Enhanced IL-6 transcriptional response to adenosine receptor ligands in horses with lower airway inflammation

Reasons for performing study: Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objectives: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL‐6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR‐subtypes in each BAL sample was determined by quantitative RT‐PCR and compared to that in 13 other tissues. Bronchoalveolar lavage cells were stimulated either with the adenosine analogue NECA, CGS‐21680 (A_2AAR selective agonist) or with a combination of NECA and SCH‐58261 (A_2AAR antagonist) and IL‐6 expression assessed. Results: Bronchoalveolar lavage cells predominantly expressed A_2BAR, with lower A_2AAR levels and marginal A₃AR expression; A₁AR was not detected. This pattern was similar to that of PBMCs but different from the other tissues tested. No significant differences in AR expression in BAL cells from both groups were detected, although a trend for decreased A_2BAR in airway‐compromised horses was observed. Treatment of BAL cells with the nonselective agonist NECA upregulated IL‐6 expression in cells from airway‐compromised horses, but levels remained unchanged in control animals. Furthermore, blockage of A_2AAR with SCH‐58261 enhanced IL‐6 mRNA induction by NECA in both groups, with higher levels in airway‐compromised horses; the amplitude of this response correlated with neutrophil count. Conclusions: These results demonstrate the presence of an adenosine/IL‐6 inflammatory axis in the bronchoalveolar milieu of airway‐compromised horses. While A_2BAR is the predominant proinflammatory AR subtype expressed, A_2AAR appears to modulate inflammatory signalling (IL‐6 expression) by adenosine. Potential relevance: This study supports selective AR targeting as a potential therapeutic approach for the modulation of inflammation in the equine lower respiratory tract.     

Changes in central venous pressure and blood lactate concentration in response to acute blood loss in horses

OBJECTIVE: To evaluate selected hemodynamic, blood gas, and biochemical responses to mild to moderate acute blood loss in standing, awake horses. DESIGN: Prospective study. ANIMALS: 7 healthy mares. PROCEDURES: Each horse was restrained in standing stocks, and its head was maintained in a neutral position; sedatives and tranquilizers were not administered. During a 1-hour period, blood was collected into collection bags by use of a suction pump. The rate of blood collection was approximately 16 mL/kg/h (7.3 mL/lb/h). Thirty minutes after blood collection, the blood was readministered at the same rate. Central venous pressure (CVP), central venous blood gas, blood lactate concentration, and heart rate were measured at baseline (after placement of catheters), after removal of blood, and after readministration of blood. RESULTS: In response to blood loss, CVP decreased and blood lactate concentration increased significantly, compared with baseline values; heart rate and results of central venous blood gas analysis did not change significantly. After readministration of blood, CVP returned to baseline value and blood lactate concentration approached baseline value. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in CVP and blood lactate concentration appear to be early indicators of hypovolemia in horses, which may represent acute blood loss in trauma patients; these variables should be monitored to assess the potential need for blood transfusions. These variables can be used to monitor responses of horses to blood transfusions when whole blood is administered as the replacement fluid.     

Repeated blood instillation into the airway of the horse does not cause pulmonary fibrosis

Reasons for performing the study: Exercise‐induced pulmonary haemorrhage (EIPH) occurs in nearly all strenuously exercising horses. Recent studies have attempted to identify the role of free blood within the airspaces, in the lung fibrosis that develops within the lungs of EIPH horses. Hypothesis: Repeated exposure of the equine lung to autologous blood results in lung fibrosis similar to that observed in spontaneous EIPH. Methods: Forty ml of autologous blood from the jugular vein was instilled into preselected lung regions of 6 horses one, 2, 3, 4 or 5 times at 2 week intervals, with 40 ml of saline instilled into the contralateral lung serving as a control. The time interval between instillation of the first blood and euthanasia ranged from 2–10 weeks. The lung from each instillation site was harvested, and the histopathology was scored from each region based upon the presence and abundance of blood, haemosiderin and interstitial collagen. Consequently, at the time of euthanasia, the time since instillation of the first blood ranged from 2–10 weeks. Results: Beyond retention of blood, and the accumulation of haemosiderin, there was no visible increase in perivascular and interstitial collagen within the blood‐instilled lung sites. In a small number of regions, there were foci of bronchiolitis obliterans organising pneumonia with collagen accumulation within these foci, but no collagen accumulation with the characteristic perivascular and interstitial histological distribution seen in EIPH. Conclusions: Free blood within the airways of horses does not result in a qualitative increase in the amount of interstitial collagen within 8–10 weeks, and is therefore an unlikely aetiological factor in the lung collagen accumulation that occurs in EIPH. Potential relevance: This study emphasises the efficiency of the equine lung in clearing blood from the airspaces. Further, it suggests that the aetiopathogenesis of EIPH is not driven by events within the airspace lumen, but rather emanates from within the vasculature and lung interstitium.     

