Animal models and mechanisms of tobacco smoke-induced chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, and its global health burden is increasing. COPD is characterized by emphysema, mucus hypersecretion, and persistent lung inflammation, and clinically by chronic airflow obstruction and symptoms of dyspnea, cough, and fatigue in patients. A cluster of pathologies including chronic bronchitis, emphysema, asthma, and cardiovascular disease in the form of hypertension and atherosclerosis variably coexist in COPD patients. Underlying causes for COPD include primarily tobacco use but may also be driven by exposure to air pollutants, biomass burning, and workplace related fumes and chemicals. While no single animal model might mimic all features of human COPD, a wide variety of published models have collectively helped to improve our understanding of disease processes involved in the genesis and persistence of COPD. In this review, the pathogenesis and associated risk factors of COPD are examined in different mammalian models of the disease. Each animal model included in this review is exclusively created by tobacco smoke (TS) exposure. As animal models continue to aid in defining the pathobiological mechanisms of and possible novel therapeutic interventions for COPD, the advantages and disadvantages of each animal model are discussed.

Ozone-induced enhancement of airway hyperreactivity in rhesus macaques: Effects of antioxidant treatment.

BACKGROUND: Ozone (O3) inhalation elicits airway inflammation and impairs treatment responsiveness in asthmatic patients. The underlying immune mechanisms have been difficult to study because of the lack of relevant experimental models. Rhesus macaques spontaneously have asthma and have a similar immune system to human subjects. OBJECTIVES: We sought to investigate mucosal immune changes after O3 inhalation in a clinically relevant nonhuman primate asthma model and to study the effects of an antioxidant synthetic lignan (synthetic secoisolariciresinol diglucoside [LGM2605]). METHODS: A cohort of macaques (n = 17) previously characterized with airway hyperreactivity (AHR) to methacholine was assessed (day 1). Macaques were treated (orally) with LGM2605 (25 mg/kg) or placebo twice per day for 7 days, exposed to 0.3 ppm O3 or air for 6 hours (on day 7), and studied 12 hours later (day 8). Lung function, blood and bronchoalveolar lavage (BAL) fluid immune cell profile, and bronchial brushing and blood cell mRNA expression were assessed. RESULTS: O3 induced significant BAL fluid neutrophilia and eosinophilia and increased AHR and expression of IL6 and IL25 mRNA in the airway epithelium together with increased BAL fluid group 2 innate lymphoid cell (ILC2s), CD1c+ myeloid dendritic cell, and CD4+ T-cell counts and diminished surfactant protein D expression. Although LGM2605 attenuated some of the immune and inflammatory changes, it completely abolished O3-induced AHR. CONCLUSION: ILC2s, CD1c+ myeloid dendritic cells, and CD4+ T cells are selectively involved in O3-induced asthma exacerbation. The inflammatory changes were partially prevented by antioxidant pretreatment with LGM2605, which had an unexpectedly disproportionate protective effect on AHR.

Nonhuman Primate Models of Respiratory Disease: Past, Present, and Future.

The respiratory system consists of an integrated network of organs and structures that primarily function for gas exchange. In mammals, oxygen and carbon dioxide are transmitted through a complex respiratory tract, consisting of the nasal passages, pharynx, larynx, and lung. Exposure to ambient air throughout the lifespan imposes vulnerability of the respiratory system to environmental challenges that can contribute toward development of disease. The importance of the respiratory system to human health is supported by statistics from the Centers for Disease Control and Prevention; in 2015, chronic lower respiratory diseases were the third leading cause of death in the United States. In light of the significant mortality associated with respiratory conditions that afflict all ages of the human population, this review will focus on basic and preclinical research conducted in nonhuman primate models of respiratory disease. In comparison with other laboratory animals, the nonhuman primate lung most closely resembles the human lung in structure, physiology, and mucosal immune mechanisms. Studies defining the influence of inhaled microbes, pollutants, or allergens on the nonhuman primate lung have provided insight on disease pathogenesis, with the potential for elucidation of molecular targets leading to new treatment modalities. Vaccine trials in nonhuman primates have been crucial for confirmation of safety and protective efficacy against infectious diseases of the lung in a laboratory animal model that recapitulates pathology observed in humans. In looking to the future, nonhuman primate models of respiratory diseases will continue to be instrumental for translating biomedical research for improvement of human health.

Aß vaccination in combination with behavioral enrichment in aged beagles: effects on cognition, Aß, and microhemorrhages.

