathology. Accordingly, we could demonstrate for the first time that T(H)17-polarized cells alone can facilitate priming toward new antigens, inducing lymphocytic airway inflammation and strong airway hyperresponsiveness. Flow cytometric analysis revealed priming of endogenous T cells for IL-17A secretion with a distinct memory/effector phenotype compared to T(H)1 cells, thus presenting an exciting model to further elucidate differentiation of T(H)17 cells., Conclusions: We show that airway inflammation mediated by T(H)17 cells facilitates sensitization to new antigens and confers increased airway responsiveness in a murine model of polysensitization, suggesting a mechanism involving IL-17A behind the increased risk for allergic sensitization in polysensitized subjects., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8(+) T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8(+) T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8(+) T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8(+) T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8(+) T cells in the lung. Notably, CD8(+) T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8(+) T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8(+) T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103(+)CD62L(lo) Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8(+) T cells.

Background: Bronchial asthma is characterized by airway inflammation and reversible obstruction. Since the gold standard of therapy, a combination of anti-inflammatory corticosteroids and bronchodilatory β(2) agonists, has recently been discussed to be related to an increased mortality, there is a need for novel therapeutic pathways., Objective: A new experimental concept that encompasses the vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide (PACAP) family of receptors by demonstrating the anti-inflammatory effects of the PACAP receptor 1 (PAC1R) in a murine model of allergic asthma is described., Methods: PAC1R expression was investigated in lung tissue and isolated dendritic cells (DCs) via real-time PCR. Ovalbumin (OVA)-induced asthma models were used in PAC1R-deficient mice and BALB/c mice treated with PAC1R agonist maxadilan (MAX). Bronchoalveolar lavages have been performed and investigated at the cellular and cytokine levels. Fluorescence staining of a frozen lung section has been performed to detect eosinophil granulocytes in lung tissue. Plasma IgE levels have been quantified via the ELISA technique. Lung function was determined using head-out body plethysmography or whole-body plethysmography., Results: Increased PAC1R mRNA expression in lung tissue was present under inflammatory conditions. PAC1R expression was detected on DCs. In OVA-induced asthma models, which were applied to PAC1R-deficient mice (PAC1R(-/-)) and to BALB/c mice treated with the specific PAC1R agonist MAX, PAC1R deficiency resulted in inflammatory effects, while agonistic stimulation resulted in anti-inflammatory effects. No effects on lung function were detected both in the gene-depletion and in the pharmacologic studies. In summary, here, we demonstrate that anti-inflammatory effects can be achieved via PAC1R., Conclusion: PAC1R agonists may represent a promising target for an anti-inflammatory therapy in airway diseases such as bronchial asthma., (© 2010 Blackwell Publishing Ltd.)

Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1β, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3(+)CD4(+) Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the context of pharmaceutical development today, studies for pediatric drug approval are requested more and more often by the regulatory authorities. The developing lung represents a potential target in juvenile toxicity studies. Due to physiological differences in prenatal and postnatal development between humans and standard animal models, experimental methods have to be modified to assess pulmonary function, and basic data on respiratory parameters need to be provided. Daily nose-only inhalation exposure from postnatal days 4 to 21 using a model substance (verapamil HCl) and plethysmographic measurements between postnatal days 2 and 50 were performed noninvasively in conscious juvenile Wistar (WU) rats. The methods proved to be feasible and did not interfere with normal growth and development of the animals. Both techniques therefore permit new insights to support human neonatal risk assessment and therefore these animal models are suitable for regulatory studies., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Inhalation is a non-invasive approach for both local and systemic drug delivery. This study aimed to define the therapeutic window for solid lipid nanoparticles (SLNs) as a drug delivery system by inhalation from a toxicological point of view. To estimate the toxic dose of SLNs in vitro, A549 cells and murine precision-cut lung slices (PCLS) were exposed to increasing concentrations of SLNs. The cytotoxic effect of SLNs on A549 cells was evaluated by MTT and NRU assays. Viability of lung tissue was determined with WST assay and by life/dead staining using calcein AM/EthD-1 for confocal microscopy (CLSM) followed by quantitative analysis with IMARIS. Inflammation was assessed by measuring chemokine KC and TNF-alpha levels. The in vivo effects were determined in a 16-day repeated-dose inhalation toxicity study using female BALB/c mice, which were daily exposed to different concentrations of SLN30 aerosols (1-200 microg deposit dose). Local inflammatory effects in the respiratory tract were evaluated by determination of total protein content, LDH, chemokine KC, IL-6, and differential cell counts, performed on days 4, 8, 12, and 16 in bronchoalveolar lavage fluid. Additionally, a histopathological evaluation of toxicologically relevant organs was accomplished. The in vitro and ex vivo dose finding experiments showed toxic effects beginning at concentrations of about 500 microg/ml. Therefore, we used 1-200 microg deposit doses/animal for the in vivo experiments. Even after 16 days of challenge with a 200-microg deposit dose, SLNs induced no significant signs of inflammation. We observed no consistent increase in LDH release, protein levels, or other signs of inflammation such as chemokine KC, IL-6, or neutrophilia. In contrast, the particle control (carbon black) caused inflammatory and cytotoxic effects at corresponding concentrations. These results confirm that repeated inhalation exposure to SLN30 at concentrations lower than a 200-microg deposit dose is safe in a murine inhalation model., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Inhibitory effects of roflumilast on responses characteristic of allergic asthma were investigated in a fungal asthma model in BALB/c mice., Methods: Mice were sensitized with Aspergillus antigen (Afu) and exposed to Afu or vehicle, and given roflumilast 1 or 5 mg/kg. Early airway response (EAR) and late airway hyperresponsiveness (AHR) to methacholine were measured via plethysmography. Bronchoalveolar lavage (BAL) was used to assess inflammatory cell count., Results: In Afu-exposed mice, roflumilast dose-dependently reduced the EAR [26% at 1 mg/kg (NS) and 94% at 5 mg/kg (p < 0.01)] and AHR [46% at 1 mg/kg (NS) and 128% at 5 mg/kg (p < 0.05)]. Roflumilast 5 mg/kg reduced neutrophil, eosinophil and lymphocyte counts [87% (p < 0.01), 40% (NS) and 67% (p < 0.01), respectively] in BAL fluid versus controls., Conclusions: In this model, roflumilast inhibited the EAR, suppressed AHR and reduced inflammatory cell infiltration., (2009 S. Karger AG, Basel.)

A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation.

We hypothesized that formation of pulmonary emphysema could be diminished after previous activation of stem cells. Animals received either a daily dose of the hematopoietic growth factors (GF; recombinant rat stem cell factor plus recombinant granulocyte colony stimulating factor; n=6, Elastase/GF group) or vehicle (n=9, Elastase/Sham group) starting 3 days before intratracheal instillation of elastase or vehicle and continued for another 25 days. Control animals were treated with NaCl (n=9, Sham/Sham group). On day 25, in all animals, a 2-mL pump was implanted subcutaneously that delivered 200 microg/h 5-bromo-2-desoxyuridine (BrdU) until study termination. Compared to controls, the Elastase/Sham group exhibited elevated total lung capacity (TLC) and functional residual capacity (FRC), significantly increased mean free alveolar pathway, alveolar volume, and decreased septal density. The Elastase/GF group showed (1) a significant increase of TLC and FRC, (2) a significant increase in alveolar size and volume, (3) a significant reduction of septal density, volume, and thickness. Proliferation in lung parenchyma and in terminal bronchioles remained significantly decreased in the Elastase/Sham group and the Elastase/GF group. Blood cell number has significantly increased in the Elastase/GF group. The application of GF-enhanced pulmonary emphysema, presumable because of increased inflammatory activity, was a result of a preventive treatment.

Neuregulin is an important growth factor in fetal surfactant synthesis, and downregulation of its receptor, ErbB4, impairs fetal surfactant synthesis. We hypothesized that pulmonary ErbB4 deletion will affect the developing lung leading to an abnormal postnatal lung function. ErbB4-deleted lungs of 11- to 14-wk-old adult HER4heart mice, rescued from their lethal cardiac defects, were studied for the effect on lung function, alveolarization, and the surfactant system. ErbB4 deletion impairs lung function and structure in HER4heart mice resulting in a hyperreactive airway system and alveolar simplification, as seen in preterm infants with bronchopulmonary dysplasia. It also leads to a downregulation of surfactant protein D expression and an underlying chronic inflammation in these lungs. Our findings suggest that this animal model could be used to further study the pathogenesis of bronchopulmonary dysplasia and might help design protective interventions.