Pulmonary function,airway cytology and bronchoalveolar lavage fluid drug concentration after aerosol administration of cefquinome to horses

The administration of antibiotics by aerosol to horses suffering from respiratory infections may partially circumvent the limitations of antimicrobial therapy, e.g. large injection volumes, low bioavailability and risk of diarrhoea. Only injectable formulations are available currently and usually contain other substances that could irritate the mucosa and induce coughing and bronchospasm. In addition, the quality of the aerosol, particularly in terms of the delivery of antibiotics to the deep parts of the lung, is unknown. Although used under field conditions, cefquinome delivered by aerosol has never been studied in horses. This study examined the safety of cefquinome injectable solution, administered by aerosol at a dose of 225 mg/inhalation to 7 healthy horses, by assessing (1) pulmonary function before and 15 min after a single inhalation, at the first day (Day 1) and the fifth day (Day 5) of a 5 day period treatment; and (2) the inflammatory status of the lung, i.e. percentage neutrophils and myeloperoxidase concentration, based on bronchoalveolar lavage (BAL) at D1 and D5. In addition, cefquinome concentrations were measured in bronchoalveolar lavage fluid after aerosol, intravenous (i.v.) and intramuscular (i.m.) administrations. A single aerosol of cefquinome injectable solution did not induce any immediate nor delayed pulmonary side effects in healthy horses and produced cefquinome concentrations in bronchoalveolar lavage (BAL) within 30 min that were higher than the minimal inhibitory concentration of the main equine respiratory pathogens. These results should stimulate further studies, especially in horses suffering from bronchial hyper‐reactivity. Aerosol delivery of antibiotics may well have a role in equine therapeutics.     

Inflammatory airway disease of horses

The purpose of this consensus statement is to provide a review of current knowledge and opinions concerning inflammatory airway disease (IAD) and to help practitioners differentiate IAD from heaves (or recurrent airway obstruction; RAO) and other inflammatory respiratory diseases of horses.     

Pulmonary epithelial lining fluid and plasma ascorbic acid concentrations in horses affected by recurrent airway obstruction

OBJECTIVE: To determine the pulmonary epithelial lining fluid (ELF) concentrations and degree of oxidation of ascorbic acid in horses affected by recurrent airway obstruction (RAO) in the presence and absence of neutrophilic airway inflammation. ANIMALS: 6 RAO-affected horses and 8 healthy control horses. PROCEDURE: Nonenzymatic antioxidant concentrations were determined in RBC, plasma, and ELF samples of control horses and RAO-affected horses in the presence and absence of airway inflammation. RESULTS: ELF ascorbic acid concentration was decreased in RAO-affected horses with airway inflammation (median, 0.06 mmol/L; 25th and 75th percentiles, 0.0 and 0.4 mmol/L), compared with RAO-affected horses without airway inflammation (1.0 mmol/L; 0.7 and 1.5 mmol/L) and control horses (2.2 mmol/L; 1.4 and 2.2 mmol/L). Epithelial lining fluid ascorbic acid remained significantly lower in RAO-affected horses without airway inflammation than in control horses. Moreover, the ELF ascorbic acid redox ratio (ie, ratio of the concentrations of dehydroascorbate to total ascorbic acid) was higher in RAO-affected horses with airway inflammation (median, 0.85; 25th and 75th percentiles, 0.25 and 1.00), compared with RAO-affected horses without airway inflammation (0.04; 0.02 and 0.22). The number of neutrophils in bronchoalveolar lavage fluid was inversely related to the ELF ascorbic acid concentration (r = -0.81) and positively correlated with the ascorbic acid redox ratio (r = 0.65). CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophilic inflammation in horses affected by RAO is associated with a reduction in the ELF ascorbic acid pool. Nutritional supplementation with ascorbic acid derivatives in horses affected by RAO is an area for further investigation.     

Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.     

