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.     

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone

REASONS FOR PERFORMING STUDY: Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES: To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS: Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS: At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS: RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE: Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.     

Assessment of leukotriene B4 production in leukocytes from horses with recurrent airway obstruction

OBJECTIVE: To determine the ex vivo leukotriene (LT) biosynthesis in peripheral blood neutrophils (PBNs) and inflammatory cells in bronchoalveolar lavage fluid (BALF) obtained from horses affected with recurrent airway obstruction (RAO). ANIMALS: 6 RAO-affected and 6 control horses. PROCEDURES: Before and 6, 24, and 48 hours after stabling, disease severity was determined subjectively by clinical and mucus scores and measurement of the maximal change in pleural pressure (deltaPpl(max)); PBNs were isolated and BALF samples were examined cytologically. The PBN and BALF cells were activated with a calcium ionophore in the presence of arachidonic acid, and production of LTC4 and LTB4 was measured per 10(6) cells. RESULTS: Clinical and mucus scores and deltaPpl(max) increased during stabling in RAO-affected horses, but not in control horses. In neutrophils and BALF cells from both groups, production of LTB4 exceeded that of LTC4. At all times, LTB4 production by PBNs was less in RAO-affected horses than it was in control horses. Before stabling, LTB4 production by cells in BALF was low in RAO-affected horses, but increased considerably after 6 hours of stabling. This increase coincided with the migration of neutrophils into the airways. In control horses, production of LTB4 did not change during stabling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested increased production of LTB4 in airways of RAO-affected horses, compared with control horses, that may contribute to the infiltration of neutrophils into the lungs and the sustained inflammation associated with RAO.     

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

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.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.     

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24 h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.     

Randomized, controlled study of inhaled fluticasone propionate, oral administration of prednisone, and environmental management of horses with recurrent airway obstruction

OBJECTIVE: To determine whether administration of glucocorticoids provides additional benefits to environmental management of horses with recurrent airway obstruction (RAO). ANIMALS: 28 horses with RAO. PROCEDURE: Horses were classified as having mild, moderate, or severe RAO. Within each category, horses were randomly assigned to receive inhaled fluticasone propionate, inhaled control substance, or oral administration of prednisone. During the 4-week study, horses were maintained outdoors and fed a pelleted feed. Clinical scores, pulmonary function, results of cytologic examination of bronchoalveolar lavage fluid (BALF), and adrenal gland function were determined before and 2 and 4 weeks after initiation of treatment. RESULTS: Clinical score and pulmonary function of all RAO-affected horses improved during the treatment period. After 4 weeks, clinical scores and pulmonary function of horses treated with a glucocorticoid were not different from those for the control treatment. In horses with severe RAO, treatment with fluticasone for 2 weeks resulted in significantly greater improvement in pulmonary function, compared with pulmonary function after treatment with prednisone or the control substance. Treatment with a glucocorticoid for 4 weeks and a low-dust environment did not have any effect on cellular content of BALF Treatment with prednisone for 2 weeks resulted in a significant decrease in serum cortisol concentration, compared with concentrations after administration of fluticasone or the control substance. CONCLUSIONS AND CLINICAL RELEVANCE: Environmental management is the most important factor in the treatment of horses with RAO. Early treatment with inhaled fluticasone can help accelerate recovery of horses with severe RAO.     

The effect of adding oral dexamethasone to feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction

BACKGROUND: Chemokine expression in airway epithelium and bronchoalveolar lavage fluid (BALF) cells of horses with recurrent airway obstruction (RAO) is increased. HYPOTHESIS: For RAO-affected horses that are stabled and fed a pelleted ration, the addition of oral dexamethasone further improves pulmonary function and reduces inflammatory gene expression in pulmonary cells. ANIMALS: Twelve RAO-affected horses. METHODS: In a randomized cross-over experiment, the effect of feeding pellets in lieu of hay to stabled, RAO-affected horses was compared with the effect of feeding pellets and administering a 21-day decreasing dose regimen of oral dexamethasone on the expression (by kinetic polymerase chain reaction) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, IL-6, and beta-actin in the BALF cells and of IL-8, CXCL2, 2 IL-1 receptor (IL-1R2), Toll-like receptor 4 (TLR4), and glyceraldehyde 3-phosphate dehydrogenase in the bronchial epithelium 2 days after the final dose. RESULTS: Both treatments reduced airway neutrophilia and breathing efforts but the addition of dexamethasone was associated with fewer treatment failures. Compared with feed changes alone, dexamethasone administration further reduced the expression of IL-8, CXCL2, and IL-1beta in the BALF cells 3.3-, 2.5-, and 4.7-fold, respectively. In the airway epithelium, both treatments were equally efficacious in reducing the expression of IL-8 and CXCL2 expression relative to pretreatment values, but either treatment failed to alter the expression of IL-1R2 and TLR4. CONCLUSIONS AND CLINICAL IMPORTANCE: For a rapid and consistent improvement in pulmonary function and a reduction in inflammatory gene expression of the BALF cells, a decreasing dose of oral dexamethasone in combination with feed alterations is more efficacious for horses that must remain stabled.     

