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1.
J Allergy Clin Immunol ; 141(2): 685-696.e6, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28601684

RESUMO

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.


Assuntos
Asma/imunologia , Linfócitos B/imunologia , Tolerância Imunológica , Troca Materno-Fetal/imunologia , Animais , Asma/genética , Asma/patologia , Asma/prevenção & controle , Linfócitos B/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Imunoglobulina G/imunologia , Troca Materno-Fetal/genética , Camundongos , Camundongos Transgênicos , Gravidez
2.
Mol Ther Nucleic Acids ; 7: 350-365, 2017 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-28624211

RESUMO

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.

3.
Am J Physiol Lung Cell Mol Physiol ; 313(2): L313-L327, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28450283

RESUMO

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-1ß, 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.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Alvéolos Pulmonares/efeitos dos fármacos , Surfactantes Pulmonares/farmacologia , Lesão Pulmonar Aguda/metabolismo , Animais , Bleomicina/farmacologia , Lavagem Broncoalveolar/métodos , Modelos Animais de Doenças , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Alvéolos Pulmonares/metabolismo , Ratos , Ratos Wistar , Respiração Artificial/métodos , Testes de Função Respiratória/métodos , Mecânica Respiratória/efeitos dos fármacos
4.
Thorax ; 70(7): 636-46, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25964315

RESUMO

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-ß1 (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 AdTGFß1-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 AdTGFß1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFß1-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.


Assuntos
Pneumonia Pneumocócica/complicações , Fibrose Pulmonar/microbiologia , Estreptolisinas/fisiologia , Animais , Antibacterianos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Líquido da Lavagem Broncoalveolar/imunologia , Toxina Diftérica , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Vacinas Pneumocócicas , Pneumonia Pneumocócica/tratamento farmacológico , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/prevenção & controle , Estreptolisinas/deficiência , Estreptolisinas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo
5.
Exp Lung Res ; 40(4): 154-63, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24712849

RESUMO

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.


Assuntos
Proteína D Associada a Surfactante Pulmonar/genética , Hipersensibilidade Respiratória/genética , Animais , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Muco/metabolismo , Ovalbumina , Fenótipo , Polimorfismo de Nucleotídeo Único , Hipersensibilidade Respiratória/metabolismo , Mucosa Respiratória/metabolismo
6.
Clin Sci (Lond) ; 126(1): 55-65, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23822145

RESUMO

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.


Assuntos
Asma/virologia , Infecções por Picornaviridae/complicações , Infecções Respiratórias/complicações , Rhinovirus/isolamento & purificação , Doença Aguda , Alérgenos/imunologia , Androstadienos/uso terapêutico , Animais , Asma/tratamento farmacológico , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/virologia , Doença Crônica , Citocinas/biossíntese , Modelos Animais de Doenças , Fluticasona , Glucocorticoides/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Pyroglyphidae/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Rhinovirus/imunologia , Carga Viral
7.
Clin Sci (Lond) ; 126(2): 155-62, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23879175

RESUMO

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.


Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Lipopolissacarídeos , Testes de Função Respiratória , Animais , Broncoconstrição/efeitos dos fármacos , Callithrix , Feminino , Masculino , Cloreto de Metacolina , Pletismografia
8.
Infect Immun ; 81(9): 3366-74, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23817611

RESUMO

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.


Assuntos
Alérgenos/imunologia , Chlamydophila pneumoniae/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pyroglyphidae/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Animais , Asma/imunologia , Asma/microbiologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/microbiologia , Infecções por Chlamydophila/patologia , Eosinofilia/imunologia , Eosinofilia/microbiologia , Eosinofilia/patologia , Feminino , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/imunologia , Pneumonia/microbiologia , Pneumonia/patologia , Infecções Respiratórias/microbiologia
9.
Pulm Pharmacol Ther ; 26(2): 281-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23266273

RESUMO

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.


Assuntos
DNA Catalítico/toxicidade , Fator de Transcrição GATA3/genética , Administração por Inalação , Animais , Encéfalo/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/imunologia , DNA Catalítico/administração & dosagem , Cães , Relação Dose-Resposta a Droga , Feminino , Coração/efeitos dos fármacos , Interferon gama/análise , Interleucina-10/análise , Pulmão/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Ratos , Ratos Wistar
10.
Front Pharmacol ; 3: 156, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22973226

RESUMO

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.

11.
J Allergy Clin Immunol ; 128(1): 168-177.e8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21459426

RESUMO

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.