Coughing,mucus accumulation,airway obstruction,and airway inflammation in control horses and horses affected with recurrent airway obstruction

OBJECTIVE: To investigate relationships between cough frequency and mucus accumulation, airway obstruction, and airway inflammation and to determine effects of dexamethasone on coughing and mucus score. ANIMALS: 13 horses with recurrent airway obstruction (RAO) and 6 control horses. PROCEDURE: 6 RAO-affected and 6 control horses were stabled for 3 days. Coughing was counted for 4 hours before and on each day horses were stabled. Before and on day 3 of stabling, tracheal mucus accumulation was scored, airway obstruction was assessed via maximal change in pleural pressure (deltaPpl(max)), and airway inflammation was evaluated by use of cytologic examination of bronchoalveolar lavage fluid (BALF). Effects of dexamethasone (0.1 mg/kg, IV, q 24 h for 7 days) were determined in 12 RAO-affected horses. RESULTS: To assess frequency, coughing had to be counted for 1 hour. In RAO-affected horses, stabling was associated with increases in cough frequency, mucus score, and deltaPpl(max). Control horses coughed transiently when first stabled. In RAO-affected horses, coughing was correlated with deltaPpl(max), mucus score, and airway inflammation and was a sensitive and specific indicator of deltaPpl(max) > 6 cm H2O, mucus score > 1.0, and > 100 neutrophils/microL and > 20% neutrophils in BALF Dexamethasone reduced cough frequency, mucus score, and deltaPpl(max), but BALF neutrophil count remained increased. CONCLUSIONS AND CLINICAL RELEVANCE: Because of its sporadic nature, coughing cannot be assessed accurately by counting during brief periods. In RAO-affected horses, coughing is an indicator of airway inflammation and obstruction. Corticosteroid treatment reduces cough frequency concurrently with reductions in deltaPpl(max) and mucus accumulation in RAO-affected horses.     

Comparison of endoscopic tracheal washing, bronchoalveolar lavage and visual detection of blood following instillation of blood into the airways of horses

In an experimental study of endoscopic tracheal washings and bronchoalvveolar lavage specimens collected prior to and following instillation of 0,250, 500 or 1000 ml of autologous citrated blood into the airways of nonexercising, adult horses, there was no apparent correlation between the amount of blood or exudate seen endoscopically, the volume of washing fluid recovered, or the cytologic evaluation of numbers of erythrophages or siderophages. Tracheal washing and bronchoalveolar lavage specimens appear to be complementary for detection of erythrophages or siderophages in cytologic specimens since these cell types may be present in one, or the other, or both types of specimens. There may be an advantage to collecting two BAL specimens since more erythrophages or siderophages were seen in the second BAL in some horses. Further studies of experimentally-instilled blood and spontaneouslyoccurring exercise-induced pulmonary hemorrhage are needed to further characterize the mechanisms by which pulmonary hemorrhage is resolved and/or characterize the cytologic methods best suited for detection and/or quantitation of pulmonary hemorrhage.     

Hormonal response to thyrotropin-releasing hormone in healthy horses and in horses with pituitary adenoma

Cortisol, triiodothyronine (T3), thyroxine (T4), insulin, and glucose responses to thyrotropin-releasing hormone (TRH) were evaluated in 12 healthy, mature horses and in 7 horses and 4 ponies with clinical signs of pituitary adenoma (PA). Within 1 hour after TRH administration, the increase in T3 and T4 was similar in healthy horses and animals with PA. Plasma cortisol in the group with PA increased (P less than 0.05) within 0.25 hours after TRH administration, and remained increased for 1.5 hours. In the control group, a significant increase in plasma cortisol concentrations did not develop after TRH administration. Plasma glucose and insulin concentrations were higher in animals with PA than in the healthy horses throughout the experiment (6 hours).     

Histamine inhalation challenge in normal horses and in horses with small airway disease

A histamine inhalation challenge (HIC) procedure was developed to assess hyperreactive states in horses. Following clinical evaluation, percutaneous lung biopsies were performed on nine light breed mares aged 6 to 15 years. Five horses, with normal small airways, were classified as group A and four subjects with small airway disease (SAD) lesions formed group B. Pulmonary mechanics parameters were monitored following an aerosol of 0.9% saline and every 5 min for up to 30 min after HIC with 0.5% w/v of histamine diphosphate, administered through a face mask for 2.5 min. Tidal volume (VT) and airflow (V) values were obtained with a pneumotachograph. Transpulmonary pressure (delta Ppl) was measured by the esophageal balloon catheter method. Dynamic compliance (Cdyn), total pulmonary resistance (RL), end expiratory work of breathing (EEW) and respiratory rate (f) were calculated by a pulmonary mechanics computer. Group A horses had increases in RL, and decreases in Cdyn whereas horses in group B were hyperreactive and showed greater changes in EEW, Cdyn, and delta Ppl but with a relatively lower variation of RL. One horse in clinical remission from SAD, but with a high biopsy score (group B), and one clinically normal horse belonging to group A showed marked hyperreactivity as shown by increases in EEW, maximum change in delta Ppl and RL and decreases in Cdyn. These results suggest that the HIC described can be used as a method to investigate airway hyperreactivity and SAD in horses.     

