Metalloproteinases and their Inhibitors under the Course of Immunostimulation by CPG-ODN and Specific Antigen Inhalation in Equine Asthma

Objectives. Inhalation of immunostimulatory bacterial DNA segments (cytosine-phosphate-guanosine-oligodeoxynucleotides, CpG-ODN) normalizes clinical and cytologic parameters in severe equine asthma. We hypothesized that CpG-ODN inhalation also reduces the misbalance of elastinolytic activity in asthmatic horses. Methods. Twenty asthmatic horses diagnosed by clinical examinations using a scoring system were included. All horses inhaled CpG-ODNs for 14 days in 2-day intervals. Matrix metalloproteinase (MMP-2/-9) and tissue inhibitors of metalloproteinase (TIMP-1/-2) concentrations were measured in tracheal aspirates using equine sandwich ELISAs before and 2 and 6 weeks after CpG-ODN inhalation. Results. MMP and TIMP concentrations correlated with the results of clinical scoring in all stages of equine asthma. Inhalation therapy led to significant reductions in clinical scores. MMP-2, MMP-9, and TIMP-2 concentrations were significantly reduced immediately, and all MMP and TIMP concentrations 6 weeks after therapy. Discussion. In equine asthma, overexpression of MMPs contributes to pathological tissue destruction, while TIMPs counteract MMPs with overexpression leading to fibrosis formation. The results of this study show that CpG-ODN inhalation may be an effective therapy to address a misbalance in equine asthma. Conclusions. Misbalance of elastinolytic activity seems to improve by CpG-ODN inhalation for at least 6 weeks posttherapy, which may reduce the remodeling of the extracellular matrix. Further studies should evaluate this effect in comparison to glucocorticoid inhalation therapy. Significance. CpG-ODN inhalation may be an effective therapy in the prevention of pulmonary fibrosis formation in equine asthma

Metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy in combination with environmental dust reduction in equine recurrent airway obstruction

Background Overexpression of matrix-metalloproteinases (MMPs) has been shown to lead to tissue damage in equine recurrent airway obstruction (RAO), as a misbalance with their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), occurs. This favors irreversible pulmonary fibrosis formation. Increased levels of MMPs, TIMPs or altered ratios between them can be used as biomarkers of respiratory disease. We hypothesized that levels of MMPs, TIMPs and their ratios correlate with improvement in clinical findings and bronchoalveolar lavage fluid (BALF) cytology after 10 days of inhalative glucocorticoid therapy and environmental dust reduction (EDR) and may be used to monitor treatment success. Ten horses with a history of RAO participated in a prospective clinical study. Clinical and cytological scoring was performed before and after inhalative therapy using budesonide (1500 μg BID over 10 days) and EDR (bedding of wood shavings and wet hay as roughage). Gelatin zymography was performed for qualitative and semi-quantitative evaluation of MMP-2 and MMP-9 in BALF supernatant, while fluorimetry was used to evaluate MMP-8 activity. Additionally, specific equine ELISA assays were used for quantitative assessment of MMP-2, MMP-9, TIMP-1 and TIMP-2. Results A significant reduction in the total and several single parameters of the clinical score were found after 10 days of inhalative therapy and EDR. The concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 (ELISA) as well as their activities (MMP-2 and MMP-9 zymography and MMP-8 fluorimetry) were significantly decreased after therapy. Significant improvements in MMP-8/TIMP-1 and MMP-8/TIMP-2 ratios were also found, differences between other ratios before and after therapy were insignificant. Conclusions Metalloproteinases and their inhibitors, in particular MMP-9 and TIMP-2, are valuable markers for clinical improvement in RAO

Metalloproteinases and Their Tissue Inhibitors in Comparison between Different Chronic Pneumopathies in the Horse

In chronic respiratory disease, matrix metalloproteinases (MMPs) contribute to pathological tissue destruction when expressed in excess, while tissue inhibitors of metalloproteinases (TIMPs) counteract MMPs with overexpression leading to fibrosis formation. They may be out of balance in equine pneumopathies and serve as biomarkers of pulmonary inflammation. We hypothesized that MMPs and TIMPs correlate to clinical findings and bronchoalveolar lavage fluid cytology in different equine chronic pneumopathies. Using a scoring system, 61 horses were classified controls as free of respiratory disease (n=15), recurrent airway obstruction (RAO, n=17), inflammatory airway disease (IAD, n=18), or chronic interstitial pneumopathy (CIP, n=11). Zymography and equine MMP and TIMP assays were used to detect MMP-2, MMP-8, MMP-9 as well as TIMP-1, and TIMP-2 in BALF supernatant. MMP-2, TIMP-1, and TIMP-2 concentrations were significantly increased in RAO and IAD compared to controls. MMP-9 concentration and MMP-8 activity evaluated by fluorimetry were significantly increased in RAO, IAD, and CIP. These results were confirmed by zymography for MMP-2 and MMP-9 activity in 52 horses. In conclusion, MMPs and TIMPs correlate well with clinical and cytologic findings. These findings support the usefulness of MMPs, TIMPs, and their ratios to evaluate the severity of respiratory disease and may help to identify subclinical cases

