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- Author or Editor: Päivi Maisi x
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SUMMARY
Objectives
To determine whether gelatinolytic activity in tracheal epithelial lining fluid (TELF), blood neutrophils, and blood lymphocytes from horses was metalloprotease activity, and to compare, for healthy horses and horses with chronic obstructive pulmonary disease, gelatinolytic activity in neutrophils, lymphocytes, and serum with activity in TELF.
Animals
7 horses with chronic obstructive pulmonary disease (COPD) and 4 healthy control horses.
Procedure
Neutrophils and lymphocytes were obtained by means of Percoll separation. Zymography was used to detect gelatinolytic activity; EDTA inhibition and 4-aminophenyl mercuric acetate activation were used to verify that gelatinolytic activity was metalloprotease activity.
Results
Gelatinolytic activity was significantly higher in TELF from horses with COPD than in TELF from healthy horses. For all samples, EDTA inhibited and APMA activated gelatinolytic activity. Gelatinolytic activity of neutrophils, lymphocytes, and serum was not significantly different between healthy horses and horses with COPD.
Conclusions
Results suggested that gelatinolytic activity in TELF from horses is metalloprotease activity. Gelatinolytic activity is increased in TELF from horses with COPD, but not in serum, neutrophils, or lymphocytes. Neutrophils and lymphocytes are possible sources of gelatinolytic activity in TELF.
Clinical Relevance
Measurements of serum, blood neutrophil, or blood lymphocyte gelatinolytic activity were of little value in distinguishing horses with COPD from healthy horses. (Am J Vet Res 1998;59:818–823)
Abstract
Objective
To establish concentration of hyaluronate (HA) in tracheal lavage fluid from healthy horses and horses with chronic obstructive pulmonary disease (COPD).
Animals and Samples
Tracheal lavage fluid samples (n = 42) from 18 horses, 11 with COPD, and 7 control horses.
Procedure
Clinical examination of the respiratory tract, tracheal lavage, and blood sample collection were performed on horses without clinical signs of respiratory tract disease and horses with clinical signs of COPD. In some horses, 1 to 5 repeated examinations were performed at 1-week intervals. Tracheal lavage fluid samples were analyzed for cell numbers, and urea concentration (made in parallel with serum samples to evaluate sample dilution effect); HA was determined by radiometric assay.
Results
Mean (± SEM) HA concentration in tracheal lavage fluid samples was significantly (P = 0.005) higher in horses with COPD (1,880 [± 309] μg/L), compared with that in control horses (256 [± 72] μg/L). The increase in HA concentration in tracheal lavage fluid of COPD-affected horses was verified by repeated sample collection and analysis.
Conclusions
In horses with chronic respiratory tract inflammation such as COPD, tracheal lavage fluid HA concentration is about 7 times higher than reference values. High HA concentration in the tracheal or bronchoalveolar lavage fluid may reflect pathophysiologic changes in connective tissue around bronchi and bronchioli, leading to continuous increased production of HA in horses with advanced forms of COPD.
Clinical Relevance
Determination of tracheal lavage fluid HA concentration may be used as a marker of chronic inflammatory changes in the COPD-affected lung. (Am J Vet Res 1997;58:729–732)
Abstract
Objective—To evaluate inhibitory effects of synthetic matrix metalloproteinase (MMP) inhibitors in vitro on gelatinolytic and collagenolytic activities in tracheal epithelial lining fluid (TELF) of horses with recurrent airway obstruction (RAO).
Animals—10 horses with RAO and 5 healthy control horses.
Procedures—Substrate-based functional assays, collagen I and gelatin degradation, were used to measure endogenous collagenolytic and gelatinolytic activities in TELF. In vitro inhibition of MMP activity in TELF with 2 chemically modified tetracyclines (CMTs; CMT-3 and CMT-8) and 2 bisphosphonates (BPs; zoledronate and pamidronate) was evaluated.
Results—CMT-3, CMT-8, zoledronate, and pamidronate in a dose-dependent manner inhibited TELF type I collagenolytic and gelatinolytic activities, although no complete inhibition of TELF type I collagenolytic and gelatinolytic activities was achieved with the inhibitor concentrations of 25 to 500μM tested. The CMTs inhibited pathologically induced collagen I degradation more effectively than BPs. Of the tested CMTs, CMT-3 was the most effective inhibitor of gelatinolytic activity, and the efficiency of CMT-3 corresponded with that of the BPs.
