RESUMO
Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.
Assuntos
Fibroblastos/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Fosfatidilinositóis/metabolismo , Fibrose Pulmonar/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Proliferação de Células , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada/genética , Fosfatidilinositóis/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/induzido quimicamenteRESUMO
Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 µM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.
Assuntos
Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Farnesiltranstransferase/antagonistas & inibidores , Inflamação/tratamento farmacológico , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Proteínas ras/metabolismo , Animais , Asma/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Hiper-Reatividade Brônquica/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Eosinófilos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Farnesiltranstransferase/metabolismo , Humanos , Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Positive-pressure mechanical ventilation is an essential therapeutic intervention, yet it causes the clinical syndrome known as ventilator-induced lung injury. Various lung protective mechanical ventilation strategies have attempted to reduce or prevent ventilator-induced lung injury but few modalities have proven effective. A model that isolates the contribution of mechanical ventilation on the development of acute lung injury is needed to better understand biologic mechanisms that lead to ventilator-induced lung injury. OBJECTIVES: To evaluate the effects of positive end-expiratory pressure and recruitment maneuvers in reducing lung injury in a ventilator-induced lung injury murine model in short- and longer-term ventilation. METHODS: 5-12 week-old female BALB/c mice (n = 85) were anesthetized, placed on mechanical ventilation for either 2 hrs or 4 hrs with either low tidal volume (8 ml/kg) or high tidal volume (15 ml/kg) with or without positive end-expiratory pressure and recruitment maneuvers. RESULTS: Alteration of the alveolar-capillary barrier was noted at 2 hrs of high tidal volume ventilation. Standardized histology scores, influx of bronchoalveolar lavage albumin, proinflammatory cytokines, and absolute neutrophils were significantly higher in the high-tidal volume ventilation group at 4 hours of ventilation. Application of positive end-expiratory pressure resulted in significantly decreased standardized histology scores and bronchoalveolar absolute neutrophil counts at low- and high-tidal volume ventilation, respectively. Recruitment maneuvers were essential to maintain pulmonary compliance at both 2 and 4 hrs of ventilation. CONCLUSIONS: Signs of ventilator-induced lung injury are evident soon after high tidal volume ventilation (as early as 2 hours) and lung injury worsens with longer-term ventilation (4 hrs). Application of positive end-expiratory pressure and recruitment maneuvers are protective against worsening VILI across all time points. Dynamic compliance can be used guide the frequency of recruitment maneuvers to help ameloriate ventilator-induced lung injury.
Assuntos
Modelos Animais de Doenças , Respiração com Pressão Positiva/métodos , Respiração Artificial/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Anesthetic protocols for murine models are varied within the literature and medetomidine has been implicated in the development of urethral plugs in male mice. Our objective was to evaluate the combination of butorphanol, dexmedetomidine, and tiletamine-zolazepam. A secondary objective was to identify which class of agent was associated with urethral obstructions in male mice. BALB/c male (n = 13) and female (n = 23) mice were assigned to dexmedetomidine and tiletamine-zolazepam with or without butorphanol or to single agent dexmedetomidine or tiletamine-zolazepam. Anesthesia was achieved in 58% (14/24) of mice without butorphanol and in 100% (24/24) of mice with butorphanol. The combination of dexmedetomidine (0.2 mg/kg), tiletamine-zolazepam (40 mg/kg), and butorphanol (3 mg/kg) resulted in an induction and anesthetic duration of 12 and 143 minutes, respectively. Urethral obstructions occurred in 66% (25/38) of trials in male mice that received dexmedetomidine with a mortality rate of 38% (5/13). Tiletamine-zolazepam, when used alone, resulted in a 0% (0/21) incidence of urethral obstructions. Combination use of dexmedetomidine, tiletamine-zolazepam, and butorphanol results in a longer and more reliable duration of anesthesia than the use of dexmedetomidine and tiletamine-zolazepam alone. Dexmedetomidine is not recommended for use in nonterminal procedures in male mice due to the high incidence of urethral obstructions and resultant high mortality rate.