Precise and repeatable measurements of pulmonary function in intact mice or rats are becoming increasingly important for experimental investigations on various respiratory disorders like asthma and for pharmacological, safety-pharmacological or toxicological testing of drugs or chemicals. This review provides a short overview of typical in-vivo measurement techniques, discusses their advantages and disadvantages and presents two of these methods in detail: the noninvasive head-out body plethysmography and an invasive but repeatable body-plethysmography in orotracheally intubated rodents. It will be demonstrated that these methods are able to monitor bronchoconstriction in safety-pharmacological tests or in asthma models showing early allergic response or late airway hyperresponsiveness in response to inhaled allergens and demonstrate drug effects on pulmonary endpoints. The changes in the respective parameters such as tidal midexpiratory flow (EF(50)) or lung resistance in typical bronchoconstriction models have been measured in the same animals and compared for validation purposes. It is concluded that both invasive and noninvasive pulmonary function tests are capable of detecting allergen-specific as well as non-allergic bronchoconstriction in intact mice or rats. The invasive determination of resistance is superior in sensitivity, whereas the noninvasive EF(50) method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of mice and rats or if the conscious animal has to be tested (e.g. safety pharmacology). The use of both techniques in a large number of studies in the last years have demonstrated that they provide useful and necessary information on pulmonary mechanics in studies of respiratory disorders including experimental models of asthma, in investigations of pulmonary pharmacology, safety pharmacology and toxicology in mice and rats.

Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Important features of this exaggerated response to bronchoconstrictive stimuli have mostly been investigated in vivo in intact animals or in vitro in isolated tracheal or bronchial tissues. Both approaches have important advantages but also certain limitations. Therefore, the aim of our study was to develop an ex vivo model of isolated lungs from sensitized mice for the investigation of airway responsiveness (AR). BALB/c mice were sensitized by intraperitoneal ovalbumin (Ova) and subsequently challenged by Ova inhalation. In vivo AR was measured in unrestrained animals by whole body plethysmography after stimulation with aerosolized methacholine (MCh) with determination of enhanced pause (P(enh)). Twenty-four hours after each P(enh) measurement, airway resistance was continuously registered in isolated, perfused, and ventilated lungs on stimulation with inhaled or intravascular MCh or nebulized Ova. In a subset of experiments, in vivo AR was additionally measured in orotracheally intubated, spontaneously breathing mice 24 h after P(enh) measurement, and lungs were isolated further 24 h later. Isolated lungs of allergen-sensitized and -challenged mice showed increased AR after MCh inhalation or infusion as well as after specific provocation with aerosolized allergen. AR was increased on days 2 and 5 after Ova challenge and had returned to baseline on day 9. AHR in isolated lungs after aerosolized or intravascular MCh strongly correlated with in vivo AR. Pretreatment of isolated lungs with the beta(2)-agonist fenoterol diminished AR. In conclusion, this model provides new opportunities to investigate mechanisms of AHR as well as pharmacological interventions on an intact organ level.

Background: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease., Objective: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model., Methods: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release., Results: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS., Conclusion: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

There are invasive and noninvasive pulmonary function tests available which are sensitive in detecting bronchoconstriction in rodents. Noninvasively measured midexpiratory flow (EF50) has been shown to be an appropriate parameter to monitor bronchoconstriction in a large number of animals, e.g. for screening purposes. Recently, a novel technique for repetitive lung function measurements in orotracheally intubated, spontaneously breathing mice has been established. Bronchoconstriction is assessed by the "gold standard" parameters airway resistance and dynamic compliance in response to aerosolized methacholine or allergens in anesthetized mice. This measurement technique has been combined with an inhalation technique which has been optimized to allow simultaneous lung function measurement in intubated animals and to obtain high aerosol concentrations. A feedback dose control system has been developed to administer a defined and constant aerosol dose to each individual animal. Using this system a prominent early allergic response and late airway hyperresponsiveness could be demonstrated in intubated mice challenged with Aspergillus fumigatus allergen. We conclude: The noninvasive EF50 method seems particularly appropriate for measurements of respiratory function in large numbers of conscious mice in assembly line fashion. The invasive technology--newly established for the mouse--is more sensitive and specific since true airway resistance and dynamic compliance are determined and allows now the adequate detection of an early allergic response in the mouse and also repetitive measurements e.g. to assess the airway hyperresponsiveness in the same animal or for monitoring purposes in chronic models.

