Reference values of two-dimensional and M-mode echocardiographic measurements as a function of body size in various equine breeds and in ponies.

INTRODUCTION: The aim of this study was to establish echocardiographic reference values for the equine species using allometric regression equations based on body weight (BW) and thoracic circumference (TC). ANIMALS: A total of 239 horses or ponies were studied, including 65 warmbloods, 33 Standardbreds, 41 Thoroughbreds, 32 Arabian horses, 28 draft horses, and 40 ponies aged from 1 day to 30 years, weighing from 18 to 890 kg, with no evidence of cardiac disease. METHODS: For each horse or pony, a two-dimensional and M-mode echocardiography was performed. Within each breed, the relationships between BW or TC and echocardiographic dimensions were examined using power regression equations. Predictions and their 95% prediction intervals were calculated for the echocardiographic measurements. RESULTS: Within each breed, all echocardiographic measurements showed a significant and positive relationship with a high coefficient of determination for the estimation of the regression equations using BW and TC as the main explanatory variables. Breed-specific power regression equations as well as the 95% prediction intervals were calculated for each echocardiographic measurement as a function of BW and TC. CONCLUSIONS: In the future, the body size-corrected and breed-specific echocardiographic reference values calculated in the present study could be used to discriminate between normal and abnormal values in a given animal.

From skeletal muscle to stem cells: an innovative and minimally-invasive process for multiple species.

Bone marrow and adipose tissue represent the two most commonly exploited sources of adult mesenchymal stem cells for musculoskeletal applications. Unfortunately the sampling of bone marrow and fat tissue is invasive and does not always lead to a sufficient number of cells. The present study describes a novel sampling method based on microbiopsy of skeletal muscle in man, pigs, dogs and horses. The process includes explant of the sample, Percoll density gradient for isolation and subsequent culture of the cells. We further characterized the cells and identified their clonogenic and immunomodulatory capacities, their immune-phenotyping behavior and their capability to differentiate into chondroblasts, osteoblasts and adipocytes. In conclusion, this report describes a novel and easy-to-use technique of skeletal muscle-derived mesenchymal stem cell harvest, culture, characterization. This technique is transposable to a multitude of different animal species.

Age-dependent expression of osteochondrosis-related genes in equine leukocytes.

INTRODUCTION: Osteochondrosis (OC) is a developmental disease in horses which has a significant impact on the horse's welfare and performance. The early disturbance in the process of endochondral ossification progresses to inflammatory and repair processes in older horses. Previously, differentially expressed genes in leukocytes of OC-affected horses have been identified. The aim of the present study is to detect age-related changes in these differentially expressed genes. MATERIALS AND METHODS: The expression of OC-related genes was analysed by real-time PCR and subsequent statistical analysis (ΔΔCT) in the leukocytes of 135 Belgian Warmblood horses divided into three different age groups: <12 months (n=47), 18-24 months (n=50) >30 months (n=38). RESULTS: Relative expression of genes of horses less than 12 months of age showed significant induction of the genes MGAT4A, PRKCG, MHCI, ApoB, ApoB3G, B4GALT6 and a significantly lower expression of the genes OAS3. Horses of 18-24 months of age, showed a significantly higher expression of the genes TBC1D9, MGAT4A, IFIH1, MHCIIa and MMP1. Horses of more than 30 months of age showed a significantly higher expression of the genes MGAT4A, HP, SECTM1 compared with their age-matched control groups. CONCLUSIONS: The study demonstrates that OC-related genes are differentially expressed in horses of different ages compared with their age-matched controls. Some of the genes may be implicated in cell signalling and differentiation as well as carbohydrate and lipid metabolism and inflammation. However, the causal relationship between the differentially expressed genes and the development and progression of the OC lesions needs to be determined.

Prevalence and risk factors for cardiac diseases in a hospital-based population of 3,434 horses (1994-2011).

