Usefulness of the assessment of discharge accumulation in the lower airways and tracheal septum thickening in the differential diagnosis of recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) in the horse.

The aim of the present study was to use endoscopic evaluation to compare the grade of accumulation of discharge in the lower airways of horses with recurrent airway obstruction (RAO) and inflammatory airway disease (IAD), and to estimate the usefulness of endoscopic evaluation in differential diagnosis of these diseases. Endoscopic evaluation consists of: the amount of discharge in the lower part of the trachea and tracheal bifurcation, and the assessment of tracheal septum thickness. This study was performed on 248 horses, 10 were healthy, 97 were diagnosed as RAO, and 141 were diagnosed as IAD. In all animals both a clinical and endoscopic examination were performed. During endoscopy, a score of mucus accumulation was estimated in 3/4 lower of the trachea and in the tracheal bifurcation. In addition to this, thickening of the tracheal septum was also assessed. According to the results of this study, it was estimated that the accumulation of mucus in the lower airways was much more advanced in RAO horses compared to horses with IAD. In horses with RAO there was a marked thickening of the tracheal septum compared to IAD horses. The results indicate the efficacy of scoring the amount of mucus in the tracheal lumen and tracheal bifurcation, as well as the efficacy of the assessment of tracheal septum thickness in the diagnosis of these diseases.

Owner assessment in judging the efficacy of airway disease treatment.

REASONS FOR PERFORMING STUDY: Efficacy of medications for recurrent airway obstruction is typically tested using clinical, cytological and lung function examinations of severely affected animals. These trials are technically challenging and may not adequately reflect the spectrum of disease and owner complaints encountered in clinical practice. OBJECTIVE: To determine if owners of horses with chronic airway disease are better able to detect drug efficacy than a veterinarian who clinically examines horses infrequently. METHOD: In a double-blinded randomised controlled trial, owners and a veterinarian compared the efficacy of dexamethasone (0.1 mg/kg bwt per os, q. 24 h, for 3 weeks; n = 9) to placebo (n = 8) in horses with chronic airway disease. Before and after treatment, owners scored performance, breathing effort, coughing and nasal discharge using a visual analogue scale (VAS). The clinician recorded vital parameters, respiratory distress, auscultation findings, cough and nasal discharge, airway mucus score, bronchoalveolar lavage fluid (BALF) cytology and arterial blood gases. RESULTS: The VAS score improved significantly in dexamethasone- but not placebo-treated horses. In contrast, the clinician failed to differentiate between dexamethasone- and placebo-treated animals based on clinical observations, BALF cytology or endoscopic mucus score. Respiratory rate (RR) and arterial oxygen pressure (PaO(2)) improved with dexamethasone but not placebo. CONCLUSIONS AND CLINICAL RELEVANCE: In the design of clinical trials of airway disease treatments, more emphasis should be placed on owner-assessed VAS than on clinical, cytological and endoscopic observations made during brief examinations by a veterinarian. Quantifiable indicators reflecting lung function such as RR and PaO(2) provide a good assessment of drug efficacy.

Efficacy and safety of sound wave treatment of recurrent airway obstruction in horses.

BACKGROUND: One proposed nonmedical therapy for recurrent airway obstruction (RAO) in horses is a handheld acoustic device that propels sound waves from the nose down the tracheobronchial tree where it is intended to dislodge mucous and relax bronchospasm, permitting clearance of mucoid secretions. OBJECTIVE: To evaluate the effectiveness and safety of this device when used as per the manufacturer's recommendations as a treatment for RAO. ANIMALS: Nine adult horses previously diagnosed with RAO. METHODS: Prospective, cross-over clinical trial. Horses were exposed to a dusty environment until airway obstruction developed as defined by standard lung mechanics (SLM). Horses were randomly assigned to receive either acoustic therapy or a sham treatment for 4 weeks while being maintained in this environment. Horses were evaluated by clinical scores, SLM, and forced expiration regularly for 4 weeks. The opposite treatment was administered after a washout period. RESULTS: Seven horses received the treatment; 9 received the sham. There were no changes (P>.05) in clinical score, maximal change in transpulmonary pressure (ΔPLmax), lung resistance (RL), or the forced expiratory flow rate averaged over the last 75-95% of expiration (FEF75-95%) over the study period. The device was determined to be safe, although several minor adverse effects were noted, including head tossing, coughing, and chewing during treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Treatment with this device did not improve clinical signs or lung function in horses with RAO kept in a dusty environment. Currently accepted treatments, including environmental management and medical therapy, should be recommended.

