Susan Skeans

Microarray profile of differentially expressed genes in a monkey model of allergic asthma.

BackgroundInhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs.ResultsOf the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only.ConclusionThis study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.

Short duration anaesthesia with medetomidine and ketamine in cynomolgus monkeys.

Cynomolgus monkeys were anaesthetized with either intramuscular ketamine (10 mg/kg or intramuscular ketamine 2 mg/kg and medetomidine 50 μg/kg. Various physiological measurements were made once the animals were safe to handle and again 10 min later. Cardiovascular and respiratory function were well maintained with both regimens but the heart rate was lower and arterial-alveolar carbon dioxide gradient was higher in the animals that received medetomidine. In those animals that received medetomidine, atipamezole was given to reverse the medetomidine but there was no difference in recovery times between the two regimens. Anaesthesia was not entirely reliable with medetomidine/ketamine and we recommend caution when using this mixture.

Eotaxin and Nitric Oxide Production as Markers of Inflammation in Allergic Cynomolgus Monkeys

Background: Cynomolgus monkeys have a natural hypersensitivity to Ascaris suum antigen. Inhalation of antigen produces immediate and delayed allergic reactions and an influx of inflammatory cells into the lungs. This study investigated the production of nitric oxide (NO) and the chemokine eotaxin during this allergic response. The effect of bronchoscopy alone on lung inflammatory cells was also investigated along with the time course of the eosinophil influx into the lung. Methods: Allergic cynomolgus monkeys were challenged with antigen. Bronchoalveolar lavage (BAL) was performed before and after challenge, and end–tidal NO was measured before and 24 h after challenge. Eotaxin was measured in the BAL fluid 6, 24 and 72 h after challenge. One group of animals was treated with dexamethasone before challenge to block the influx of cells into the lung. Results: BLA alone induced an influx of neutrophils, but not eosinophils, into the lung 24 h later. A single antigen challenge produced a marked increase in BAL eosinophils that was apparent at 6 h but increased at 72 h after challenge. The increase at 6 h was largely blocked by dexamethasone. Three antigen challenges produced elevated BAL eosinophil levels that persisted for at least 8 weeks. Eotaxin levels rose dramatically 6 h after challenge and remained the same after 24 h. By 72 h, the eotaxin levels had returned to baseline. The increase in eotaxin at 6 h was nonsignificantly reduced by dexamethasone. Exhaled NO levels doubled 24 h after challenge and were not affected by dexamethasone. Conclusions: Eotaxin and NO production were increased after airway challenge in allergic monkeys. The rise in NO was not blocked by dexamethasone. The effects of bronchoscopy on the BAL can be avoided by using alternate lungs on consecutive occasions. Eosinophils persist in the BAL for many weeks after antigen challenge.

The effects of body fat on pulmonary function and gas exchange in cynomolgus monkeys.

Obesity adversely affects lung function in humans often reducing arterial blood oxygenation. To determine if obesity adversely affects lung function in cynomolgus monkeys, which is a species that is often used for pulmonary research, pulmonary mechanics, ventilation, functional residual capacity (FRC), and arterial blood gases were measured using spontaneous respiration and on mechanical ventilation with room air or 100% O(2). Body fat percentage was measured by dual energy X-ray absorption. Blood leptin levels were measured by radioimmune assay. Obese monkeys breathed faster with lower tidal volume, but pulmonary resistance and dynamic lung compliance did not change with body fat. FRC and blood leptin were, respectively, negatively and positively correlated with percent body fat. FRC correlated moderately with ventilatory parameters and strongly with arterial oxygen tension, alveolar-arterial oxygen difference and venous admixture. Therefore, obesity in cynomolgus monkeys had marked, deleterious effects on FRC, ventilation and arterial oxygenation. Obesity may be an important confounding variable in lung function studies in primates.

A simple noninvasive method to measure the cough reflex in dogs

INTRODUCTION This study describes a method to measure the cough reflex in dogs that is simple to perform, requires no surgical intervention and can be used to profile efficacy and side-effect liabilities of antitussive drugs. METHODS Experiments were performed in propofol-anesthetized dogs in which cardiopulmonary functions were non-invasively monitored before and after the induction of cough produced by spraying 0.75 ml of distilled water into the trachea. RESULTS The magnitude of the cough response, measured by the frequency and amplitude was not different for individual dogs performed with repeated trials on different days. Treatment with the opioid antitussive drug, torbutrol (0.055-0.0055 mg/kg, s.c.) inhibited the cough frequency but not the amplitude induced by the water challenge. Furthermore, side effects of torbutrol were identified as mild respiratory depression and an anesthetic-sparing effect with propofol. DISCUSSION This method offers many distinct advantages to evaluate efficacy of antitussive drugs including the fact that no surgery is required, it takes only 15-20 min to complete an experiment, and it can be used to simultaneously profile antitussive and side effect liabilities of drugs developed for the treatment of cough.