Mucin genes in horse airways: MUC5AC,but not MUC2, may play a role in recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.     

Time-dependent changes of cytokines mRNA in bronchoalveolar lavage fluid from symptomatic recurrent airway obstruction-affected horses

During an 18 day test, we measured the cytokine mRNA expression (Interleukin-1beta [IL-1beta], Interleukin-8 [IL-8], Interferon-gamma [IFN-gamma], Tumor Necrosis Factor-alpha [TNF-alpha]) of cells from bronchoalveolar lavage fluid [BALF] in five horses previously diagnosed with RAO, before and during challenge exposure, and after the desensitization phase which involved dexamethasone treatment and environmental modification. Simultaneously, the same cytokine mRNA expression of cells from BALF in four asymptomatic RAO-affected horses maintained outdoors was analyzed. An evident respiratory distress was observed in the challenge group within 3 days, with a significant overexpression of IL-8 and TNF-alpha mRNA on the ninth day. The pharmacological and environmental desensitization provided a down regulation of all the cytokines. No statistical modification characterized the cytokine kinetics of the asymptomatic horses maintained outdoors. A comparison for each time point of the cytokines between the exposed and unexposed horses showed no significant differences. The study suggested that a standardized exposure protocol and sampling time in experimental studies of RAO is mandatory for a correct comparison of the results obtained by different Authors. However, the absence of significant changes between the exposed and unexposed horses could depend on the lack of the sample uniformity since the evolution of the disease represents a continuum from a healthy to a pathological condition.     

Lower shedding of strongylid eggs by Warmblood horses with recurrent airway obstruction compared to unrelated healthy horses

An association between equine recurrent airway obstruction (RAO) and increased resistance to intestinal parasites has been demonstrated in descendants of an RAO-affected stallion. It was hypothesised that members of another high-incidence RAO family (F) and unrelated RAO-affected Warmblood horses (UA) would shed fewer strongylid eggs than unrelated RAO-unaffected pasture mates (PM) under the same environmental conditions. Faecal worm egg counts were performed on faecal samples (63 F, 86 UA, 149 PM) and classified into three categories: 0, 1-100 and >100 eggs per gram. While results for F did not differ from PM, UA were 2.5-times less likely to shed strongylid eggs than PM. RAO-affected Warmblood horses may be more resistant to strongylid nematodes than unrelated unaffected pasture mates and a family history of RAO does not necessarily confer protection against helminth infections.     

Proteomic analysis in canine leishmaniasis

In horses, Recurrent Airway Obstruction (RAO) is an allergic disease that involves IgE mediated Type I Hypersensitivity responses. The development of this type of allergy involves a series of events that begins with reaginic antibodies, mainly IgE and some IgG subclasses. These reaginic antibodies bind with high affinity, via the Fc portion, to FcεRI receptors on the membrane of mast cells and basophils. Once bound, environmental allergens cross-link the antibodies, which results in mast cell degranulation leading to the production of histamine and other chemical mediators that act together to induce airway inflammation. RAO-affected horses present with coughing, respiratory distress, airway obstruction and poor performance. The aspect of the RAO has been extensively studied, yet the precise sequence of events is still not well understood. Therefore, this study proposes a bioassay for reaginic antibody detection from horse serum of RAO-affected individuals, in order to determine the etiology of disease, which mediate immediate type reactions. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from affected or unaffected horses and one of the RAO antigens (Aspergillus fumigatus). The results presented here demonstrate that 30% of RAO-affected horses react positively in this in vitro bioassay, whereas unaffected horses do not. This bioassay may facilitate further research on RAO and other allergic diseases in horses.     