Thoracoscopic lung biopsies in heaves-affected horses using a bipolar tissue sealing system.

OBJECTIVE: To validate the use of the LigaSure™ Vessel Sealing System (LVSS) to perform thoracoscopic lung tissue biopsies in heaves-affected horses. STUDY DESIGN: Prospective clinical study. ANIMALS: Heaves-affected horses (n=12). METHODS: Lung biopsies (n=34) were collected with the LVSS (2-4 biopsies/horse) in horses with and without clinical signs of heaves. Thoracoscope (13th intercostal space [ICS]) and 2 instruments (between the 12-15th ICS) portals were used. Selected clinical and arterial blood gas variables were monitored. Postoperative pneumothorax was evaluated. Depth of thermal injury to the surrounding tissue and representativeness of the biopsies were determined. RESULTS: Mean surgical time was 22.9±8.0 minutes. The complication rate was 5.6%, and primarily related to a focal inadequate sealing of the biopsy margin. Five horses in exacerbation required intraoperative intranasal O(2) . Mean PaO(2) was significantly lower in heaves-affected horses with clinical signs compared with those without clinical signs. Postoperative pneumothorax was detected radiographically after 20 of the 34 procedures. One horse with clinical signs of heaves developed a fatal tension pneumothorax 5 days postoperatively despite close radiographic monitoring. CONCLUSION: Thoracoscopic lung biopsy using LVSS is a rapid and effective technique to harvest peripheral lung tissues from heaves-affected horses. Although the complication rate was tolerable, tension pneumothorax was a potential life-threatening complication because of incomplete lung sealing. CLINICAL RELEVANCE: LVSS can be used with relative safety to perform thoracoscopic lung biopsy, but close postoperative monitoring is necessary to avoid tension pneumothorax.

Adenosine receptor modulation: potential implications in veterinary medicine.

Adenosine is a purine nucleoside whose concentration increases during inflammation and hypoxia and the many roles of this molecule are becoming better understood. Increased reactivity to adenosine of the airways of asthmatic but not of normal subjects underlines the role of adenosine in airway inflammation. The identification and pharmacological characterisation of different adenosine receptors have stimulated the search for subtype-specific ligands able to modulate the effects of this molecule in a directed way. Several compounds of different chemical classes have been identified as having potential drawbacks, including side effects resulting from the broad distribution of the receptors across the organism, have prevented clinical application. In this article, the effects of adenosine's different receptors and the intracellular signalling pathways are reviewed. The potential of adenosine receptor modulation as a therapeutic target for chronic airway inflammation is considered, taking equine recurrent airway disease and feline asthma as examples of naturally occurring airway obstructive diseases. Other potential applications for adenosine receptor modulation are also discussed. As the intrinsic molecular events of adenosine's mechanism of action become uncovered, new concrete therapeutic approaches will become available for the treatment of various conditions in veterinary medicine.

Effects of in vitro exposure to hay dust on the gene expression of chemokines and cell-surface receptors in primary bronchial epithelial cell cultures established from horses with chronic recurrent airway obstruction.

OBJECTIVE: To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on chemokine and cell-surface receptor (CSR) gene expression in primary bronchial epithelial cell cultures (BECCs) established from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION: BECCs established from bronchial biopsy specimens of 6 RAO-affected horses and 6 healthy horses. PROCEDURES: 5-day-old BECCs were treated with PBS solution, hay dust solutions, LPS, or beta-glucan for 6 or 24 hours. Gene expression of interleukin (IL)-8, chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, toll-like receptor 2, toll-like receptor 4, IL-1 receptor 1, and glyceraldehyde 3-phosphate dehydrogenase was measured with a kinetic PCR assay. RESULTS: Treatment with PBS solution for 6 or 24 hours was not associated with a significant difference in chemokine or CSR expression between BECCs from either group of horses. In all BECCs, treatment with hay dust or LPS for 6 hours increased IL-8, CXCL2, and IL-1beta gene expression > 3-fold; at 24 hours, only IL-1beta expression was upregulated by > 3-fold. In all BECCs, CSR gene expression was not increased following any treatment. With the exception of a 3.7-fold upregulation of CXCL2 in BECCs from RAO-affected horses (following 6-hour hay dust treatment), no differences in chemokine or CSR gene expression were detected between the 2 groups. At 24 hours, CXCL2 gene expression in all BECCs was downregulated. CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial CXCL2 upregulation in response to hay dust particulates may incite early airway neutrophilia in horses with RAO.