Beta-amyloid (Aß) immunotherapy is a promising intervention to slow Alzheimer's disease. Aging dogs naturally accumulate Aß and show cognitive decline. An active vaccine against fibrillar Aß 1-42 (VAC) in aged beagles resulted in maintenance but not improvement of cognition along with reduced brain Aß. Behavioral enrichment (ENR) led to cognitive benefits but no reduction in Aß. We hypothesized cognitive outcomes could be improved by combining VAC with ENR in aged dogs. Aged dogs (11-12 years) were placed into 4 groups: (1) control/control (C/C); (2) control/VAC (C/V); (3) ENR/control (E/C); and (4) ENR/VAC (E/V) and treated for 20 months. VAC decreased brain Aß, pyroglutamate Aß, increased cerebrospinal fluid Aß 42 and brain-derived neurotrophic factor RNA levels but also increased microhemorrhages. ENR reduced brain Aß and prevented microhemorrhages. The combination treatment resulted in a significant maintenance of learning over time, reduced Aß, and increased brain-derived neurotrophic factor mRNA despite increased microhemorrhages; however, there were no benefits to memory. These results suggest that the combination of immunotherapy with behavioral enrichment leads to cognitive maintenance associated with reduced neuropathology that may benefit people with Alzheimer's disease.

VTX-1463, a novel TLR-8 agonist, attenuates nasal congestion after ragweed challenge in sensitized beagle dogs.

VTX-1463 is a selective toll-like receptor (TLR) 8 agonist that activates a subset of innate immune cells to produce a unique cytokine profile. Delivery of VTX-1463 via nasal spray may modulate the nasal response in allergic rhinitis. The aim of this study was to determine the effects of VTX-1463 on the nasal response in a dog model of allergic rhinitis. Ragweed (RW)-sensitized dogs were pretreated with increasing doses of VTX-1463 1 day prior to RW challenge or with two doses (4 or 8 days and 1 day prior to RW). Changes in nasal cavity volume (NV) were determined by acoustic rhinometry and nasal lavage fluid was assessed for histamine, lipid mediators, and cellular infiltrates at sequential times following RW challenge. VTX-1463 pretreatment significantly preserved NV during the acute response to RW challenge for all doses tested. The area under the curve (AUC) for NV over the 1.5 h assessment period in RW challenged vehicle controls averaged 51.5% (SEM: ±2.12%) of the baseline NV over all studies. A single 100 µg dose of VTX-1463 given 1 day prior to RW yielded an AUC for NV of 69.3% (±6.59%) of baseline, while a 1000 µg dose administered twice (8 days and 1 day prior to RW) resulted in an AUC for NV of 85.4% (±4.74%, P < 0.05) of baseline. For a single 1000 µg VTX-1463 dose 1 day prior to RW, multiple mediators produced by mast cells, including histamine, PGE2, PGD2, and cysteinyl LTs, were significantly reduced relative to the vehicle control. The selective TLR8 agonist, VTX-1463, preserved NV in a dose-dependent manner in the acute phase of a nasal allergic response. The therapeutic effect appears to result from attenuated mast cell mediator release. Modulating the local cytokine response via TLR8 agonism appears to have a therapeutic effect on the acute allergic nasal response.

The in vivo efficacy and side effect pharmacology of GS-5759, a novel bifunctional phosphodiesterase 4 inhibitor and long-acting ß 2-adrenoceptor agonist in preclinical animal species.

Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, with inhaled ß 2-adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)-6-[[3-[[4-[5-[[2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino]pent-1-ynyl]phenyl]carbamoyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), a novel bifunctional compound with both phosphodiesterase 4 (PDE4) inhibitor and long-acting ß 2-adrenoceptor agonist (LABA) activity, which has been optimized for inhalation delivery. GS-5759 dose-dependently inhibited pulmonary neutrophilia in a lipopolysaccharide (LPS) aerosol challenge model of inflammation in rats with an ED50 ≤ 10 µg/kg. GS-5759 was also a potent bronchodilator with an ED50 of 0.09 µg/kg in guinea pigs and 3.4 µg/kg in dogs after methylcholine (MCh) and ragweed challenges respectively. In cynomolgus monkeys, GS-5759 was dosed as a fine-particle dry powder and was efficacious in the same dose range in both MCh and LPS challenge models, with an ED50 = 70 µg/kg for bronchodilation and ED50 = 4.9 µg/kg for inhibition of LPS-induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS-5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED50 observed in the rat LPS inflammation model. GS-5759 is a bifunctional molecule developed for the treatment of COPD, which has both bronchodilator and anti-inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

The neonatal susceptibility window for inhalant allergen sensitization in the atopically predisposed canine asthma model.

