The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction.

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.

Early onset airway obstruction in response to organic dust in the horse.

Equine recurrent airway obstruction (RAO) has been used as a naturally occurring model of human asthma. However, it is unknown whether there is an early-phase response in RAO. The aim of this study was to determine whether exposure to organic dust induces immediate changes in lung function in RAO-affected horses, which could be mediated by airway mast cells. Six RAO-affected horses in remission and six control horses were challenged with hay-straw dust suspension by nebulization. Total respiratory resistance at 1 Hz, measured by forced oscillation, was increased from 0.62 +/- 0.09 cmH(2)O.l(-1).s (mean +/- SE) to 1.23 +/- 0.20 cmH(2)O.l(-1).s 15 min after nebulization in control horses (P = 0.023) but did not change significantly in the RAO group. Total respiratory reactance at 1 Hz (P = 0.005) was significantly lower in the control horses (-0.77 +/- 0.07 cmH(2)O.l(-1).s) than in the RAO group (-0.49 +/- 0.04 cmH(2)O.l(-1).s) 15 min after nebulization. Bronchoalveolar lavage fluid (BALF) histamine concentration was significantly elevated 10 and 20 min postnebulization in control horses but not in RAO horses. Minimum reactance at 1 Hz in the early postnebulization period significantly correlated with both prechallenge BALF mast cell numbers (r = -0.65, P = 0.02) and peak BALF histamine concentration postnebulization (r = -0.61, P = 0.04). In conclusion, RAO horses, unlike human asthmatic patients, do not exhibit an early-phase response. However, healthy control horses do demonstrate a mild but significant early (<20 min) phase response to inhaled organic dust. This response may serve to decrease the subsequent dose of dust inhaled and as such provide a protective mechanism, which may be compromised in RAO horses.

The role of oxidative stress in an equine model of human asthma.

Equine recurrent airway obstruction (RAO) is a naturally occurring respiratory disease in horses with many similarities to human asthma and, as a result, has been used as an animal model of this disease. Oxidative stress has been demonstrated to occur in a range of respiratory diseases in human beings including asthma. Quantitatively, horses have a greater non-enzymatic antioxidant capacity in the pulmonary epithelial lining fluid compared to human beings due to high ascorbic acid concentrations, which reflects their ability to synthesise ascorbic acid. Consequently, a greater oxidative load is likely to be required to induce oxidative stress in horses compared to human beings. Induction of acute neutrophilic airway inflammation in RAO horses by exposure to organic dust does not result in marked pulmonary oxidative stress. However, with a more prolonged inflammatory response, the antioxidant capacity is depleted and oxidative stress occurs. Despite the clear evidence of oxidative stress in RAO, there is currently limited data linking oxidative stress with a causal role in the development of the pathophysiological features of RAO, namely airway obstruction, airway hyper-responsiveness, airway inflammation and mucus accumulation. However, pathways do exist whereby oxidants could potentially augment the production of important mediators in RAO. Further work is required to ascertain the benefits of antioxidant supplementation in RAO and to determine the role of oxidative stress in the pathogenesis of the disease. Given the similarities with human asthma, results from RAO horses could enhance the understanding of the role of oxidative stress in human asthma.

Effect of acute airway inflammation on the pulmonary antioxidant status.

Effects of acute airway inflammation induced by organic dust inhalation on pulmonary antioxidant status were investigated in healthy horses and horses affected by recurrent airway obstruction. Exposure to organic dust induced acute airway neutrophilia, which was associated with increases in elastase and decreases in ascorbic acid concentrations in bronchoalveolar lavage fluid. However, markers of oxidative stress were unaffected, as was hydrogen peroxide in breath condensate. Decreases in ascorbic acid correlated with increased respiratory resistance (P = .001) when both groups were combined. In conclusion, acute neutrophilic airway inflammation does not result in significant evidence of oxidative stress in horses affected by recurrent airway obstruction.

Breath condensate hydrogen peroxide correlates with both airway cytology and epithelial lining fluid ascorbic acid concentration in the horse.

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and alpha-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0 +/- 0.5 micromol/l. 0.4 +/- 0.2 micromol/l and 0.9 +/- 0.2 micromol/l H2O2, respectively; p < 0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r = -0.80; p < 0.0001) and correlated positively with TW inflammation score (r = 0.76, p < 0.0001) and BAL neutrophil count (r = 0.80, p < 0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.

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.

Pulmonary bioavailability of ascorbic acid in an ascorbate-synthesising species, the horse.

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.