Airway Closure after Antigen Challenge in Cynomolgus Monkeys: Effect of the Histamine H1 Receptor Antagonist, Chlorpheniramine Maleate

Background: Airway closure is frequently observed in human asthma. However, limited information exists on the factors that cause this condition. In this study, an allergic cynomolgus monkey model was used to characterize the condition of airway closure and assess the contribution of histamine H1 receptors to this response. Methods: Oscillatory lung mechanics, arterial blood gases during ventilation on 100% O2 and functional residual capacity (FRC) assessed by helium dilution were measured before and then 10 min and 24 h after Ascaris aerosol challenge in 12 male Ascaris-sensitive cynomolgus monkeys. The monkeys were pretreated with intravenous saline or chlorpheniramine maleate (0.3 mg/kg) in a randomized crossover design. Results: Ascaris challenge produced a large increase in airway resistance, an increase in lung tissue damping (G) that measures ventilation inhomogeneity in the lung, a reduction in arterial oxygen tension (PaO2) during ventilation on 100% O2 and a reduction in FRC. These effects were seen 10 min after the Ascaris challenge, but by 24 h, these parameters had returned close to the baseline values. Chlorpheniramine maleate (0.3 mg/kg, i.v.) produced a 12-fold shift in the histamine bronchoconstrictor dose-response curve. Pretreatment of monkeys with chlorpheniramine maleate (0.3 mg/kg, i.v.) attenuated the increase in airway resistance induced by Ascaris challenge, but had only a small effect on the increase in G and the reductions in PaO2 and FRC after antigen. Conclusions: These results demonstrate that airway closure occurs immediately after the antigen challenge in allergic cynomolgus monkeys and that histamine H1 receptors contribute very minimally to this response.

Differential Effects of Dexamethasone on the Proximal and Distal Lung Response to Antigen Challenge in Allergic Cynomolgus Monkeys

The proximal and distal portions of the lungs may respond differently to antigen challenge and bronchodilator treatment. This difference may contribute to differences in actual and perceived efficacy of therapies. In this study we used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and discern the effects of antigen challenge on proximal (large airway) and distal (small airway and lung parenchyma) portions of the lung. In addition we treated the animals with two i.m. injections of either a saline control or dexamethasone (0.5 mg/kg) 18 and 1 hour(s) before the antigen challenge. The FOT technique was used to measure indices of proximal airway status, Newtonian airway resistance (RN), and distal airway status, including tissue damping (G) and tissue elastance (H). Challenging the animals with Ascaris Suum antigen caused a significant increase in both the proximal and distal lung measures. Pretreatment with dexamethasone significantly reduced the peak increase in RN but not G or H. In addition, the area under the curve (AUC) of the FOT response over 60 minutes was significantly reduced for the RN but again, G and H were not significantly reduced. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre-treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on the proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to provide a mechanism for evaluation of novel therapies for small airway dysfunction.

Agreement of SpO2, SaO2 and ScO2 in anesthetized cynomolgus monkeys (Macaca fascicularis)

OBJECTIVE To assess the agreement between three measurements of arterial oxygen saturation (SpO2, SaO2 and ScO2) in anesthetized cynomolgus monkeys. STUDY DESIGN Prospective study. ANIMALS Eleven mature, male cynomolgus monkeys (Macaca fasicularis). METHODS Monkeys were anesthetized with intramuscular ketamine followed by intravenous propofol. The trachea of each was intubated and the lungs ventilated. Arterial oxygen saturation was measured with a Nonin 8500 V pulse oximeter, using a lingual clip on the cheek. Arterial blood samples were taken from an indwelling catheter. Inspired oxygen concentration was varied from 12 to 20%, and 88 paired arterial blood samples and saturation measurements were taken. Arterial oxygen saturation in the blood samples was measured using a cooximeter. The saturation was also calculated from the arterial oxygen tension using the Adair equation. The results were compared using Bland and Altmans method. RESULTS The pulse oximeter readings were 2.7% higher than that of the cooximeter, with a limit of agreement of -3.9 to 9.3%. The pulse oximeter readings were 1.8% higher than the calculated saturation, with a limit of agreement of -6.5% to 10.1%. The cooximeter readings were 0.9% lower than the calculated saturation, with a limit of agreement of -5.6% to 3.8%. CONCLUSIONS The agreement between SpO2 and other measurements of arterial oxygen saturation in this study is typical for this technique. The bias and limits of agreement are consistent with reports in other species. CLINICAL RELEVANCE The Nonin 8500 V is a useful pulse oximeter for clinical use in primates.