Conclusions and Clinical Relevance—An increase in MMP activity in the equine respiratory tract may potentially be inhibited by administration of CMTs or BPs. Distinct synthetic MMP inhibitors may eventually provide an additional means for pharmacologic treatment by decreasing ongoing active tissue destructive inflammation associated with chronic lung disease. The MMP inhibitors such as CMTs and BPs that are targeted to solely inhibit a pathologic increase in MMP activities provide the advantage of minimal adverse effects that are characteristics of other excessively potent MMP inhibitors.
Abstract
Objectives—To determine whether samples of tracheal epithelial lining fluid (TELF) obtained from horses have elastinolytic activity characteristic of metalloproteinases, to compare elastinolytic activity in TELF obtained from healthy horses and horses with chronic obstructive pulmonary disease (COPD), and to determine whether chemically modified tetracycline-3 (CMT-3) inhibits elastinolytic activity in TELF.
Animals—10 horses with COPD and 10 healthy control horses.
Procedure—Zymography and fluorometry were used to measure elastinolytic activity, and EDTA was used to inhibit elastinolytic activity and verify that the activity was attributable to metalloproteinases. Possible inhibition of elastinolytic activity with CMT-3 was studied in vitro.
Results—Elastinolytic activity was found in TELF obtained from all horses, and this activity was significantly higher in TELF obtained from horses with COPD than in TELF obtained from healthy horses. For all samples, EDTA and CMT-3 inhibited elastinolytic activity.
Conclusions and Clinical Relevance—Elastinolytic activity is detectable in TELF obtained from horses and seems to be attributable to metalloproteinases. Elastinolytic activity in TELF is significantly inhibited by CMT-3. Elastinolytic activity in TELF can be detected by means of zymography or fluorometry. Increased elastinolytic activity may reflect destruction of pulmonary tissue in horses with COPD. Chemically modified tetracyclines such as CMT-3 may provide an additional treatment possibility for horses with COPD. (Am J Vet Res 2000;61:1067–1073)
Abstract
Objectives—To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD).
Animals—12 horses with COPD and 12 healthy control horses.
Procedure—Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage fluid (BALF).
Results—Collagenase activity was approximately 7 times higher in samples obtained from horses with COPD, compared with control horses. During stabling, horses with COPD had significantly higher collagenase activity than after being maintained on summer pasture, when activity was similar to that of control horses. Immunoreactivity of MMP-8 and MMP-13 was significantly increased in TELF of horses with COPD, compared with healthy horses. In TELF, a positive correlation was detected between immunoreactivity of MMP-8 and MMP-13 and the amount of degradation of type-I collagen. Macrophages and epithelial cells were the major cellular sources of MMP-8 and MMP-13.
Conclusions and Clinical Relevance—Increased collagenase activity in TELF indicates active ongoing disease and, thus, may reflect lung tissue changes in horses with COPD. Measurements of collagenase activity and MMP immunoreactivity may provide additional diagnostic tools to identify the active phase of chronic lung disease. (Am J Vet Res 2001;62:1142–1148)
Abstract
Objectives
To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process.
Design
Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases.
Sample Population
Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD.
Procedure
Caseinolytic agar-diffusion assay.
Results
The protease-inhibition profile of tracheal aspirates differed from horse to horse. The profiles did not resemble that of any of the pure proteases. Acetylcysteine, pentamidine, and diminazene were most effective in inhibiting proteolytic activity in tracheal aspirates in vitro.
Conclusions
A mixed type of proteolytic activity is present in the respiratory tract secretions of horses with COPD.