The prevalence of asthma continues to increase. Asthma is caused by a Th2 cell-driven immune response. Its optimal treatment remains a challenge, and a sufficient immunotherapeutic approach to treating asthma has yet to be found. Using a murine asthma model, we show that a single injection of an anti-CD137 (4-1BB) mAb prevents the development of airway hyperreactivity, eosinophilic airway inflammation, excessive mucus production, and elevated IgE during the observation period of 7 weeks. Most importantly, even established disease is completely reversed by anti-CD137 mAb administration. The protection is associated with markedly reduced Th2 cytokine production and increased secretion of the Th1 cytokine IFN-gamma. While B lymphocytes are partly depleted, the number of CD8+ T cells is increased. Blockade of IFN-gamma and depletion of CD8+ T cells during treatment with anti-CD137 mAb reduces in part but does not abrogate the protective effect of CD137 mAb. In contrast, CD137 mAb-mediated CD4+ T cell anergy is critical for the observed effects, since transfer of CD4+ T cells from CD137 mAb-treated mice conveyed protection. These data demonstrate, for the first time to our knowledge, the capacity of anti-CD137 mAb to ameliorate allergic asthma, and they indicate CD137 as a possible target for therapeutic intervention in this disease.

Aim: Non-invasive analysis of tidal expiratory flow parameters such as Tme/TE (time needed to reach peak expiratory flow divided by total expiratory time) or midexpiratory tidal flow (EF50) has been shown useful for phenotypic characterization of lung function in humans and animal models. In this study, we aimed to compare the utility of two non-invasive measures, EF50 and Tme/TE, to monitor bronchoconstriction to inhaled cholinergic and allergic challenges in Brown-Norway rats., Methods: Non-invasive measurements of Tme/TE and EF50 were paralleled by invasive recordings of Tme/TE, EF50 and pulmonary conductance (GL)., Results: First, dose-response studies with acetylcholine were performed in naive rats, showing that EF50 better than Tme/TE reflected the dose-related changes as observed with the classical invasive outcome parameter GL. The subsequent determination of allergen-specific early airway responsiveness (EAR) showed that ovalbumin-sensitized and -challenged rats exhibited airway inflammation and allergen-specific EAR. Again, EF50 was more sensitive than Tme/TE in detecting the allergen-specific EAR recorded with invasive and non-invasive lung function methods and agreed well with classical GL measurements., Conclusion: We conclude that non-invasive assessment of EF50 is significantly superior to Tme/TE and serves as a suitable and valid tool for phenotypic screening of cholinergic and allergic airway responsiveness in rats.

Background: This study seeks to compare the ability of repeatable invasive and noninvasive lung function methods to assess allergen-specific and cholinergic airway responsiveness (AR) in intact, spontaneously breathing BALB/c mice., Methods: Using noninvasive head-out body plethysmography and the decrease in tidal midexpiratory flow (EF50), we determined early AR (EAR) to inhaled Aspergillus fumigatus antigens in conscious mice. These measurements were paralleled by invasive determination of pulmonary conductance (GL), dynamic compliance (Cdyn) and EF50 in another group of anesthetized, orotracheally intubated mice., Results: With both methods, allergic mice, sensitized and boosted with A. fumigatus, elicited allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response studies to aerosolized methacholine (MCh) were performed in the same animals 48 h later, showing that allergic mice relative to controls were distinctly more responsive (p < 0.05) and revealed acute airway inflammation as evidenced from increased eosinophils and lymphocytes in bronchoalveolar lavage., Conclusion: We conclude that invasive and noninvasive pulmonary function tests are capable of detecting both allergen-specific and cholinergic AR in intact, allergic mice. The invasive determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive EF50 method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of conscious mice.

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.