RESUMO
Proposition 2, which requires that egg-laying hens be confined only in ways that allow these animals to lie down, stand up, fully extend their limbs and turn around freely, was passed by the voters of California in 2008. These new housing requirements were introduced in the USA and European Union without considering the potential impact of changes in layer hen housing on the health of poultry workers in the new facilities. Particles were collected from ambient air inside a large layer hen complex featuring separate barns with conventional battery caging, enriched caging, or 'free range' (aviary) housing during winter, spring, and summer seasons over one year. Toxicity of the particles was evaluated by analysis of inflammatory cell influx into lung lavage fluid after intratracheal instillation into mice. Capacity of the particles to elicit oxidative stress was evaluated using a macrophage cell line engineered with a reporter gene sensitive to nuclear factor κB activation. We observed similar pro-inflammatory and pro-oxidant effects of the particles collected from different types of barns and over different seasons, suggesting that standard industrial hygiene techniques for evaluating respirable particles in ambient air can adequately monitor worker risk. Based on particle concentrations found in ambient air in the barns, we can rank the facilities for worker exposure to particles as conventional caging (now banned) approximately equal to enriched caging (permitted under Proposition 2). Aviary housing is associated with increased exposure of workers to particulate matter and, therefore, to greater risk of allergic reactions and/or decreased respiratory function.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Galinhas , Abrigo para Animais , Pulmão/efeitos dos fármacos , Material Particulado/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Criação de Animais Domésticos , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , California , Linhagem Celular , Endotoxinas/química , Endotoxinas/toxicidade , Abrigo para Animais/legislação & jurisprudência , Abrigo para Animais/normas , Humanos , Exposição por Inalação/efeitos adversos , Pulmão/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Camundongos Endogâmicos BALB C , NF-kappa B/agonistas , NF-kappa B/metabolismo , Exposição Ocupacional/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Mucosa Respiratória/imunologia , Estações do Ano , Testes de Toxicidade Aguda , Recursos HumanosRESUMO
Systemic treatment with statins mitigates allergic airway inflammation, TH2 cytokine production, epithelial mucus production, and airway hyperreactivity (AHR) in murine models of asthma. We hypothesized that pravastatin delivered intratracheally would be quantifiable in lung tissues using mass spectrometry, achieve high drug concentrations in the lung with minimal systemic absorption, and mitigate airway inflammation and structural changes induced by ovalbumin. Male BALB/c mice were sensitized to ovalbumin (OVA) over 4 weeks, then exposed to 1% OVA aerosol or filtered air (FA) over 2 weeks. Mice received intratracheal instillations of pravastatin before and after each OVA exposure (30 mg/kg). Ultra performance liquid chromatography - mass spectrometry was used to quantify plasma, lung, and bronchoalveolar lavage fluid (BALF) pravastatin concentration. Pravastatin was quantifiable in mouse plasma, lung tissue, and BALF (BALF > lung > plasma for OVA and FA groups). At these concentrations pravastatin inhibited airway goblet cell hyperplasia/metaplasia, and reduced BALF levels of cytokines TNFα and KC, but did not reduce BALF total leukocyte or eosinophil cell counts. While pravastatin did not mitigate AHR, it did inhibit airway hypersensitivity (AHS). In this proof-of-principle study, using novel mass spectrometry methods we show that pravastatin is quantifiable in tissues, achieves high levels in mouse lungs with minimal systemic absorption, and mitigates some pathological features of allergic asthma. Inhaled pravastatin may be beneficial for the treatment of asthma by having direct airway effects independent of a potent anti-inflammatory effect. Statins with greater lipophilicity may achieve better anti-inflammatory effects warranting further research.