Assuntos
Hiper-Reatividade Brônquica/imunologia , Ativação Linfocitária/imunologia , Pneumonia/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Separação Celular , Citometria de Fluxo , Inalação , Interleucina-17/imunologia , Interleucina-17/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pneumonia/metabolismo , Pneumonia/patologia
12.
J Immunol ; 186(11): 6106-18, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21518973

RESUMO

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.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Pneumonia/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Tolerância Imunológica/imunologia , Memória Imunológica/imunologia , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/imunologia , Cadeias alfa de Integrinas/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Selectina L/genética , Selectina L/imunologia , Selectina L/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Pneumonia/genética , Pneumonia/metabolismo , Testes de Função Respiratória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/metabolismo
13.
Pulm Pharmacol Ther ; 24(2): 203-14, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21195789

RESUMO

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-1ß, 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.


Assuntos
Inflamação/etiologia , Interleucina-10/genética , Receptores Toll-Like/agonistas , Resistência das Vias Respiratórias/imunologia , Animais , Asma/tratamento farmacológico , Asma/imunologia , Relação Dose-Resposta a Droga , Eosinofilia/imunologia , Feminino , Inflamação/imunologia , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Ovalbumina/imunologia , Fatores de Tempo , Receptor 7 Toll-Like/agonistas
14.
Reprod Toxicol ; 30(3): 422-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20493942

RESUMO

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.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Pulmão/efeitos dos fármacos , Preparações Farmacêuticas , Administração por Inalação , Envelhecimento/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Masculino , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Pletismografia Total , Ratos , Ratos Wistar , Verapamil/administração & dosagem , Verapamil/efeitos adversos , Verapamil/sangue
15.
Pharmacology ; 83(3): 188-95, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19176983

RESUMO

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.


Assuntos
Aminopiridinas/farmacologia , Antígenos de Fungos/imunologia , Aspergillus fumigatus/imunologia , Benzamidas/farmacologia , Inibidores da Fosfodiesterase 4 , Mecânica Respiratória/efeitos dos fármacos , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Asma/fisiopatologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Líquido da Lavagem Broncoalveolar/citologia , Ciclopropanos/farmacologia , Modelos Animais de Doenças , Eosinófilos/citologia , Feminino , Contagem de Leucócitos , Linfócitos/citologia , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/citologia , Pneumonia/metabolismo , Hipersensibilidade Respiratória/tratamento farmacológico
16.
Respir Res ; 9: 54, 2008 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-18638383

RESUMO

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.


Assuntos
Catepsinas/metabolismo , Colágeno/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Antibióticos Antineoplásicos , Bleomicina , Catepsina K , Catepsinas/genética , Modelos Animais de Doenças , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
17.
J Pharmacol Toxicol Methods ; 55(1): 16-26, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16793289

RESUMO

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.


Assuntos
Testes de Função Respiratória/métodos , Resistência das Vias Respiratórias , Animais , Asma/fisiopatologia , Modelos Animais de Doenças , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Intubação Intratraqueal , Complacência Pulmonar , Camundongos , Pletismografia/instrumentação , Pletismografia/métodos , Pletismografia Total/métodos , Ratos , Resultado do Tratamento
18.
Exp Toxicol Pathol ; 57 Suppl 2: 5-11, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16618537

RESUMO

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.


Assuntos
Asma/fisiopatologia , Modelos Animais de Doenças , Pulmão/fisiopatologia , Testes de Função Respiratória/métodos , Animais , Asma/diagnóstico , Hiper-Reatividade Brônquica/fisiopatologia , Camundongos , Ratos
19.
J Clin Invest ; 116(4): 1025-36, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16528411

RESUMO

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.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos CD/fisiologia , Asma/terapia , Receptores de Fator de Crescimento Neural/imunologia , Receptores de Fator de Crescimento Neural/fisiologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Especificidade de Anticorpos , Antígenos CD/metabolismo , Asma/imunologia , Asma/metabolismo , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Anergia Clonal , Colágeno/imunologia , Colágeno/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Imunoglobulina E/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral
20.
J Immunol ; 174(9): 5507-15, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15843549

RESUMO

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.


Assuntos
Alérgenos/imunologia , Brônquios/patologia , Epitopos de Linfócito T/imunologia , Interleucina-15/antagonistas & inibidores , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Animais , Brônquios/imunologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/prevenção & controle , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Inibidores do Crescimento/fisiologia , Imunoglobulina E/biossíntese , Memória Imunológica/imunologia , Imunossupressores/farmacologia , Inflamação/imunologia , Inflamação/prevenção & controle , Interleucina-15/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/antagonistas & inibidores , Ovalbumina/imunologia , Subunidades Proteicas/fisiologia , Receptores de Interleucina-15 , Receptores de Interleucina-2/fisiologia , Hipersensibilidade Respiratória/patologia , Solubilidade , Células Th2/citologia , Células Th2/imunologia
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