Precise and repeatable measurements of pulmonary function in intact mice are becoming increasingly important for experimental investigations on various respiratory disorders including asthma. Here, we present validation of a novel in vivo method that, for the first time, combines direct and repetitive recordings of standard pulmonary mechanics with cholinergic aerosol challenges in anesthetized, orotracheally intubated, spontaneously breathing mice. We demonstrate that, in several groups of nonsensitized BALB/c mice, dose-related increases in pulmonary resistance and dynamic compliance to aerosolized methacholine are reproducible over short and extended intervals without causing detectable cytological alterations in the bronchoalveolar lavage or relevant histological changes in the proximal trachea and larynx regardless of the number of orotracheal intubations. Moreover, as further validation, we confirm that allergic mice, sensitized and challenged with Aspergillus fumigatus, were significantly more responsive to cholinergic challenge (P < 0.01) and exhibited marked eosinophilia and lymphocytosis in bronchoalveolar lavage fluids as well as significant pathological alterations in laryngotracheal histology compared with nonsensitized mice. We suggest that this approach will provide useful and necessary information on pulmonary mechanics in studies of various respiratory disorders in mice, including experimental models of asthma and chronic obstructive pulmonary disorder, investigations of pulmonary pharmacology, or more general investigations of the genetic determinants of lung function.

Keratinocyte growth factor (KGF) is a mitogen for pulmonary epithelial cells. Intratracheal administration of KGF to adult rats results in alveolar epithelial type II and bronchiolar epithelial cell proliferation. While cellular responses to KGF have been intensively studied, functional consequences regarding lung function are unknown. Therefore, in this study, we sought to investigate whether KGF alters pulmonary function variables. Rats received either recombinant human KGF (rHuKGF) (5 mg/kg) or vehicle intratracheally. Before and on days 3 and 7 after treatment, pulmonary function was determined by body plethysmography. Subsequently, lung histological changes were quantified. rHuKGF induced a transient proliferation of alveolar and bronchiolar epithelial cells. The extent of type II cell hyperplasia was significantly correlated with a transient reduction in tidal volume and an increase in breathing frequency. In addition, quasi-static compliance, total lung capacity, and vital capacity were reduced after rHuKGF instillation, suggesting the development of a transitory restrictive lung disorder. Moreover, reduced expiratory flow rates and forced expiratory volumes, as well as increased functional residual capacity after rHuKGF but not vehicle, suggest obstructive lung function changes. In conclusion, the induction of alveolar and bronchiolar epithelial cell proliferation by KGF is paralleled by moderate functional consequences that should be taken into account when the therapeutic potential of KGF is tested.

Background: The increased production of nerve growth factor (NGF) has been associated with allergen-induced airway hyperresponsiveness and enhanced airway inflammation in experimental models of asthma. The aim of this study was to investigate whether a local application of anti-NGF to the lungs may affect the allergen-specific early (EAR) and late (LAR) airway responses to ovalbumin (Ova) of Ova-sensitized brown Norway rats., Methods: Rats were sensitized systemically with Ova and were boosted twice intratracheally with Ova aerosol using a microsprayer. Two hours before every boost, the animals were pretreated either with aerosolized anti-NGF or with a control antibody. On day 21, all animals were challenged with inhalational Ova aerosol and pulmonary resistance was recorded in anesthetized, orotracheally intubated animals during the early and late asthmatic responses. In addition, differential cell counts from bronchoalveolar lavage and serum immunoglobulin E (IgE) levels were determined 48 h post-Ova challenge., Results: Pretreatment with anti-NGF significantly attenuated the EAR but had no significant effect on the LAR. Serum IgE levels and inflammatory cell influx into the lungs were not affected by anti-NGF pretreatment., Conclusion: The data from this study suggest that NGF is directly involved in the development of the EAR without affecting the inflammatory airway response or LAR.

Keratinocyte growth factor (KGF) induces transient proliferation of alveolar type II cells (AEII) associated with surfactant alterations. To test the hypothesis that homeostasis of intracellular phospholipid stores is maintained under KGF-induced hyperplasia, we (1) collected tissue from adult rat lungs, fixed for light and electron microscopy 3 days after intratracheal instillation of 5 mg recombinant human (rHu) KGF/kg body weight or phosphate-buffered saline (PBS), and from untreated control animals (five animals/group) for design-based stereology of AEII and lamellar body (LB) ultrastructure; and (2) we analyzed uptake and distribution of instilled radiolabeled phospholipids. After rHuKGF, AEII-coverage of alveolar walls (PBS:8.3 +/- 3.0%; rHuKGF:30.6 +/- 4.8%) and number of AEII/ml lung volume (PBS:28.5 +/- 6.5 x 10(6); rHuKGF:48.2 +/- 5.8 x 10(6)) were increased (p < 0.008). Number (PBS:97 +/- 25; rHuKGF:54 +/- 7) and volume (PBS:45.3 +/- 13.8 microm(3); rHuKGF:21.0 +/- 4.7 microm(3)) of LBs per cell were decreased (p < 0.008), but not total amount/ml lung volume (PBS:128 +/- 46. 4 x 10(7) microm(3); rHuKGF:103 +/- 34. 7 x 10(7) microm(3)). This was paralleled by a shift to larger LBs. After rHuKGF, radiolabeled phospholipids accumulated in whole lung tissue relative to lavage fluid (p < 0.01). However, less radiolabel was incorporated per cell (p < 0.01). We conclude that under rHuKGF-induced AEII proliferation intracellular surfactant was decreased per single cell, whereas a constant amount was maintained per unit lung volume. We suggest that surfactant homeostasis is regulated at the level of phospholipid transport processes, for example, secretion and reuptake.