BACKGROUND: Risk factors for cardiac diseases in horses have not been explored in a large population of animals. OBJECTIVES: To describe risk factors for various cardiac diseases in a hospital-based population of horses. ANIMALS: Files of 3,434 horses admitted at the Internal Medicine Department of the Liege Equine Teaching Hospital between 1994 and 2011 were reviewed and of those, 284 were categorized as having moderate-to-severe cardiac disease. METHODS: Observational study. After calculating prevalence for each cardiac disease, we tested whether breed (chi-square test) or sex, age, body weight (BW), and other cardiac diseases (logistic regressions) were risk factors (p < .05 significant). RESULTS: Mitral regurgitation (MR, 4.4%), atrial fibrillation (AF, 2.3%), aortic regurgitation (AR, 2.1%), and tricuspid regurgitation (TR, 1.7%) were the most common cardiac abnormalities detected. Determinants were male sex and increasing age for AR (OR = 2.03, CI = 1.07-4.94), racehorses breed and middle-age for TR (OR = 4.36; CI = 1.10-17.24), and high BW for AF (OR = 3.54; CI = 1.67-7.49). MR was the most common valvular disease associated with AF, clinically important ventricular arrhythmia, pulmonary regurgitation (PR), and congestive heart failure (CHF). TR was also associated with AF, PR, and CHF; AR was not associated with CHF. CONCLUSIONS AND CLINICAL IMPORTANCE: Several previously suspected risk factors for a variety of equine cardiac diseases are statistically confirmed and other risk factors are highlighted in the studied hospital-based population. These observations should be taken into account in health and sport's monitoring of horses presenting predisposing factors.

Sensitivity and specificity of blood leukocyte counts as an indicator of mortality in horses after colic surgery.

The objectives of this study were to describe and relate perioperative changes in blood leukocyte counts to the outcome of surgical colic horses, determine a cut-off value in the early postoperative period to obtain an indicator of the outcome, and compare the obtained value to a validation population of horses. Fifty-three horses undergoing colic surgery were included in the descriptive part of the study. Total leukocyte counts were performed before, during and serially after surgery. A receiver operating characteristic analysis was performed on the leukocyte counts of 45 of these horses to determine a cut-off value for the outcome. The results obtained were validated on a second set of 50 horses that underwent colic surgery in similar conditions. The kinetics of blood leukocytes in survivors was higher than in non-survivors during the first days. Non-survivor horses were more likely to have at least one blood leukocyte count ≤ 3.9 × 10(3)/mm(3) between 28 and 60 hours after surgery than survivor horses. This cut-off value was confirmed in the validation population. These results suggest that routine values of blood leukocyte counts can be used as an additional prognostic indicator after colic surgery alongside other predictors previously associated with the outcome.

Effect of breed, sex, age and body weight on echocardiographic measurements in the equine species.

Little is known about the effect of various animal's signalment variables on echocardiographic reference values in the equine species. This study was performed to evaluate the effect of sex, breed, age and body weight (BW) on echocardiographic measurements in the equine species. Echocardiography was performed on 212 ponies or horses of various breeds, aged from 1 day to 37 years old (mean±SD: 7.8 ± 5.8 years), BW 38-890 kg (mean ± SD: 421 ± 133 kg), and free of cardiac disease. Fifty of those animals aged from 2 months to 35 years old (mean ± SD: 11.6 ± 6.4 years old); BW 77-662 kg (mean ± SD: 436 ± 135 kg) were also examined using the pulsed-wave Doppler mode. Standard two-dimensional and M-mode echocardiography were performed on all animals. Standard pulsed-wave Doppler examination of each cardiac valve was performed on the 50 first examined animals. Data were analysed using a general linear model including the effect of sex, age, breed and BW after logarithmic transformation of the data. Therefore, the same analysis was performed separately on animals aged ≤ 2 years-old and on older animals. All dimensional echocardiographic measurements were significantly affected by BW and most of them were significantly affected by breed, but not by sex. Only the aortic and the pulmonary artery internal diameter were significantly affected by age. None of the Doppler measurements were significantly affected by the tested variables. In conclusion, in the equine species, dimensional echocardiographic reference values should be established using regression equations as a function of BW, which could increase the diagnostic value of this leading technique in equine cardiology. Breed could also have an effect on those measurements.