Clinical Relevance
Acetylcysteine, pentamidine, and diminazene seem to have potential to be used in vivo to protect the lungs of horses with COPD from proteolytic damage. (Am J Vet Res 1996; 57:603–607)
Summary
Plasma concentrations of hypoxanthine, uric acid, and allantoin, which are breakdown products of adenine nucleotides, were measured in Standardbred and Finnhorse trotters during and after an exercise test on a high-speed treadmill, after an incremental exercise test performed on a racetrack, and after a racing competition. Fiber-type composition of the middle gluteal muscle and the muscle concentrations of adenine nucleotides and inosine monophosphate were measured after the racetrack test. Changes in the concentration of hypoxanthine were not observed in any of the tests. Peak concentration of uric acid was measured between 5 and 30 minutes after exercise, and it was three- to tenfold higher than the value at rest. The variability can be explained by intensity of the exercise test and variation among horses. The concentration of allantoin after exercise was 2 to 3 times as high as that at rest, depending on the intensity of the exercise, although the absolute increase was about 10 times as high as the increase in the concentration of uric acid. Peak values of allantoin for the treadmill and the racetrack tests were obtained 4 to 6 minutes after exercise and < 30 minutes after the races. Peak concentration of allantoin correlated positively with the percentage of type-II (IIA + IIB) fibers in the middle gluteal muscle. Significant correlations were not observed between plasma concentration of uric acid or allantoin and muscle concentrations of adenosine triphosphate (atp) or inosine monophosphate. It can be concluded that in horses, breakdown of atp during and after exercise continues until allantoin is produced. The peak concentration of allantoin increases with the intensity of exercise, is reached rapidly after exercise, and the variation in the time to the peak value is small among horses. It is suggested that the main source of allantoin is the fast-twitch, type-II fibers and that the mixed muscle concentrations of adenine nucleotides are of limited value when estimating the effects of exercise on atp content of the muscle tissue.
Abstract
Objective—To determine reference values for cytologic examination results of bronchoalveolar lavage fluid (BALF) and to investigate effects of repeated lavages on pulmonary health and on results of cytologic examination of BALF in dogs.
Animals—16 healthy adult Beagles.
Procedure—All dogs underwent pulmonary lavage to obtain BALF. Eleven dogs were repeatedly lavaged 6 times at 5– to 7–week intervals. Analyses for total and differential cell counts and for viability of cells before and after cell processing were performed. Arterial blood gas analysis before and after bronchoalveolar lavage was used to study the safety of the lavage procedure. Histologic and radiologic examinations were used to study effects of repeated lavages on pulmonary health.
Results—Mean (± SD) cell count was 104 ± 69 cells/µl, comprising 75 ± 7% alveolar macrophages, 13 ± 6% lymphocytes, 5 ± 4% neutrophils, 4 ± 5% eosinophils, 2 ± 2% mast cells, 0.6 ± 0.7% epithelial cells, and 0.3 ± 0.4% plasma cells. Centrifugation of samples and washing of cells caused significant cell loss (59 ± 13%). Repeated lavages did not cause significant variations in cell counts of BALF or results of arterial blood gas analysis, thoracic radiography, or histologic examination of pulmonary specimens. Only a moderate, although significant, decrease in arterial oxygen content was observed after bronchoalveolar lavage.
Conclusions and Clinical Relevance—Analysis indicated that several lavages performed at 5– to 7–week intervals can safely and reliably be used to study the kinetics of pathologic processes in pulmonary tissues or for evaluation of therapeutic efficacy. (Am J Vet Res 2001;62:13–16)
Abstract
Objective—To study gelatinolytic matrix metalloproteinases (MMPs) in tracheobronchial lavage fluid (TBLF) obtained from clinically normal calves and calves with Pasteurella multocida infection.
Sample Population—Samples of TBLF obtained from 11 calves with clinical signs of respiratory tract disease and growth of P multocida and Mycoplasma spp during culture of TBLF and samples of TBLF from 6 clinically normal calves with no bacterial growth during culture of TBLF.
Procedure—MMPs in TBLF were analyzed by use of gelatin zymography. Gelatinases were identified on the basis of molecular weights and inhibition by EDTA.
Results—The main gelatinolytic MMPs detected were the proform (90 to 110 kd) and active form (75 to 85 kd) of MMP-9 (gelatinase B) and the proform (67 to 75 kd) and active form (< 65 kd) of MMP-2 (gelatinase A). Increased amounts of active MMP-2 and MMP-9 were detected in TBLF of calves with respiratory tract disease, compared with amounts of active MMP-2 and MMP-9 in TBLF of clinically normal calves. Concurrent infection with Mycoplasma bovirhinis in calves with pneumonia attributable to P multocida was associated with higher concentrations of MMP-9.
Conclusions and Clinical Relevance—The host response to P multocida includes increases in MMP-2 and MMP-9 concentrations in TBLF. Greater amounts of MMPs detected in calves with concurrent M bovirhinis and P multocida infection indicates synergism between these organisms. (Am J Vet Res 2005;66:2101–2106)