Bestimmung von Metalloproteinasen und Interleukin-8 in bronchoalveolären Lavageflüssigkeit bei Pferden mit verschiedenen Pneumopathien

The aim of the present study was to investigate the clinical importance of inflammatory markers in the pathogenesis of RAO, chronic interstitial pneumopathy and acute to subacute respiratory infections in horses in parallel with the clinical examination scores, TBS and BALF cytology and pulmonary function testing. To our knowledge, the detection of the concentration of MMP-2 and MMP-9 in BALF by ELISA was the first trial performed in our study and was found to be an easy, accurate and reliable method. In addition, a semi-quantitative densitometry method based on the gelatin zymography bands for estimating the MMP-2 and MMP-9 activity were performed. As the collagen zymography for measuring MMP-8 was impossible to perform; the concentration of MMP-8 was detected in BALF by a fluorescent method. A total of 64 warmblood horses, admitted to the Equine Clinic, Freie Universität Berlin, were used in the present study. Fifteen clinically healthy horses were used as the healthy standard which had no history of respiratory disease and BALF neutrophils percentage ≤8%. Clinical history, physical examination, endoscopy, radiography of the lung and percentage of neutrophils in BALF and TBS were used as the basis for grouping of diseased horses. RAO-affected horses in exacerbation (n=17), in remission (n=18), horses with chronic interstitial pneumopathy (n=11) and horses with acute to subacute respiratory infection (n=3) are the main disease groups studied. The total examination score were increased significantly in diseased horses compared to controls. The percentage of neutrophils in BALF and TBS was correlated with the concentration of MMP-2, MMP-8, MMP-9 and IL8 and this support the role of these inflammatory markers in the pathogenesis of lung disease. MMP-2 concentration was significantly increased in RAO-affected horses in exacerbation (5.21±0.77 ng/ml) and remission (7.67±15.5 ng/ml) compared to clinically healthy horses (2.49±0.83 ng/ml). MMP-9 concentration was significantly increased in RAO-affected horses in exacerbation (433.34±89.05 pg/ml), remission (312.06±23.92 pg/ml) and horses with chronic interstitial pneumopathy (263.2±23.85 pg/ml) compared to healthy horses (176.29±60.22 pg/ml). These results were confirmed by zymography for MMP-2 and MMP-9 activity in 55 horses. MMP-8 concentration was also significantly increased in RAO-affected horses in exacerbation (0.84±1 μmol/l), remission (0.1±0.06 μmol/l) and horses with chronic interstitial pneumopathy (0.02±0.01 μmol/l) compared to healthy individuals (0.01±0.01 μmol/l) as evaluated by fluorescent method. BALF neutrophil percentage showed a high positive correlation with the concentration of MMP-2, MMP-8 and MMP-9. Also, a highly positive correlation was found with the MMP-2 and MMP-9 activity measured by gelatin zymography. These results were suggestive for its role in the pathogenesis of lung disease. In addition, IL-8 was significantly increased in RAO-affected horses in exacerbation and remission. The observations of the present study provide new insights into the diagnostic potential of various biomarkers in equine airway disease and could be used as an important tool for detecting lung tissue remodeling. Further studies should be focused on the role of TIMPs as a therapeutic approach.Ziel der vorliegenden Studie war es, die klinische Bedeutung verschiedener Entzündungsmarker in der Pathogenese der Recurrent Airway Obstruction (RAO) in Exazerbation und Remission, der chronischen interstitiellen Pneumopathie und akuter bis subakuter Infektionen der tiefen Atemwege bei Pferden auf eine Korrelation zu den Ergebnissen der klinischen Untersuchung, Tracheobronchialsekret (TBS) und bronchoalveolärer Lavageflüssigkeit (BALF) Zytologie und Tests zur Lungenfunktion zu untersuchen. Soweit wir wissen, wurden die Konzentrationen von MMP-2 und MMP-9 im Rahmen dieser Studie erstmals mittels ELISA ermittelt und diese Methode erwies sich als einfach, akkurat und zuverlässig. Außerdem erfolgte eine semi-quantitative densitometrische Auswertung der Banden der Gelatin-Zymographie zur Abschätzung der Aktivitäten von MMP-2 und MMP-9. Da sich die Kollagen-Zymographie zur Bestimmung der MMP-8 als wenig praktikabel erwies, erfolgte die Auswertung der MMP-8 in der BALF fluorimetrisch. Insgesamt wurden 64 Warmblüter an der Klinik für Pferde, Allgemeine Chirurgie und Radiologie der Freien Universität Berlin im Rahmen dieser Studie untersucht. 15 dieser Pferde waren vorberichtlich und klinisch gesund, der Anteil der neutrophilen Granulozyten in der BALF lag ≤ 8% und sie dienten somit als gesunde Kontrollgruppe. Die Ergebnisse der Vorberichtserhebung, der klinischen, endoskopischen und röntgenologischen Untersuchungen, der Lungenfunktionstests und der zytologischen Untersuchunen von TBS und BALF dienten der Einordnung der Probanden in die verschiedenen Erkrankungsgruppen: RAO in Exazerbation (n=17), RAO in Remission (n=18), chronisch interstitelle Pneumopathie (n=11) und akute bis subakute Atemwegsinfektionen (n=3) Der Gesamtuntersuchungsscore war bei den erkrankten Pferden gegenüber den gesunden Kontrolltieren signifikant erhöht. Der Anteil neutrophiler Granulozyten in TBS und BALF korrelierte mit den Konzentrationen von MMP-2, -8, -9 und IL-8, was die Rolle dieser Entzündungsmarker in der Pathogenese equiner Pneumopathien unterstützt. Die MMP-2 Konzentration war bei RAO in Exazerbation (5,21 ± 0,77 ng/ml) und Remission (7,67 ± 15,5 ng/ml) gegenüber der Kontrollgruppe (2,49 ± 0,83 ng/ml) signifikant erhöht. Die MMP-9 Konzentration war bei RAO in Exazerbation (433,34 ± 89,05 pg/ml), RAO in Remission (312,06 ± 23,92 pg/ml) sowie den chronisch interstiellen Pneumopathien (263,2 ± 23,85 pg/ml) gegenüber der Kontrollgruppe (176,29 ± 60,22 pg/ml) signifikant erhöht. Diese Ergebnisse wurden für die MMP-2 und -9 in 55 BALF Proben zymographisch bestätigt. Die MMP-8 Konzentration war bei RAO in Exazerbation (0,84 ± 1 μmol/l), RAO in Remission (0,1 ± 0,06 μmol/l) sowie den chronisch interstiellen Pneumopathien (0,02 ± 0,01 μmol/l) gegenüber der Kontrollgruppe (0,01 ± 0,01 μmol/l) fluorimetrisch signifikant erhöht. Der Anteil neutrophiler Granulozyten in der BALF zeigte eine hochsignifikante positive Korrelation zu den zymographisch gemessenen Aktvititäten von MMP-2 und -9, was die Bedeutung der gelatinolytischen Aktvität bei verschiedenen Pneumopathien des Pferdes unterstützt. Außerdem war IL-8 signifikant erhöht bei RAO in Exazerbation und Remission. Die Ergebnisse der vorliegenden Arbeit ermöglichen neue Einblicke in eine mögliche diagnostische Verwendung verschiedener Biomarker bei equinen Atemwegserkrankungen und diese könnten genutzt werden, um Umbauprozesse in der pulmonalen extrazellulären Matrix besser einschätzen zu können. Weitere Studien sollten sich mit der Rolle der natürlichen Inhibitoren der MMPs (tissue inhibitors of metalloproteinases, TIMPs) als mögliche Therapieoption beschäftigen