Association of breeding conditions with prevalence of osteochondrosis in foals.

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet elucidated, but it is accepted that OC lesions are the result of intrinsic genetic and external factors. The aim of the present work was to evaluate the relationship between breeding management and OC. Breeding conditions were recorded, and a radiological examination was performed in 223 foals. Feeding practice and housing management were analysed in a multivariate model to determine risk factors for OC in three periods: gestation, birth to weaning and weaning to one-year-old. The major breakthrough of this study is the significant relationship between OC development and (1) the maternal nutrition during gestation and (2) the type of housing of the foals during their first year. It appears that mares fed with concentrates during gestation are more likely to produce foals that are subsequently affected by OC compared with other mares (P<0.05). Foals housed exclusively at pasture until one year of age are significantly less affected than foals exclusively housed in box or, alternatively, in box and at pasture (P<0.05). These results underline the role of the energy metabolism and the level of exercise in the aetiologic process of the disease, and help to develop preventive strategies during the crucial period of gestation to one year of age of the foal.

Two-dimensional and M-mode echocardiographic reference values in healthy adult Saanen goats.

Echocardiography has become a routine non-invasive cardiac diagnostic tool in most species. Accurate measurement of cardiac dimensions requires reference values, which are poorly documented in goats. The aim of the present study was to test the inter-day repeatability and to establish the reference values of two-dimensional (2D-) and time-motion (M-) mode echocardiographic variables in healthy adult Saanen goats. Six goats were investigated three times by the same observer at one-day interval using a standardised 2D- and M-mode echocardiographic protocol. The intra-observer inter-day repeatability was tested using analysis of variance, calculation of the coefficient of variation and confidence intervals. A single echocardiographic examination was performed in six other goats, and values obtained in the 12 goats were used to establish the 2D- and M-mode echocardiographic reference values in healthy adult female Saanen goats. Statistical analysis revealed a good inter-day repeatability of the echocardiographic cardiac measurements. Echocardiographic reference values obtained in healthy adult Saanen goats seemed slightly higher than those reported in healthy Swedish domestic goats and were similar to those reported in healthy adult sheep.

Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses.

REASONS FOR PERFORMING STUDY: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. OBJECTIVE: To compare plasmatic NE concentrations before and after different forms of intensive exercise. MATERIALS AND METHODS: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. RESULTS: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P < 0.05). NE was significantly (P < 0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P < 0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. CONCLUSIONS: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.

Effect of a 120 km endurance race on plasma and muscular neutrophil elastase and myeloperoxidase concentrations in horses.

REASONS FOR PERFORMING STUDY: Intense physical exercise can induce the degranulation of neutrophils leading to an increase in plasma concentration of the neutrophil marker enzymes myeloperoxidase (MPO) and elastase (ELT). These enzymes have pro-oxidative and pro-inflammatory properties and may play a role in the exercised-induced muscular damage. OBJECTIVES: To measure MPO and ELT concentrations in plasma and muscles of endurance horses and to correlate them to the extent of exercise-induced muscular damage. METHODS: Seven endurance horses qualified on 120 km races were tested in this study. Neutrophil count, serum creatine kinase (CK), plasmatic and muscular MPO and ELT concentrations were measured before and 2 h after a 120 km endurance race. RESULTS: The race produced a significant increase of neutrophils, CK, and plasma MPO and ELT levels. A significant correlation was observed between the MPO and ELT values in plasma (r(2) = 0.92, P < 0.01) and in muscles (r(2) = 0.89, P < 0.01) while plasmatic concentrations of MPO and ELT were not significantly correlated to muscular ones. An increase of mean concentrations (± s.e.) of MPO (T0: 9.85 ± 3.9, T1: 228.9 ± 95.9 ng/mg proteins) and ELT (T0: 8.4 ± 2.4, T1: 74.5 ± 39.7 ng/mg proteins) in the muscles were observed after the race. Interestingly, the individual data showed large differences between the horses. Muscular MPO and ELT concentrations were significantly correlated to plasma CK levels. The coefficient of correlation (r(2)) was 0.69 (P < 0.01) for MPO and 0.66 (P < 0.01) for ELT, respectively. CONCLUSIONS: Results underline the possible role of MPO and ELT in exercise-induced muscular damage. POTENTIAL RELEVANCE: Further studies should investigate the effect of exercise type and intensity, as well as the role of the training state on MPO and ELT involvement in muscular damage. The assessment of the intensity of exercise-induced neutrophilic degranulation may have a potential role in the monitoring of the athletic career.