Allergic asthma often begins in early life and, although many risk factors have been enumerated, the specific factors that initiate disease progression in an individual remain unclear. Using our dog model of early life allergen inhalation, we tested the hypothesis that the atopically biased neonatal immune system would exhibit tolerance to ragweed if allowed to mature normally before exposure or artificially through innate immune stimulation with early life exposure. Dogs were subjected to a series of inhalational ragweed exposures from 1 to 20 weeks old, with or without inhalation of a Toll-like receptor 4 (TLR4) agonist (CRX-527), or from 13 to 31 weeks old. Serum allergen-specific antibody response was assessed at 4, 8 and 20 weeks after the last sensitizing exposure. At 24 or 35 weeks old, airway hyper-responsiveness to methacholine and ragweed challenges and pulmonary inflammation by bronchoalveolar lavage were tested 1 and 4 days after ragweed challenge at 28 or 39 weeks old. Allergen-free immune maturation resulted in no airway hyper-responsiveness and very little ragweed-specific IgE relative to the control group, but eosinophilia developed upon ragweed challenge. TLR4 agonism yielded no airway hyper-responsiveness, but a strong airway neutrophilia developed upon ragweed challenge. Our data indicate that an atopic predisposition creates a critical window in which allergen exposure can lead to an asthmatic phenotype. Allergen-free immune maturation may lead to allergen tolerance. TLR4 agonism before early life allergen exposure may abrogate the development of allergen-specific bronchonconstriction, but allergen-specific pulmonary inflammation remains a strong concern.

Evaluation of inhaled carbon monoxide as an anti-inflammatory therapy in a nonhuman primate model of lung inflammation.

Carbon monoxide (CO) confers anti-inflammatory protection in rodent models of lung injury when applied at low concentration. Translation of these findings to clinical therapies for pulmonary inflammation requires validation in higher mammals. We have evaluated the efficacy of inhaled CO in reducing LPS-induced lung inflammation in cynomolgus macaques. LPS inhalation resulted in profound neutrophil influx and moderate increases in airway lymphocytes, which returned to baseline levels within 2 wk following exposure. CO exposure (500 ppm, 6 h) following LPS inhalation decreased TNF-α release in bronchoalveolar lavage fluid but did not affect IL-6 or IL-8 release. Lower concentrations of CO (250 ppm, 6 h) did not reduce pulmonary neutrophilia. Pretreatment with budesonide, a currently used inhaled corticosteroid, decreased LPS-induced expression of TNF-α, IL-6, and IL-8, and reduced LPS-induced neutrophilia by ∼84%. In comparison, CO inhalation (500 ppm, for 6 h after LPS exposure) reduced neutrophilia by ∼67%. Thus, inhaled CO was nearly as efficacious as pretreatment with an inhaled corticosteroid at reducing airway neutrophil influx in cynomolgus macaques. However, the therapeutic efficacy of CO required relatively high doses (500 ppm) that resulted in high carboxyhemoglobin (COHb) levels (>30%). Lower CO concentrations (250 ppm), associated with anti-inflammatory protection in rodents, were ineffective in cynomolgus macaques and also yielded relatively high COHb levels. These studies highlight the complexity of interspecies variation of dose-response relationships of CO to COHb levels and to the anti-inflammatory functions of CO. The findings of this study warrant further investigations for assessing the therapeutic application of CO in nonhuman primate models of tissue injury and in human diseases. The study also suggests that akin to many new therapies in human diseases, the translation of CO therapy to human disease will require additional extensive and rigorous proof-of-concept studies in humans in the future.

Efficacy of famotidine for the prevention of exercise-induced gastritis in racing Alaskan sled dogs.

BACKGROUND: Omeprazole reduces the severity of exercise-induced gastritis but not the prevalence of gastric lesions in sled dogs. The frequent feeding of sled dogs during competition likely results in decreased absorption of omeprazole and, thereby, decreased efficacy. HYPOTHESIS: Famotidine, a histamine-2 blocker with good bioavailability in the presence of food, would reduce the incidence and severity of exercise-induced gastric disease in sled dogs. ANIMALS: Sixteen fit Alaskan sled dogs (4 female, 12 male, all intact, age 2-6 years). METHODS: Dogs were randomly assigned to treatment (22 mg famotidine PO q24h) or control groups (n = 8 per group). Famotidine was administered with a meal to the treatment group once daily for 7 days before a challenge and once during exercise. Control dogs were fed an identical diet as the principal group. The 16 dog team completed a 100-mile exercise challenge in 18 hours. A gastroscopy was performed 24 hours after the challenge. The appearance of the mucosa was scored by an individual by using a scoring system. RESULTS: Treatment with famotidine significantly reduced the severity score compared with control (P = .0004). No adverse effects of treatment were reported. CONCLUSIONS AND CLINICAL RELEVANCE: Famotidine is effective in reducing the severity of exercise-induced gastric disease in racing Alaskan sled dogs, with minimal to no adverse effects, and may be recommended for prophylactic use in short distance races.