Lung Inflammatory Response in Neonatal Diarrheic Bovine Calves with Respiratory Disease Syndrome (RDS)

To investigate the lung inflammatory response during diarrheic episodes in neonatal calves with respiratory disease syndrome, 27 newly born bovine Friesian calves of both sexes (up to 30 days old) were studied, of which 17 diarrheic calves with RDS admitted to the Hospital of the Faculty of Veterinary Medicine Zagazig University and ten apparently healthy calves with no history of any previous illness kept as a control group in the same age range. The examined calves were admitted with a history of diarrhea with respiratory disease syndrome and presented with a variety of clinical signs, including anorexia, diarrhea, cough, dyspnea, and variable degrees of nasal discharge dehydration, weight loss, dullness and pale mucous membranes. The laboratory findings reveal significant changes in diarrheic calf blood parameters, with the RDS group showing significantly lower pH, PaO2, HCO3, BE, and significantly higher PaCO2 and blood lactate values compared to healthy group. Serum glucose, Na, and Cl levels were also significantly lower, whereas serum K levels were significantly higher compared to healthy group. There was a positive correlation between pH and PaO2, HCO3, and BE concentrations, but a negative correlation between pH and PaCO2 and lactate concentrations. The lung-specific epithelial and endothelial biomarkers in healthy and diarrheic neonatal calves with ARD included in the present study revealed that, serum ADMA and SP-D levels were significantly lower, while ET-1 concentrations increased significantly in diarrheic calves with RDS compared to control group measurements. The relationship between ADMA, ET-1 and SP-D concentrations, showed a very strong negative correlation between ADMA and SP-D concentrations and the concentration of ET-1. Whereas, a strong positive correlation between ADMA and SP-D concentrations.