We tested the hypothesis whether allergic airway inflammation in ovalbumin sensitized and challenged Brown Norway rats is associated with intrinsic surfactant alteration and dysfunction. The determination of intra-alveolar surfactant subtypes and alveolar edema within their original microenvironment is only possible using an ultrastructural stereological approach. Therefore both lungs of control and asthmatic rats were fixed by vascular perfusion. The volume fractions of surfactant subtypes and the epithelial surface fraction covered with alveolar edema were determined by point and intersection counting. Furthermore, lung resistance was measured by means of whole-body plethysmography. The surface activity of surfactant from bronchoalveolar lavage was determined as minimum surface tension at minimal bubble size with a pulsating bubble surfactometer. Compared with controls, in asthmatics (1) the fraction of inactive unilamellar forms was significantly increased from 56% to 66%, (2) the fraction of alveolar epithelium covered with alveolar edema visible by light microscopy was significantly increased from 0.7% to 5.0%, (3) the fraction of alveolar epithelium covered with fluid seen by electron microscopy expanded significantly from 5% to 21%, (4) lung resistance was significantly elevated from 14% to 86% and (5) surface tension was enhanced from 6 mN/m to 12 mN/m. Thus, the inflammatory process after allergen challenge of sensitized Brown Norway rats causes intra-alveolar surfactant alterations. These surfactant alterations might contribute to small airway dysfunction.

This study was designed to evaluate the value and applicability of tidal breathing pattern analysis to assess bronchoconstriction in conscious rats. Using noninvasive, head-out body plethysmography and the decrease in tidal midexpiratory flow (EF(50)), we measured airway responsiveness (AR) to inhaled acetylcholine and allergen in conscious Brown-Norway rats, followed by invasive determination of pulmonary conductance (GL) and EF(50) in anesthetized rats. Dose-response studies to acetylcholine showed that noninvasively recorded EF(50) closely reflected the dose-dependent decreases observed with the invasive monitoring of simultaneously measured GL and EF(50). After sensitization and intratracheal boost to ovalbumin or saline, rats were assessed for early and late AR to aerosolized ovalbumin. Ovalbumin aerosol challenge resulted in early and late AR in allergen-sensitized rats, whereas controls were unresponsive. The allergen-specific AR, as measured noninvasively by EF(50), was similar in degree compared with invasively recorded EF(50) and GL and was associated with enhanced IgE and airway inflammation. We conclude that EF(50) is a noninvasive and physiologically valid index of bronchoconstriction in a rat model of asthma.

Asthma is a major cause of morbidity worldwide with prevalence and severity still increasing at an alarming pace. Hallmarks of this disease include early-phase bronchoconstriction with subsequent eosinophil infiltration, symptoms that may be mimicked in vivo by the complement-derived C3a anaphylatoxin, following its interaction with the single-copy C3aR. We analyzed the pathophysiological role of the C3a anaphylatoxin in a model of experimental OVA-induced allergic asthma, using an inbred guinea pig strain phenotypically unresponsive to C3a. Molecular analysis of this defect revealed a point mutation within the coding region of the C3aR that creates a stop codon, thereby effectively inactivating gene function. When challenged by OVA inhalation, sensitized animals of this strain exhibited a bronchoconstriction decreased by approximately 30% in comparison to the corresponding wild-type strain. These data suggest an important role of C3a in the pathogenesis of asthma and define a novel target for drug intervention strategies.