Influx of neutrophils and persistence of cytokine expression in airways of horses after performing exercise while breathing cold air.

OBJECTIVE: To determine effects of exercise performed while breathing cold air on expression of cytokines and influx of neutrophils in airways of horses. ANIMALS: 9 adult horses. PROCEDURES: In a crossover study, bronchoalveolar lavage fluid (BALF) was obtained 24 and 48 hours after each of 2 submaximal exercise sessions performed by horses while breathing warm (25 degrees C) or cold (-5 degrees C) air. Total and differential nucleated cell counts were determined for each BALF sample. Relative mRNA expression of cytokines in BALF cells was quantified by use of a reverse transcription-PCR assay. RESULTS: Horses had a modest but significant influx of neutrophils into the airways 24 hours after a single exercise session while breathing cold air. No other cell types were increased at 24 or 48 hours after exercising while breathing cold air. Continued increases in expression of cytokines interleukin (IL)-5 and-10 as well as proinflammatory cytokines IL-1, -6, and -8 were detected 24 hours after exercising while breathing cold air. Forty-eight hours after exercising while breathing cold air, expression of IL-10 was still higher than that for IL-10 after horses exercised while breathing warm air. Expression of tumor necrosis factor-alpha was significantly increased at 48 hours after exercising while breathing cold air. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of intrapulmonary airways to cold air alters immunologic responses of horses for at least 48 hours. The increased expression of cytokines that suppress cell-mediated immunity may predispose athletes to viral infections of the respiratory tract following exercise in cold weather.

Pharmacokinetics of acyclovir after single intravenous and oral administration to adult horses.

The purpose of the study reported here was to describe the bioavailability and pharmacokinetics of acyclovir after intravenous and oral administration to horses. Six healthy adult horses were used in a randomized cross-over study with a 3 x 3 Latin square design. Three treatments were administered to each horse: 10 mg of injectable acyclovir/kg of body weight in 1 L of normal saline delivered as an infusion over 15 minutes; 10 mg of acyclovir/kg in tablets by nasogastric intubation; and 20 mg of acyclovir/kg in tablets by nasogastric intubation. A 2-week washout period was provided between each treatment. Serum samples were obtained for acyclovir assay using reversed-phase, high-performance liquid chromatography with fluorescence detection. Deproteinated serum was injected onto a C18 column, and elution occurred under isocratic conditions. The limit of quantification was 0.04 microg/mL. The assay exhibited suitable accuracy, precision, and recovery. The IV data were analyzed by a 3-compartment model, and oral data were analyzed noncompartmentally. Intragastric acyclovir administration at either dose was associated with high variability in serum acyclovir-time profiles, low Cmax, and poor bioavailability. The dosage of 20 mg/kg was associated with mean (+/- SD) Cmax of 0.19 +/- 0.10 microg/mL, and bioavailability was 2.8%. Inhibition of equine herpesvirus has been reported to require significantly higher acyclovir concentrations than those obtained here. The results of this study do not support a therapeutic benefit for the oral administration of acyclovir up to doses of 20 mg/kg.

Cold weather exercise and airway cytokine expression.

Athletes who perform repeated exercise while breathing cold air have a high prevalence of asthmalike chronic airway disease, but the mechanism linking such activity to airway inflammation is unknown. We used a novel animal model (exercising horses) to test the hypothesis that exercise-induced chronic airway disease is caused by exposure of intrapulmonary airways to unconditioned air, resulting in the upregulation of cytokine expression. Bronchoalveolar lavage fluid (BALF) was obtained from eight horses 5 h after submaximal exercise while they breathed room temperature or subfreezing air in a random crossover design. BALF total and differential nucleated cell counts were determined, and relative cytokine mRNA expression in BALF nucleated cells was quantified by real-time RT-PCR using primer and probe sequences specific for equine targets. There were no significant changes in total or differential cell concentrations between BALF recovered after warm and cold air exercise, although there was a strong trend toward increased concentrations of airway epithelial cells after cold air exercise (P = 0.0625). T(H)2 cytokines IL-4, IL-5, and IL-10 were preferentially upregulated after cold air exercise 12-, 9-, and 10-fold, respectively, compared with warm air exercise. Other cytokines (IL-2 and IL-6) were upregulated to a lesser extent (6- and 3-fold, respectively) or not at all (IL-1, IL-8, IFN-gamma, and TNF-alpha). These results suggest that cold weather exercise can lead to asthmalike airway disease through the local induction of cytokines typical of the T(H)2 phenotype.