RESUMO
OBJECTIVES: We hypothesised that severe asthmatics taking a statin drug, in addition to inhaled corticosteroids/long-acting ß-agonist inhaler therapy, would have better asthma symptom control and improved lung function compared to their controls. STUDY DESIGN: A retrospective, cross-sectional study of 165 patients with severe asthma seen from 2001-2008. Hierarchical linear and logistic regression models were used for modelling fitting. SETTING: University of California, Davis Medical Center (Sacramento, California, USA). Academic, single-centre, severe asthma subspecialty clinic. PARTICIPANTS: 612 screened, 223 eligible and 165 adult patients were included in the final study (N=165; 31 statin users and 134 non-users). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was asthma control as measured by the Asthma Control Test (ACT). The secondary endpoints included lung function, symptoms and the need for corticosteroid burst and peripheral eosinophil count. RESULTS: At baseline, statin users compared to non-users were older, had lower lung function (FEV1% predicted, FEV1, forced vital capacity and FEF25-75%) and had a higher prevalence of comorbid conditions. Statin use was associated with more aspirin and ipratropium inhaler use than in non-users. Patients in both groups were obese (body mass index ≥ 30). Statin users had better asthma symptom control compared to non-users (higher adjusted mean ACT score by 2.2±0.94 points, p<0.02). Median statin use was for 1 year. There were no statistically significant differences in lung function, corticosteroid or rescue bronchodilator use or peripheral eosinophilia between the two groups. CONCLUSIONS: In our severe asthma referral population, statin users already taking inhaled controller therapy achieved better asthma control compared to non-users. The implications of this study is that patients with severe asthma could potentially benefit from added statin treatment. Because our study population was on average obese, the obese severe asthmatic may be a viable asthma subphenotype for further studies. Prospective randomised clinical trials evaluating the safety and efficacy of statins in severe asthma are warranted.
RESUMO
Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.
Assuntos
Anticorpos Monoclonais , Antígenos de Helmintos/análise , Doenças do Cão/diagnóstico , Equinococose/veterinária , Fezes/parasitologia , Parasitologia/métodos , Medicina Veterinária/métodos , Animais , Doenças do Cão/parasitologia , Cães , Equinococose/diagnóstico , Equinococose/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/química , Feminino , Masculino , Organização Pan-Americana da Saúde , Peru , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In recent years, the international demand for commodities has prompted enormous growth in agriculture in most South American countries. Due to intensive use of fertilizers, cyanobacterial blooms have become a recurrent phenomenon throughout the continent, but their potential health risk remains largely unknown due to the lack of analytical capacity. In this paper we report the main results and conclusions of more than five years of systematic monitoring of cyanobacterial blooms in 20 beaches of Montevideo, Uruguay, on the Rio de la Plata, the fifth largest basin in the world. A locally developed microcystin ELISA was used to establish a sustainable monitoring program that revealed seasonal peaks of extremely high toxicity, more than one-thousand-fold greater than the WHO limit for recreational water. Comparison with cyanobacterial cell counts and chlorophyll-a determination, two commonly used parameters for indirect estimation of toxicity, showed that such indicators can be highly misleading. On the other hand, the accumulated experience led to the definition of a simple criterion for visual classification of blooms, that can be used by trained lifeguards and technicians to take rapid on-site decisions on beach management. The simple and low cost approach is broadly applicable to risk assessment and risk management in developing countries.
Assuntos
Toxinas Bacterianas/análise , Cianobactérias , Monitoramento Ambiental/métodos , Proliferação Nociva de Algas , Toxinas Marinhas/análise , Microcistinas/análise , Rios/microbiologia , Microbiologia da Água , Clorofila/análise , Clorofila A , Toxinas de Cianobactérias , Países em Desenvolvimento , Monitoramento Ambiental/economia , Ensaio de Imunoadsorção Enzimática , Gestão de Riscos , UruguaiRESUMO
Our previous work has shown that coarse particulate matter (PM(10-2.5)) from wildfire smoke is more toxic to lung macrophages on an equal dose (by mass) basis than coarse PM isolated from normal ambient air, as evidenced by decreased numbers of macrophages in lung lavage fluid 6 and 24hours after PM instillation into mouse lungs in vivo and by cytotoxicity to a macrophage cell line observed directly in vitro. We hypothesized that pulmonary macrophages from mice instilled with wildfire coarse PM would undergo more cytotoxicity than macrophages from controls, and that there would be an increase in oxidative stress in their lungs. Cytotoxicity was quantified as decreased viable macrophages and increased percentages of dead macrophages in the bronchoalveolar lavage fluid (BALF) of mice instilled with wildfire coarse PM. At 1hour after PM instillation, we observed both decreased numbers of viable macrophages and increased dead macrophage percentages as compared to controls. An increase in free isoprostanes, an indicator of oxidative stress, from control values of 28.1±3.2pg/mL to 83.9±12.2pg/mL was observed a half-hour after PM instillation. By 1hour after PM instillation, isoprostane values had returned to 30.4±7.6pg/mL, not significantly different from control concentrations. Lung sections from mice instilled with wildfire coarse PM showed rapid Clara cell responses, with decreased intracellular staining for the Clara cell secretory protein CCSP 1hour after wildfire PM instillation. In conclusion, very rapid cytotoxicity occurs in pulmonary macrophages and oxidative stress responses are seen 0.5-1hour after wildfire coarse PM instillation. These results define early cellular and biochemical events occurring in vivo and support the hypothesis that oxidative stress-mediated macrophage toxicity plays a key role in the initial response of the mouse lung to wildfire PM exposure.