Vegetative endocarditis in equids (1994--2006).

BACKGROUND: Endocarditis is a rare heart condition with variable clinical expressions in equids. Risk factors for this disease are incompletely understood. OBJECTIVE: Describe risk factors for endocarditis in equids. ANIMALS: One hundred and fifty-three equids admitted to Liège University, 9 diagnosed with endocarditis and 144 free from endocarditis but admitted to the hospital with a differential diagnosis including this disease. METHODS: Retrospective case-control study. RESULTS: Equids with endocarditis were significantly younger (mean age = 4.84 +/- 5.74 years) than control equids (mean age = 10.8 +/- 7.73 years) (P = .01). No sex or breed predisposition was observed. Animals with hyperthermia (odds ratio [OR] = 24.4; confidence interval [CI] = 1.40-428), synovial distension (OR = 13.4; CI = 3.00-59.8), lameness (OR = 6.52; CI = 1.63-26.1), hyperglobulinemia (OR = 26.4; CI = 3.03-229), hypoalbuminemia (OR = 11.4; CI = 1.34-96.8), hyperfibrinogenemia (OR = 9.81; CI = 1.16-82.7), or leukocytosis (OR = 7.12; CI = 1.40-36.4) presented a significantly higher risk of having endocarditis than control horses. The presence of two of the clinical signs mentioned above significantly increased the probability of a diagnosis of endocarditis (P< or = .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Age is associated with equine endocarditis. The diagnostic value of certain clinical signs and abnormalities in blood parameters in this disease are described.

The use of radial extracorporeal shockwave therapy in the treatment of urethral urolithiasis in the horse: a preliminary study.

BACKGROUND: Radial extracorporeal shockwave therapy (ESWT) is widely used in equine practice for the treatment of orthopedic problems. However, its original use as a lithotripsy device in human and canine urology led us to postulate that it could be used as an alternative to the surgical treatment of urethral calculi in horses. HYPOTHESIS: Radial ESWT can easily and safely fragment calculi in the distal urethra of the horse. ANIMALS: Two postmortem cases and 1 live case of obstructive urinary disease admitted at the Veterinary Teaching Hospital of Liege. METHODS: A radial shockwave device was directly applied to the urethra in an attempt to fragment calculi. An ex vivo trial was performed on the same retrieved calculi to investigate pressure settings in order to obtain complete fragmentation of the calculus. RESULTS: In all cases, radial ESWT was able to fragment the calculus partially, enabling retrieval of the remaining fragments via the urethra. Much higher pressure settings than those used for in vivo partial fragmentation were necessary to obtain complete destruction of the calculi ex vivo. CONCLUSIONS AND CLINICAL IMPORTANCE: This brief report suggests the use of radial ESWT as a safe and useful alternative to more invasive surgical management of urethral calculi in horses.

Tetanus in the equine species: a retrospective study of 31 cases.

