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.

A Novel Nonhuman Primate Model of Nonatopic Asthma.

Nonhuman primate models have an essential role in understanding progressive respiratory disease pathogenesis. Immune and physiologic parameters in the nonhuman primate closely reflect the complexity of human systems and provide an exceptional translational impact for the investigation of the mucosal immune changes in response to environmental exposures. This potential warrants the development of novel models that will clarify the interaction of respiratory disease and the inhalable environment and the potential of novel therapies to alleviate the untoward results of these interactions. Nonhuman primate models of asthma can be spontaneous, induced, or experimentally manipulated by various exposures. Here we describe a model of exacerbation of airway hyperreactivity induced by exposure to an air pollutant, ozone, in a cohort of young adult asthmatic rhesus macaques.

LRRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

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.

A structural and functional analysis of the glycosyltransferase BshA from Staphylococcus aureus: Insights into the reaction mechanism and regulation of bacillithiol production.

Bacillithiol is a glucosamine-derived antioxidant found in several pathogenic Gram-positive bacteria. The compound is involved in maintaining the appropriate redox state within the cell as well as detoxifying foreign agents like the antibiotic fosfomycin. Bacillithiol is produced via the action of three enzymes, including BshA, a retaining GT-B glycosyltransferase that utilizes UDP-N-acetylglucosamine and l-malate to produce N-acetylglucosaminyl-malate. Recent studies suggest that retaining GT-B glycosyltransferases like BshA utilize a substrate-assisted mechanism that goes through an SN i-like transition state. In a previous study, we relied on X-ray crystallography as well as computational simulations to hypothesize the manner in which substrates would bind the enzyme, but several questions about substrate binding and the role of one of the amino acid residues persisted. Another study demonstrated that BshA might be subject to feedback inhibition by bacillithiol, but this phenomenon was not analyzed further to determine the exact mechanism of inhibition. Here we present X-ray crystallographic structures and steady-state kinetics results that help elucidate both of these issues. Our ligand-bound crystal structures demonstrate that the active site provides an appropriate steric and geometric arrangement of ligands to facilitate the substrate-assisted mechanism. Finally, we show that bacillithiol is competitive for UDP-N-acetylglucosamine with a Ki value near 120-130 µM and likely binds within the BshA active site, suggesting that bacillithiol modulates BshA activity via feedback inhibition. The work presented here furthers our understanding of bacillithiol metabolism and can aid in the development of inhibitors to counteract resistance to antibiotics such as fosfomycin.

Cigarette smoke and HIV synergistically affect lung pathology in cynomolgus macaques.

In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and chronic obstructive pulmonary disease (COPD) are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced lung function and increased extrathoracic tracheal ring thickness. Interestingly, SHIV infection generated significant numbers of HIV-gp120+ epithelial cells (HGECs) in small airways and alveoli, and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung, and the effects are exacerbated by CS.

Structure and function of the bacillithiol-S-transferase BstA from Staphylococcus aureus.

Bacillithiol is a low-molecular weight thiol produced by many gram-positive organisms, including Staphylococcus aureus and Bacillus anthracis. It is the major thiol responsible for maintaining redox homeostasis and cellular detoxification, including inactivation of the antibiotic fosfomycin. The metal-dependent bacillithiol transferase BstA is likely involved in these sorts of detoxification processes, but the exact substrates and enzyme mechanism have not been identified. Here we report the 1.34 Å resolution X-ray crystallographic structure of BstA from S. aureus. Our structure confirms that BstA belongs to the YfiT-like metal-dependent hydrolase superfamily. Like YfiT, our structure contains nickel within its active site, but our functional data suggest that BstA utilizes zinc for activity. Although BstA and YfiT both contain a core four helix bundle and coordinate their metal ions in the same fashion, significant differences between the protein structures are described here.

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.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated ß-galactosidase (SA-ß-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

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.

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the "recovery stroke" transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

Mild Traumatic Brain Injury and Post-concussion Syndrome: Treatment and Related Sequela for Persistent Symptomatic Disease.

Sport-related concussion typically resolves within a few weeks of the injury; however, persistent symptoms have been reported to occur in 10% to 15% of concussions. These ongoing symptoms can cause significant disability and be frustrating for the patient and family. In addition, factors other than brain injury can cause complications for these patients, such as adjustment disorder or exacerbation of preexisting conditions such as depression or migraine. Individuals with prolonged symptoms of concussion may be classified as having post-concussion syndrome. A careful and thoughtful evaluation is important, as the clinician must determine whether these prolonged symptoms reflect brain injury pathophysiology versus another process. Although there have been numerous studies on the acute management of concussion, much less is available on the treatment of persistent disease. This review will provide an evaluation approach for the patient with prolonged concussion symptoms and review recent literature on treatment strategies.

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.

Myocyte repolarization modulates myocardial function in aging dogs.

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

A novel nonhuman primate model of cigarette smoke-induced airway disease.

Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)-exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD.

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.

Serum chemistry alterations in Alaskan sled dogs during five successive days of prolonged endurance exercise.

OBJECTIVE: To determine the impact of successive days of endurance exercise on select serum chemistry values in conditioned Alaskan sled dogs. DESIGN: Prospective cohort study. ANIMALS: 10 conditioned Alaskan sled dogs. PROCEDURES: All dogs ran 160 km/d for 5 consecutive days. Serum was obtained prior to exercise and immediately after each exercise run; all samples were obtained before dogs were fed. Serum electrolyte, mineral, protein, total bilirubin, urea nitrogen, creatinine, and cardiac troponin-I concentrations and serum alkaline phosphatase, alanine aminotransferase, creatine kinase, and aspartate aminotransferase activities were measured. Data were analyzed by means of analysis of covariance for a randomized complete block design with dog as a blocking variable, time as a covariate, and distance run as the treatment of interest. Least square mean values were compared with values obtained prior to exercise, and linear and quadratic contrasts were examined. RESULTS: Serum globulin concentration was low prior to exercise (mean +/- SD, 2.2 +/- 0.3g/dL) and progressively decreased as exercise continued. Exercise was associated with increases in serum chloride, urea nitrogen, and cardiac troponin-I concentrations and serum alanine aminotransferase, creatine kinase, and aspartate aminotransferase activities and with progressive decreases in serum potassium, total protein, and albumin concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that multiple successive days of endurance exercise resulted in mild aberrations in serum chemistry variables in conditioned sled dogs. Changes likely reflected the metabolic stresses of prolonged endurance exercise as well as dietary composition. Hypoglobulinemia in resting, conditioned sled dogs may reflect the immunosuppressive or catabolic effects of intense endurance training.