Cellular basis of chronic obstructive pulmonary disease in horses.

Chronic obstructive pulmonary disease (COPD) is an inflammatory obstructive disease of the airways characterized with hypersensitivity of the airway tissues to various allergens, most commonly the fungi contained in the poor-quality hay and straw bedding-Saccharopolyspora rectivirgula, Aspergillus fumigatus, and Thermoactinomyces vulgaris. It is manifested clinically in middle-aged horses with recurrent episodes of dyspnea, chronic cough, and their reduced athletic and working capacity. Pulmonary emphysema and lack of pulmonary collapse are the most common gross lesion. Pathohistological findings in horses with COPD are chronic bronchitis/bronchiolitis, with characteristic changes in lumen, mucosa, submucosa, and smooth muscle layer and alveolar emphysema, both distensive and destructive form. Increased immunoreactivity in lungs and tracheobronchial lymph nodes is also noted. Most common lesions seen on cytology imprint smears from tracheal bifurcation is thick, viscous, PAS-positive mucus that forms Curschmann's spirals. Dominant cell population consists of desquamated airway epithelial cells, as well as eosinophils, neutrophils, mast cells, erythrocytes, and alveolar macrophages. Primary pulmonary pathogens as well as potential contaminants and secondary infection agents were isolated bacteriologically from lung samples. All of the aforementioned findings correlate pointing to the fact that chronic bronchitis/bronchiolitis represents a basic substrate of COPD, which have combined inflammatory and immunological etiology, and emphysema is secondary to airway obstruction.

Pulmonary dysfunction and skeletal muscle changes in horses with RAO.

BACKGROUND: Chronic pulmonary diseases (recurrent airway obstruction [RAO]) have been reported to alter skeletal muscle cells in humans. The purpose of this study was to evaluate a potential relationship between pulmonary and muscle variables in horses with a clinical diagnosis of RAO. Muscle biopsies from healthy horses and from horses with RAO were investigated and the relationship between the severity of lung disease and the degree of muscular changes was determined. HYPOTHESIS: We hypothesized that chronic pulmonary disease can lead to changes of the skeletal muscle in horses. ANIMALS: Fifteen healthy horses (control) and 50 horses with RAO were examined. METHODS: In a prospective clinical trial, a complete lung examination was performed in all horses. In all horses, muscle enzyme activity at rest and after exercise and muscle biopsies from the M. gluteus medius were examined. RESULTS: None of the horses had clinical or histologic signs of primary or neurogenic myopathies. According to the clinical, endoscopic, and radiographic findings and with a scoring system, the horses with RAO were grouped according to the severity of pulmonary findings (15 horses mild, 24 horses moderate, 11 horses severe RAO). Pathologic changes of the skeletal muscle (fiber atrophy or fiber hypertrophy, myofibrillar degeneration, hyperplasia of mitochondria, and ragged-red-like fibers) were identified in most horses with RAO but in only a few individual control horses. In addition, a marked depletion of muscle glycogen storage was evident in the RAO horses but not in the control group. Other pathologic changes of skeletal muscle such as centralized nuclei and regenerating fibers were rare, but were more frequent in horses with lung diseases than in the control group. The degree of muscle cell changes was also graded with a scoring system and correlated with the severity of pulmonary disease (r= 0.55). CONCLUSION: Chronic pulmonary disease in horses is associated with structural changes in skeletal muscle. CLINICAL IMPORTANCE: Because chronic pulmonary disease may affect muscles, early and effective therapy may prevent these changes. This finding could be of clinical importance but requires further studies.

Association between cough and cytology of bronchoalveolar lavage fluid and pulmonary function in horses diagnosed with inflammatory airway disease.