BACKGROUND: Few studies exist about factors affecting the outcome of horses with tetanus. ANIMALS: 31 equids (30 horses and 1 donkey) with a clinical diagnosis of tetanus admitted to the Equine Clinic of the University of Liege between 1991 and 2006. The cases were divided into two groups according to the outcome (survivors and non-survivors). METHODS: The clinical data of survivors and non-survivors were compared using an ANOVA (continuous data) or a Fisher's test (discrete data). RESULTS: The survival rate was 32%. Most animals were 5 years or younger, and none had been appropriately vaccinated. The non-survivors were significantly younger than the survivors. The development of dyspnoea, recumbency, and the combination of dysphagia, dyspnoea, and recumbency was observed significantly more in the non-survivors than in the survivors. The timing of tetanus antitoxin administration (either immediately after the onset of suggestive signs or after a delay) was not different between the two groups. The time between the occurrence of a wound and the first signs ranged from 2 days to 2 months and was not significantly different between groups. All non-survivors died within 8 days of the first signs. CONCLUSIONS & CLINICAL IMPORTANCE: This study suggests that young animals are affected more often and more severely by tetanus than older animals. Dyspnoea, recumbency, and the combination of dysphagia, dyspnoea, and recumbency can be considered as indicators of a poor prognosis in equids suffering from tetanus.

The composition of the inflammatory infiltrate in three cases of polyneuritis equi.

Polyneuritis equi (PNE) is a rare neurological disease in the horse. Because of the suspicion in PNE of a T-lymphocyte mediated immune response against the myelin, the objective of this study was to determine the composition of the inflammatory infiltrate in the involved nerves of 3 horses with PNE, studied retrospectively. T-lymphocytes were demonstrated in the lesions, which suggests a T-lymphocyte mediated immune response against myelin. In addition, the presence of B-lymphocytes indicated a local production of antibodies. More research, involving a prospective study, is needed to determine whether the T-lymphocytes are cytotoxic or T-helper lymphocytes.

Determination of lidocaine and its two N-desethylated metabolites in dog and horse plasma by high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry.

A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry is described. The sample preparation includes a liquid-liquid extraction with methyl tert-butylmethyl ether after addition of 2M sodium hydroxide. Ethylmethylglycinexylidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and acetonitrile as mobile phases. Good linearity was observed in the range of 2.5-1000 ng ml(-1) for lidocaine in both dog and horse plasma. For MEGX, linear calibration curves were obtained in the range of 5-1000 ng ml(-1) and 20-1000 ng ml(-1) for dog and horse plasma, respectively. In dog and horse plasma good linearity was observed in the range of 200-1500 ng ml(-1) for GX. The limit of quantification (LOQ) in dog plasma for lidocaine, MEGX and GX was set at 2.5 ng ml(-1), 20 ng ml(-1) and 200 ng ml(-1), respectively. For horse plasma a limit of quantification of 2.5 ng ml(-1), 5 ng ml(-1) and 200 ng ml(-1) was achieved for lidocaine, MEGX and GX, respectively. In dog plasma, the limit of detection (LOD) was found to be 0.8 ng ml(-1), 2.3 ng ml(-1) and 55 ng ml(-1) for lidocaine, MEGX and GX, respectively. In horse plasma the LOD's found for lidocaine, MEGX and GX, were 1.1 ng ml(-1), 0.5 ng ml(-1) and 13 ng ml(-1), respectively. The method was shown to be of use in pharmacokinetic studies after application of a transdermal patch in dogs and after an intravenous infusion in horses.

Exercise and pharmacological stress echocardiography in healthy horses.