Assuntos
Poluentes Atmosféricos/toxicidade , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Estresse Oxidativo/fisiologia , Material Particulado/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/citologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Material Particulado/administração & dosagemRESUMO
BACKGROUND: The Fogarty International Center (FIC) of the United States National Institutes of Health includes the International Training and Research in Environmental and Occupational Health (ITREOH) Program. The "International Training Program in Environmental Toxicology and Public Health" Center, funded in 2002 is based at the University of California, Davis, and is part of the ITREOH group of Centers. It has major efforts focused at the public universities in Montevideo, Uruguay, and Salta, Argentina. RESULTS: Training and research efforts in Salta begun in 2005 in the College of Engineering. A donated used real-time PCR machine was the starting point and the initial FIC support was instrumental to face other problems including physical space, research projects and grants, trainees, training, networking, and distractions/opportunities in order to develop local capacities in Environmental Engineering using modern methodology. After 6 years of successful work, the Salta center has become a reference Center in the field, and is still growing and consolidating. CONCLUSIONS: This program has had a significant impact locally and regionally. The model used in Argentina could be easily adapted to other fields or types of projects in Argentina and in other developing countries.
Assuntos
Fortalecimento Institucional , Engenharia/educação , Saúde Ambiental/educação , Universidades , Argentina , Engenharia/organização & administração , Saúde Ambiental/organização & administração , Financiamento Governamental , Humanos , Cooperação Internacional , National Institutes of Health (U.S.) , Avaliação de Programas e Projetos de Saúde , Saúde Pública , Estados UnidosRESUMO
The mechanistic basis of the high toxicity to lung macrophages of coarse PM from the California wildfires of 2008 was examined in cell culture experiments with mouse macrophages. Wildfire PM directly killed macrophages very rapidly in cell culture at relatively low doses. The wildfire coarse PM is about four times more toxic to macrophages on an equal weight basis than the same sized PM collected from normal ambient air (no wildfires) from the same region and season. There was a good correlation between the extent of cytotoxicity and the amount of oxidative stress observed at a given dose of wildfire PM in vitro. Our data implicate NF-κB signaling in the response of macrophages to wildfire PM, and suggest that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. The relative ratio of toxicity and of expression of biomarkers of oxidant stress between wildfire PM and "normal" PM collected from ambient air is consistent with our previous results in mice in vivo, also suggesting that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. Our findings from this and earlier studies suggest that the active components of coarse PM from the wildfire are heat-labile organic compounds. While we cannot rule out a minor role for endotoxin in coarse PM preparations from the collected wildfire PM in our observed results both in vitro and in vivo, based on experiments using the inhibitor Polymyxin B most of the oxidant stress and pro-inflammatory activity observed was not due to endotoxin.
Assuntos
Poluentes Atmosféricos/toxicidade , Incêndios , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Material Particulado/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação de Macrófagos/fisiologia , Macrófagos Alveolares/metabolismo , Camundongos , Tamanho da PartículaRESUMO
Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.