BACKGROUND: The objective of this study was to examine the relationship between chronic cough, pulmonary mechanical function, bronchial hyper-responsiveness, and peripheral airway inflammation in adult performance horses with nonseptic inflammatory airway disease (IAD). HYPOTHESIS: We hypothesized that horses with a presenting complaint of cough have a higher percentage of inflammatory cells on bronchoalveolar lavage (BAL), greater airway obstruction, and airway hyper-responsiveness (AHR) than do horses without cough. ANIMALS/SAMPLE POPULATION: Adult performance horses (n = 137) referred for evaluation of signs of IAD including cough or exercise intolerance (university hospital patients) and BAL samples (n = 142) taken in first opinion practice. METHODS: A descriptive, retrospective cohort analysis was performed to evaluate the association between chronic cough and pulmonary mechanical function, AHR, BAL cytology, patient signalment, and comorbid features (multivariable logistic regression). RESULTS: Cough was significantly more prevalent in horses >7 years, and best characterized by a high BAL neutrophil count (>5%) and nasal discharge. Lung function mechanics, abnormal thoracic auscultation, and exercise intolerance did not retain statistical significance in the logistical regression analysis of cough. Although AHR was not related to neutrophilic airway inflammation (BAL neutrophils >5%), it was significantly associated with BAL mast cells >2%. CONCLUSIONS AND CLINICAL IMPORTANCE: Our data support that neutrophilic airway inflammation may potentiate cough without further changing respiratory mechanical function in IAD. In contrast, mast cell release increased AHR without affecting the incidence of cough. Cough may be used as an indicator of neutrophilic airway inflammation in the presence of low-grade nonseptic respiratory disease.

Evaluation of a thoracoscopic technique using ligating loops to obtain large lung biopsies in standing healthy and heaves-affected horses.

OBJECTIVE: To evaluate use of pre-tied ligating loop to perform thoracoscopic, large lung biopsy in normal and heaves-affected horses. STUDY DESIGN: Prospective clinical study. ANIMALS: Normal (n=5) and heaves-affected (n=6) horses. METHODS: Lung biopsies, 1 from each hemithorax, were collected thoracoscopically using a pre-tied ligating loop. Horses were either normal (C) or heaves-affected with the latter being in remission (Ha) for the initial biopsy and in exacerbation (Hs) for the 2nd biopsy. Clinical variables, PaO(2), and PaCO(2) were used to determine the effect of surgical biopsy. Postoperative pneumothorax was monitored by serial thoracic radiographic examinations. RESULTS: Thoracoscopic lung biopsy (n=29, 22 procedures) was well tolerated by all horses. Complication rate was 31%, including 8 ligature slippage and 1 pulmonary hemorrhage. Intranasal oxygen was administered intraoperatively to 6 horses (2 C, 1 Ha, 3 Hs) with severe hypoxemia or labored breathing. There was a significant decrease in PaO(2) during surgery in horses not supplemented with oxygen. Postoperative pneumothorax (21/22 procedures) detected radiographically resolved within 3 weeks. CONCLUSION: Thoracoscopic lung biopsy using pre-tied ligating loops was minimally invasive, relatively inexpensive, and fairly efficient. Heaves-affected horses tolerated the surgery well, even when in exacerbation; however, the technique was associated with non life-threatening complications in 31% of the biopsies, most of which required correction with additional ligating loops or more sophisticated instrumentation. CLINICAL RELEVANCE: Using laparoscopic pre-tied ligating loop for thoracoscopically-assisted lung biopsy can be considered in horses with normal and impaired lung function but alternative instrumentation and access to intranasal oxygen must be available to the surgeon in case of complications.

Effect of antigen challenge on dynamics of CD62P and CD41/61 expression on platelets in horses with recurrent airway obstruction (RAO).

Recurrent airway obstruction (RAO), also known as heaves, is an allergic respiratory condition that develops in horses following an exposure to aeroallergens in hay and straw. This is manifested by airway hyperreactivity, inflammation, bronchoconstriction, as well as a leukocyte and platelet infiltration into the airways. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to explore the effect of seven-day antigen challenge on dynamics of platelet indices and CD41/61 and CD62 P expression on platelets in horses with RAO. Ten RAO-affected horses and ten healthy horses were included in this study. All horses were exposed to 7 days hay and straw challenge. Blood samples were collected prior to the challenge (Pre-challenge) and 1, 2, 3, 7 and 14 days after the initiating the antigen challenge. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62 P and CD41/61 was detected by flow cytometry on activated platelets. Antigen challenge resulted in a significant gradual decrease of PLT in RAO horses, but not in controls. MPV and P-LCR in control and RAO-affected horses remained unchanged after antigen challenge. The expression of CD62 P and CD41/61 in RAO horses was significantly higher compared to control horses. The antigen challenge resulted in an increase expression of CD62 P and CD41/61 on the platelets of RAO-affected horses, while did not lead to significant changes in the control group. An increased expression of CD62 P and CD41/61 indicates platelet activation what may contribute to the formation of platelet aggregates in their respiratory system.

Lung function and airway cytologic profiles in horses with recurrent airway obstruction maintained in low-dust environments.