Chronic toxic and carcinogenic effects of gasoline engine exhaust inhalation were investigated in rats. The exhaust from the combustion of commercial fuel containing 30 ppm ferrocene additive was compared to exhaust from the same fuel without ferrocene. This study was part of a procedure to get a special authorization for the use of ferrocene as gasoline additive according to the German Gasoline Lead Act. To generate the exhausts, pairs of engines of the same type and age were operated on computer-controlled test benches in a combined urban-freeway driving cycle. The engines were equipped with three-way catalysts and lambda sensors. Rats inhaled the exhausts after dilution at ratios of about 1.20 and 1:40 for 18 h/day, 5 days/wk for 12 mo (chronic toxicity study) or for 24 mo followed by 6 mo of clean air (carcinogenicity study). The limiting factor for the exhaust concentration was the relative humidity of the exposure atmosphere. At defined intervals, body weight and food consumption, parameters of clinical chemistry, hematology, bronchoalveolar lavage (BAL), and mechanical lung function were measured, as well as lung clearance and particle retention in the lungs. In the high-dose groups and the controls the complete organ/tissue spectrum was investigated histopathologically, and in the low-dose groups the respiratory tract. Only slight exposure-related effects could be detected, like a loss in the background iron content of the cell pellet of the bronchoalveolar lavage fluid and cytoplasmic inclusions and goblet-cell hyperplasias in the nasal cavity. Between the clean-air controls and the exhaust-exposed groups, no exposure-related differences occurred in body weight development, mortality incidences, or any of the clinical investigations. Ninety-two to 94% of the animals developed age-related tumors, predominantly in the mammary glands, uterus, adrenals, thyroid, and pituitary. In the respiratory tract a total of five tumors was found: one in the controls and four in the low-dose groups. No physical, chemical, or toxicological differences between the exhausts from fuel without or with ferrocene were demonstrated.

Increased airway resistance in asthma may be partly due to poor function of pulmonary surfactant. This study investigated the inflammatory changes of bronchoalveolar lavage fluid (BALF) and the performance of BALF surfactant in healthy control subjects (n = 9) and patients with mild allergic asthma (n = 15) before and after segmental challenge. BALF was obtained for baseline values, and 24 h after challenge with saline solution in one lung segment and with allergen in another. Cell counts, phospholipid and protein concentrations, and ratios of small to large surfactant aggregates (SA/LA) were analyzed. Surface tension was determined with a pulsating bubble surfactometer, and the ability of the BALF surfactant to maintain airway patency was assessed with a capillary surfactometer. Baseline values of control subjects and asthmatics were not different. Challenge with saline and antigen raised total inflammatory cells in both control subjects and asthmatics. Allergen challenge of asthmatics, but not of healthy volunteers, significantly increased eosinophils, proteins, SA/ LA, and surface tension at minimum bubble size, and diminished the time the capillary tube is open. In conclusion, allergen challenge in asthmatics induced surfactant dysfunction, probably mainly because of inhibiting proteins. During an asthma attack, narrow conducting airways may become blocked, which might contribute to an increased airway resistance.

Impaired graft function in the postoperative course after lung transplantation (LTx) may in part be due to alterations in pulmonary surfactant. Animal data provide increasing evidence for surfactant abnormalities in the early course after graft reperfusion. However, little is known about the integrity of the surfactant system in human lung transplant recipients. We therefore investigated surfactant properties in bronchoalveolar lavage fluid (BALF) of patients with lung transplants (n = 60) in comparison to that of healthy subjects (n = 10). The phospholipid concentrations of BALF and of surfactant subfractions were measured, and total protein was analyzed. Surface activity was measured with a pulsating bubble surfactometer (PBS). Minimum surface tension was 15.8 +/- 1.1 mN/m in lung transplant recipients, whereas healthy subjects had minimum surface tensions of 3.4 +/- 1.9 mN/m (p = 0.0004). As a marker for potential surfactant inhibition, protein-to-phospholipid (PL) ratios showed no significant differences in the two major study groups. The ratio of small surfactant aggregates to large surfactant aggregates was increased in patients with lung transplants (p = 0.043). Episodes of infection or rejection did not change surface activities, nor did they induce altered ratios of protein to PL or of small to large surfactant aggregates. Surfactant activity did not correlate with pulmonary-function data. Moreover, surface tension showed no correlation with the time after transplantation. Our results suggest a persistent impairment of biophysical surfactant properties after LTx, possibly due to type-II-cell malfunction.