Pulmonary function and adrenal gland suppression with incremental doses of aerosolized beclomethasone dipropionate in horses with recurrent airway obstruction

OBJECTIVE: To evaluate clinical response, pulmonary function, and adrenal gland response to incremental doses of beclomethasone dipropionate in horses with recurrent airway obstruction. DESIGN: Crossover trial. ANIMALS: 8 horses with recurrent airway obstruction. PROCEDURE: Horses randomly assigned to 4 groups were treated twice daily via aerosol administration of placebo or 500, 1,000, or 1,500 micrograms of beclomethasone dipropionate in a crossover design with a 10-day minimum washout period. Subjective assessment of airway obstruction, serum cortisol concentration, and maximum change in pleural pressure during tidal breathing (delta Pplmax) were determined daily prior to morning drug administration, and delta Pplmax was reevaluated 15 minutes after morning drug administration. Pulmonary resistance and dynamic compliance were determined at baseline and approximately 12 hours after the final treatment. RESULTS: An immediate treatment effect was not identified. Within 24 hours, delta Pplmax and airway obstruction were lower in horses receiving beclomethasone. Onset and magnitude of response was similar among the 3 beclomethasone dose regimens. Pulmonary resistance was improved only after administration of all 3 doses of beclomethasone, whereas dynamic compliance was improved after administration of 1,000 micrograms and 1,500 micrograms of beclomethasone. Reduction in serum cortisol concentration occurred with all 3 beclomethasone dose regimens; however, the magnitude of adrenal gland suppression was greater in horses receiving 1,000 or 1,500 micrograms of beclomethasone. CONCLUSIONS AND CLINICAL RELEVANCE: Low-dose (500 micrograms) beclomethasone administration caused similar, improvement in pulmonary function, compared with high-dose beclomethasone (1,000 and 1,500 micrograms), with the exception of dynamic compliance, and caused less suppression of endogenous cortisol production.     

Pulmonary haemorrhage in standardbred horses after racing

SUMMARY Endoscopic examinations of the upper respiratory tract were done on 92 of 314 Standardbred horses that raced one or more times at 4 consecutive, weekly race meetings. Although participation was voluntary, the characteristics of the population of horses examined were not statistically different from those of all horses that raced. No horse showed epistaxis, but 34 (32.4%) examinations of the trachea revealed blood that ranged from a trace in the tracheal mucus to large amounts scattered over the tracheal walls. Forty-four horses exhibited minor degrees of pharyngeal lymphoid hyperplasia, 2 had asynchronous movement of the left arytenoid cartilage and 15 had grains of sand in the respiratory tract. There was no association between bleeding and age, sex, distance of race, place in race or date of race. Mucus and mucopurulent material occurred less often after longer races and more often on the last 3 race nights.     

Pulmonary fibrosis and gammaherpesvirus infection in horses

Pulmonary fibrosis is a devastating and often progressive condition leading to exercise intolerance and frequently the demise of the animal. Although uncommonly encountered in horses, the condition is intensely researched both in human medicine and animal models. Viral infections have long been suspected to play a part in the development of pulmonary fibrosis and neoplastic conditions in other species. In 2007, an association between equine herpesvirus 5 (EHV‐5) infection and nodular pulmonary fibrosis in horses was suggested and the name equine multinodular pulmonary fibrosis (EMPF) was introduced. Recently, the presence of EHV‐5 in equine lymphoma has also emerged. The case report by Schwarz et al. in this issue describes a horse suffering from concurrent T cell leukaemia and EMPF in association with EHV‐5. This article summarises current knowledge about EMPF and EHV‐5 infections in horses, recent developments in the understanding of pulmonary fibrosis in man and the proposed contribution of viral infections to pulmonary fibrosis and neoplastic conditions.