BACKGROUND: The effects of long-term environmental management on airway obstruction and inflammation in horses with recurrent airway obstruction (RAO) are unknown. HYPOTHESIS: Horses with RAO maintained in low-dust environments have persistent airway obstruction and neutrophilic inflammation. ANIMALS: Study horses were treated for RAO and then maintained in low-dust environments with no medical management. Horses were classified into 3 groups by years after diagnosis: 1 year (time 1, n = 9), 2-3 years (time 2, n = 7), and 5-6 years (time 3, n = 8). The comparison groups were age-matched healthy horses. METHODS: In this cross-sectional study, a clinical examination was performed, and the clinical score was calculated. Standard lung function, forced expiratory maneuvers, and the cytology of bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The clinical scores of the RAO horses were higher than those of the non-RAO horses at time 2 (P = .018). Standard lung function data were not different between the groups at any time point. The forced expiratory flow between 75-95% of exhaled vital capacity was lower in RAO horses than in non-RAO horses at all time points (P < .02), indicating persistent peripheral airway obstruction. Cytologic evaluation of BALF revealed no difference in total nucleated cell numbers or differential cell counts between RAO and non-RAO horses at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: The peripheral airway obstruction detected in horses with RAO maintained in low-dust environments likely is due to irreversible airway remodeling but is not associated with cytologic evidence of airway inflammation.

Plethysmographic comparison of breathing pattern in heaves (recurrent airway obstruction) versus experimental bronchoconstriction or hyperpnea in horses.

BACKGROUND: Horses with recurrent airway obstruction (heaves) are described as exhibiting "increased abdominal effort," but it is unknown whether this translates to an effective contribution to ventilation. HYPOTHESIS: We hypothesized that heaves is characterized by asynchrony between rib cage and abdominal motions, and that the abdominal component is the major contributor to ventilation. ANIMALS: The rib cage versus abdominal motion in naturally occurring heaves (n = 15) was compared to controls at rest (n = 7) and during hyperpnea because of lobeline treatment, and the effects of histamine-induced bronchoconstriction in controls (n = 10). METHODS: Flow patterns, phase angle (theta) between the rib and abdominal compartments, abdominal (Vabd) contribution to tidal volume (VT), and lung mechanics were measured. RESULTS: Findings unique to the heaves group included the loss of biphasic expiratory flow, severely increased theta with the abdomen consistently lagging behind the rib cage, and a reduced contribution of the abdomen to ventilation. A subgroup of heaves (n = 5) with abdominal paradox showed a significant (P < .05) reduction in tidal volume, and increased respiratory rate. Bronchodilation reduced theta in heaves (P = .06), but theta remained significantly higher after bronchodilation than found in controls. CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that breathing pattern in horses with heaves is characterized by severe rib cage/abdominal asynchrony, with the rib cage motion in synchrony with flow, therefore dominating ventilation. In a subset of heaves, the abdominal compartment (diaphragm, abdominal muscles) was completely out of synchrony with flow ("abdominal paradox") despite the clinical appearance of "increased abdominal effort."

Segment-dependent expression of muscarinic acetylcholine receptors and G-protein coupling in the equine respiratory tract.

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720+/-59 nmol/L) than in bronchi (438+/-48 nmol/L) or lung (22 +/- 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24-28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87-90%) predominated over M3 (14-22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31-36%) or presence of guanosine 5'-triphosphate (GTP) (approximately 100%). Western blotting for G-protein alpha-subunits showed a much more robust expression of G(alphai1/2) in the trachea (with highest receptor density) than in the lung or bronchi, whereas G(alphas)-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.

Adiponectin attenuates endoplasmic reticulum stress and alveolar epithelial apoptosis in COPD rats.

OBJECTIVE: The present study was designed to evaluate the effect of Adiponectin (APN) against alveolar epithelial apoptosis in chronic obstructive pulmonary disease (COPD) rat models. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley (SD) rats were randomly assigned to three groups: Sham group, COPD group, and COPD + APN group (2.5 ug/kg/day). To assess the effect of APN, histopathological evaluations, lung function, and the apoptotic index (AI) of alveolar septal cells, were performed. In addition, the levels of oxidative stress and endoplasmic reticulum stress were measured. RESULTS: HE staining demonstrated that APN inhibited pathological injury in COPD rats. In addition, APN could restore the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum. APN also inhibited the levels of endoplasmic reticulum stress pathway including CHOP, phospho-JNK and Caspase-12 in alveolar epithelial cell. Furthermore, APN significantly inhibited the protein levels of Caspase-3 and apoptosis in alveolar epithelial cell of COPD rats. CONCLUSIONS: Our findings suggested that APN might effectively ameliorate the progression of COPD via inhibiting the endoplasmic reticulum stress-induced alveolar epithelial apoptosis in rats.

Effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of COPD.

STUDY OBJECTIVES: The inflammatory responses and associated clinical severity of COPD exacerbations are greatly variable, and the determinants of these factors are poorly understood. We examined the hypothesis that bacteria and viruses may modulate this heterogeneity and that interactions between bacterial and viral infection may affect changes in airway bacterial load and the clinical features and inflammatory responses of exacerbations in patients with COPD. DESIGN: Prospective cohort study. SETTING: Outpatient Department, London Chest Hospital, London, UK. PATIENTS: Thirty-nine patients with COPD. MEASUREMENTS: We prospectively studied 56 COPD exacerbations, obtaining clinical data and paired sputum and serum samples at baseline and exacerbation. Qualitative and quantitative microbiology, polymerase chain reaction detection for rhinovirus, and estimation of cytokine levels by enzyme-linked immunosorbent assay were performed. RESULTS: A total of 69.6% of exacerbations were associated with a bacterial pathogen, most commonly Haemophilus influenzae. Rhinovirus was identified in 19.6% of exacerbations. The rise in bacterial load at exacerbation correlated with the rise in sputum interleukin (IL)-8 (r = 0.37, p = 0.022) and fall in FEV1 (r = 0.35, p = 0.048). Exacerbations with both rhinovirus and H. influenzae had higher bacterial loads (10(8.56) cfu/mL vs 10(8.05)cfu/mL, p = 0.018) and serum IL-6 (13.75 pg/mL vs 6.29 pg/mL, p = 0.028) than exacerbations without both pathogens. In exacerbations with both cold symptoms (a marker of putative viral infection) and a bacterial pathogen, the FEV1 fall was greater (20.3% vs 3.6%, p = 0.026) and symptom count was higher (p = 0.019) than those with a bacterial pathogen alone. CONCLUSIONS: The clinical severity and inflammatory responses in COPD exacerbations are modulated by the nature of the infecting organism: bacterial and viral pathogens interact to cause additional rises in inflammatory markers and greater exacerbation severity.

Cloning and expression of the extra-cellular part of the alpha chain of the equine high-affinity IgE receptor and its use in the detection of IgE.

The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the baculovirus expression system. The recombinant proteins differed in size between the two systems, presumably due to differences in the extent of glycosylation. However, recombinant protein from both cell systems bound equine IgE present in bronchoalveolar lavage fluid from horses with Heaves. These results suggest that the recombinant extra-cellular part of FcepsilonRI should be a useful tool with which to study equine IgE responses.

Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction.

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. OBJECTIVE: To identify components of organic dust responsible for elevated MMP levels in equine airways. METHODS: Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. RESULTS: HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. CONCLUSIONS: The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. POTENTIAL RELEVANCE: Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.

Dose responses to inhalation of endotoxin, hay dust suspension and Aspergillus fumigatus extract in horses as measured by levels and activation of matrix metalloproteinase-9.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase after endotoxin (LPS) exposure, but there are no reports describing dose-dependent increases or activation following exposure. OBJECTIVES: To study matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) responses in bronchoalveolar lavage fluid (BALF) from heaves-susceptible and control horses following inhalation of hay dust suspension (HDS), LPS and Aspergillus fumigatus extract (AFE). METHODS: Heaves-susceptible (n = 7) and control (n = 6) horses received inhalation challenges with 3 different doses of HDS and LPS. Heaves-susceptible horses (n = 6) also received 3 different doses of AFE and one dose of AFE depleted of endotoxin (AFE-LPS). BALF collected following inhalation challenges was analysed using gelatin zymography. Gelatinolytic bands were identified as complex, pro-MMP-9, active MMP-9, pro-MMP-2 and active MMP-2 based on molecular weights. RESULTS: Each challenge substance induced a dose-dependent elevation in gelatinolytic activity. The dose-dependency was most evident for pro-MMP-9 and total MMP-9 levels in heaves-susceptible horses following LPS challenges. CONCLUSIONS: There is a dose-dependent elevation in MMP-9 in BALF of heaves-susceptible and control horses following inhalation challenge with organic dust and some of its components, elevation being more marked in heaves-susceptible horses. Organic dust components vary in their pro-inflammatory potential. POTENTIAL RELEVANCE: This study supports the role of MMPs in the pathogenesis of heaves and highlights the potential value of protease inhibitors in attenuating the airway inflammatory response to inhaled organic dust.