Assuntos
Anticorpos/isolamento & purificação , Haptenos/imunologia , Anticorpos de Cadeia Única/química , Animais , Anticorpos/imunologia , Sítios de Ligação , Camelídeos Americanos/imunologia , Carbanilidas/imunologia , Masculino , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Exhaled breath nitric oxide (NO) is an accepted asthma biomarker. Lung concentrations of NO and its amino acid precursor, L-arginine, are regulated by the relative expressions of the NO synthase (NOS) and arginase isoforms. Increased expression of arginase I and NOS2 occurs in murine models of allergic asthma and in biopsies of asthmatic airways. Although clinical trials involving the inhibition of NO-producing enzymes have shown mixed results, small molecule arginase inhibitors have shown potential as a therapeutic intervention in animal and cell culture models. Their transition to clinical trials is hampered by concerns regarding their safety and potential toxicity. In this review, we discuss the paradigm of arginase and NOS competition for their substrate L-arginine in the asthmatic airway. We address the functional role of L-arginine in inflammation and the potential role of arginase inhibitors as therapeutics.
RESUMO
Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) pathway. These drugs have been associated with improved respiratory health, and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin (OVA)-exposed mice would attenuate early features of airway remodeling by a mevalonate-dependent mechanism. BALB/c mice initially were sensitized to OVA and then exposed to 1% OVA aerosol for 2 weeks after sensitization for 6 exposures. Simvastatin (40 mg/kg) or simvastatin plus MA (20 mg/kg) were injected intraperitoneally before each OVA exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but it did not alter airway hydroxyproline content or transforming growth factor-ß1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, which are indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Additional studies using subchronic or chronic allergen exposure models are needed to extend these initial findings.
Assuntos
Asma/imunologia , Células Caliciformes/patologia , Pulmão/metabolismo , Sinvastatina/uso terapêutico , Aerossóis , Animais , Arginase/antagonistas & inibidores , Arginina/metabolismo , Asma/tratamento farmacológico , Asma/enzimologia , Asma/patologia , Western Blotting , Corantes , Modelos Animais de Doenças , Células Caliciformes/efeitos dos fármacos , Hidroxiprolina/metabolismo , Hiperplasia/prevenção & controle , Imuno-Histoquímica , Inflamação/patologia , Inflamação/fisiopatologia , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Nitratos/metabolismo , Nitritos/metabolismo , Ovalbumina/administração & dosagem , Ovalbumina/farmacologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVES AND DESIGN: The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. MATERIALS OR SUBJECTS: Mice from a C57BL/6 wild-type, NOS1(-/-), NOS2(-/-), and NOS3(-/-) genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. METHODS: We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. RESULTS: Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3(-/-) strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1(-/-) animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2(-/-), and NOS3(-/-) allergen-exposed mice. CONCLUSION: The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This "homeostatic" mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo I/genética , Ovalbumina/química , Animais , Deleção de Genes , Células Caliciformes/citologia , Inflamação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Isoformas de ProteínasRESUMO
The authors have previously demonstrated that wildfire-derived coarse or fine particulate matter (PM) intratracheally instilled into lungs of mice induce a strong inflammatory response. In the current study, the authors demonstrate that wildfire PM simultaneously cause major increases in oxidative stress in the mouse lungs as measured by decreased antioxidant content of the lung lavage supernatant fluid 6 and 24 h after PM administration. Concentrations of neutrophil chemokines/cytokines and of tumor necrosis factor (TNF)-alpha were elevated in the lung lavage fluid obtained 6 and 24 h after PM instillation, consistent with the strong neutrophilic inflammatory response observed in the lungs 24 h after PM administration, suggesting a relationship between the proinflammatory activity of the PM and the measured level of antioxidant capacity in the lung lavage fluid. Chemical analysis shows relatively low levels of polycyclic aromatic hydrocarbons compared to published results from typical urban PM. Coarse PM fraction is more active (proinflammatory activity and oxidative stress) on an equal-dose basis than the fine PM despite its lower content of polycyclic aromatic hydrocarbons. There does not seem to be any correlation between the content of any specific polycyclic aromatic hydrocarbon (or of total polycyclic aromatic hydrocarbon content) in the PM fraction and its toxicity. However, the concentrations of the oxidation products of phenanthrene and anthracene, phenanthraquinone and anthraquinone, were several-fold higher in the coarse PM than the fine fraction, suggesting a significant role for atmospheric photochemistry in the formation of secondary pollutants in the wildfire PM and the possibility that such secondary pollutants could be significant sources of toxicity in the wildfire PM.