Genetics of recurrent airway obstruction (RAO)

Recurrent airway obstruction (RAO) is a multifactorial and polygenic disease. Affected horses are typically 7 years of age or older and show exercise intolerance, increased breathing effort, coughing, airway neutrophilia, mucus accumulation and hyperreactivity as well as cholinergic bronchospasm. The environmental factors responsible are predominantly allergens and irritants in haydust, but the immunological mechanisms underlying RAO are still unclear. Several studies have demonstrated a familiar predisposition for RAO and it is now proven that the disease has a genetic basis. In offspring, the risk of developing RAO is 3-fold increased when one parent is affected and increases to almost 5-fold when both parents have RAO. Segregation analysis in two high-prevalence families demonstrated a high heritability and a complex inheritance with several major genes. A whole genomescan showed chromosome-wide significant linkage of seven chromosomal regions with RAO. Of the microsatellites, which were located near atopy candidate genes, those in a region of chromosome 13 harboring the IL4R gene were strongly associated with the RAO phenotype in the offspring of one RAO-affected stallion. Furthermore, IgE-levels are influenced by hereditary factors in the horse, and we have evidence that RAO-affected offspring of the same stallion have increased levels of specific IgE against moldspore allergens. The identification of genetic markers and ultimately of the responsible genes will not only allow for an improved prophylaxis, i.e. early identification of susceptible individuals and avoidance of high-risk matings, but also improve our ability to find new therapeutic targets and to optimize existing treatments.     

Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.     

Abnormalities in lung surfactant in horses clinically affected with recurrent airway obstruction (RAO)

Background: Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses. Hypothesis: Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO). Animals: Six healthy horses and 5 horses with RAO. Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell‐free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high‐performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer. Results: Compared with healthy horses, surfactant from RAO‐affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7–62.2 μg/mL BALF versus 172; 111–267 μg/mL BALF, P= .0062) and CSP (20.2; 6.4–48.9 μL/mL BALF versus 155; 94.4–248 μg/mL BALF, P= .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6–5.6% versus 6.6; 4.1–7.6%, P= .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO‐affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8–22.8, P= .045). Conclusions and Clinical Importance: This study demonstrates that surfactant from RAO‐affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO‐affected horses.     

Mast cells and IgE-bearing cells in lungs of RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation and obstruction following exposure of susceptible horses to mouldy hay and straw. The aim of the present study was to investigate whether lung tissue from horses with RAO contains higher numbers of IgE-protein positive (+) cells and mast cells compared to controls after mouldy hay challenge. Furthermore, mast cell subtypes in lung tissue were investigated. IgE+ cells were detected in most lung tissue samples but no significant differences between RAO-affected and control horses were found. In the wall of the bronchi and bronchioli of both RAO-affected and control horses, mainly chymase+ mast cells (MC(C)) were present (85% in the bronchial wall and 77% in the wall of the bronchioli), while 73% of the mast cells (MC) around blood vessels were tryptase+ mast cells (MC(T)). No double stained MCs were detected. RAO-affected horses had significantly more MC(C) than controls in the wall of the bronchi (median=7.6 and 1.7 cell/mm(2), respectively, P< or =0.05). They also showed a tendency for more MC(C) in the wall of the bronchioli than controls (median=21 and 2.9 cells/mm(2), respectively, P=0.07) but there were no differences in MC(T) numbers. The data suggest an involvement of MC(C) in the pathogenesis of RAO. Independently of the clinical diagnosis, there was a significant relationship between high MC(C) numbers in the bronchial wall and lung fibrosis, suggesting that these MC(C) may be involved in tissue remodelling. Furthermore, high MC(C) numbers were also associated with increased infiltration with lymphocytes and neutrophils.     

Comparison of the antioxidant status in tracheal and bronchoalveolar epithelial lining fluids in recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Following a period of airway inflammation the clearance of inflammatory cells along the mucociliary escalator may impose a considerable oxidant load on the trachea. OBJECTIVES: To determine the degree of oxidative stress in tracheal epithelial lining fluid (ELF) in comparison to that present in peripheral airways after an acute exposure to organic dust. METHODS: Tracheal wash fluid and bronchoalveolar lavage fluid (BALF) were collected for cytology and antioxidant analyses from 6 recurrent airway obstruction (RAO)-affected horses and 6 healthy control horses before and after stabling on straw bedding for 24 h. RESULTS: In RAO-affected horses, organic dust exposure resulted in a significant decrease in ascorbic acid concentration in tracheal ELF (P<0.0001), which was greater than the decrease in bronchoalveolar ELF (P = 0.0003). The percentage decrease in tracheal ELF ascorbic acid correlated with the percentage decrease in bronchoalveolar ELF ascorbic acid (r = 0.76; P = 0.004) following exposure. CONCLUSIONS: Acute organic dust exposure results in significant antioxidant depletion in the trachea, which may reflect inflammation and oxidative processes in peripheral airways. POTENTIAL RELEVANCE: Further work is required to evaluate the role of ascorbic acid depletion in the pathogenesis of RAO.