Assuntos
Antioxidantes/metabolismo , Citocinas/metabolismo , Incêndios , Pulmão/metabolismo , Tamanho da Partícula , Material Particulado/efeitos adversos , Poluentes Atmosféricos/efeitos adversos , Animais , Antioxidantes/análise , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , California , Citocinas/biossíntese , Pulmão/química , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Material Particulado/administração & dosagem , Hidrocarbonetos Policíclicos Aromáticos/administração & dosagem , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Meio SelvagemRESUMO
The environmental impact of rice agriculture is poorly studied in developing countries, mainly due to limitations of the analytical capacity. Here, we report the development of a clomazone enzyme-linked immunosorbent assay as a fast and cost-effective tool to monitor the dissipation of this herbicide along the harvest. Antibodies were prepared using different strategies of hapten conjugation, and the best hapten/antibody pair was selected. It proved to be a reliable tool to measure the herbicide in the 2.0-20 ng/mL range in field samples, with excellent correlation with high-performance liquid chromatography results. The assay was used to study the dissipation of the herbicide in the floodwater of experimental rice paddies in Uruguay. Large differences in the residual amounts of herbicide were observed depending on the flooding practices. Because of its robustness and simplicity, the assay may be useful to delineate and monitor management practices that can contribute to minimizing the release of the herbicide in the environment.
Assuntos
Monitoramento Ambiental/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Herbicidas/análise , Isoxazóis/análise , Oryza/efeitos dos fármacos , Oxazolidinonas/análise , Agricultura , Oryza/químicaRESUMO
Arginase1 and nitric oxide synthase2 (NOS2) utilize l-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA) significantly increased total l-arginine content in the airway compartment. We hypothesized that such an increase in l-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that l-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2, which is a major source of NO production in the inflamed airway, although arginase inhibition may also be affecting the turnover of arginine by the other NOS isoforms, NOS1 and NOS3. The increased l-arginine content in the airway compartment of mice treated with nor-NOHA may directly or indirectly, through NOS2, control arginase expression both in response to OVA exposure and at a basal level.
Assuntos
Arginase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/fisiologia , Ovalbumina/imunologia , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/genética , Aerossóis , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Arginase/biossíntese , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Pulmão/patologia , Complacência Pulmonar/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Ovalbumina/administração & dosagem , Pneumonia/patologiaRESUMO
BACKGROUND: During the last week of June 2008, central and northern California experienced thousands of forest and brush fires, giving rise to a week of severe fire-related particulate air pollution throughout the region. California experienced PM(10-2.5) (particulate matter with mass median aerodynamic diameter > 2.5 mum to < 10 mum; coarse ) and PM(2.5) (particulate matter with mass median aerodynamic diameter < 2.5 mum; fine) concentrations greatly in excess of the air quality standards and among the highest values reported at these stations since data have been collected. OBJECTIVES: These observations prompt a number of questions about the health impact of exposure to elevated levels of PM(10-2.5) and PM(2.5) and about the specific toxicity of PM arising from wildfires in this region. METHODS: Toxicity of PM(10-2.5) and PM(2.5) obtained during the time of peak concentrations of smoke in the air was determined with a mouse bioassay and compared with PM samples collected under normal conditions from the region during the month of June 2007. RESULTS: Concentrations of PM were not only higher during the wildfire episodes, but the PM was much more toxic to the lung on an equal weight basis than was PM collected from normal ambient air in the region. Toxicity was manifested as increased neutrophils and protein in lung lavage and by histologic indicators of increased cell influx and edema in the lung. CONCLUSIONS: We conclude that the wildfire PM contains chemical components toxic to the lung, especially to alveolar macrophages, and they are more toxic to the lung than equal doses of PM collected from ambient air from the same region during a comparable season.