REASONS FOR PERFORMING STUDY: Stress echocardiography could be a useful diagnostic test in horses suspected to suffer from exercise-induced myocardial dysfunction as a cause of exercise intolerance. OBJECTIVE: To evaluate the effect of treadmill exercise and pharmacological stress test on left ventricular echocardiographic parameters. METHODS: Echocardiography was performed in 2 groups of 5 healthy horses, either immediately after a near-maximal treadmill exercise (Group EXE) or during a pharmacological challenge (Group DOB) consisting of 35 microg/kg atropine, followed by incremental dobutamine infusion rates of 2-6 microg/kg bwt/min for 5 min duration each, which led to a cumulative dobutamine dose of 100 microg/kg. Left ventricular M-mode parameters were compared at rest and at heart rates of 80, 100, 110, 120 130, and 140 beats/min, within each group. RESULTS: In 2 horses of Group EXE, echocardiographic measurements were impossible at 140 and 130 beats/min, as their heart rates dropped too fast in the immediate post exercise period. In the remaining 3 horses image quality was not always satisfactory at heart rates of 130 and 140 beats/min. Systolic left ventricular parameters and fractional shortening measured at 130 and 140 beats/min were significantly different from values obtained at lower heart rates. Horses in Group DOB reached expected heart rates of 80 and 100 beats/min, after the administration of atropine and during a dobutamine infusion rate of 2 microg/kg bwt/min, respectively. Heart rates targets of 110, 120, 130, and 140 beats/min were reached at mean (+/- s.d.) dobutamine infusion rates of 2.8 +/- 0.4, 3.2 +/- 0.4, 4.0 +/- 0.7, 5 +/- 0.7 microg/kg bwt/min, respectively. Systolic left ventricular parameters and fractional shortening at heart rates of 110, 120, 130, and 140 beats/min, were significantly different from values obtained at lower heart rates. CONCLUSION: The pharmacological stress test induced changes in ventricular dimensions at heart rates of 80 to 140 beats/min. Using this test, high quality images can easily be obtained at heart rates of 140 beats/min. Conversely, in post exercise echocardiography, obtaining good quality images at heart rates of 130 and 140 beats/min is difficult, which limits use of the technique in routine clinical settings. POTENTIAL RELEVANCE: Further studies should demonstrate the potential of pharmacological stress test as a diagnostic tool in horses suffering from exercise-induced myocardial dysfunction.

Atropine reduces dobutamine-induced side effects in ponies undergoing a pharmacological stress protocol.

REASONS FOR PERFORMING STUDY: High-dose dobutamine stress echocardiography has been shown to be cardiotoxic and arrhythmogenic in horses. However, the test may have benefit in practice as a pharmacological challenge of exercise without the treadmill being required. OBJECTIVES: To investigate the effect of low-dose dobutamine on cardiac performance in ponies previously treated with atropine, in order to develop a pharmacological protocol that allows examination of the equine heart under stimulation. METHODS: In 13 healthy Shetland ponies, heart rate (HR), stroke index (SI) and cardiac index (CI) were calculated from pulsed-wave Doppler ultrasound measurements performed at rest and during incremental steps of dobutamine infusion. Group 1 (n = 7) received dobutamine infusion at 2 microg/kg bwt/min for 5 mins followed by incremental rates of 5 microg/kg bwt/min every 5 mins, from 5 to 40 microg/kg bwt/min. Group 2 (n = 6) received dobutamine infusion in incremental rates of 1 microg/kg bwt/min, every 5 mins, from 2 microg/kg bwt/min to 5 microg/kg bwt/min, after premedication with 2 injections of 25 microg/kg bwt of atropine 5 mins apart. RESULTS: The increase in CI during the pharmacological challenge was higher in Group 2 and reached about 2.5 times the resting value. This increase in CI was mediated by a significant increase in HR in both groups, while SI significantly decreased in Group 1 and did not change significantly in Group 2. Ponies of Group 1, but not those of Group 2, showed excessive restlessness and cardiac arrhythmias during the pharmacological challenge and a high intragroup variability in cardiac response. CONCLUSIONS: The results of this study suggest that a low dose of dobutamine in ponies previously given atropine could be a helpful pharmacological protocol to perform stress echocardiography in equids. POTENTIAL RELEVANCE: Further studies should evaluate left ventricular wall motion in horses undergoing low-dose dobutamine protocol after pretreatment with atropine.

Enhanced survival of lung granulocytes in an animal model of asthma: evidence for a role of GM-CSF activated STAT5 signalling pathway.

BACKGROUND: As granulocyte/macrophage colony stimulating factor (GM-CSF) mediated delay of granulocyte apoptosis contributes to the accumulation of inflammatory cells at the site of inflammation in many diseases, we sought to determine whether asthma is also associated with a GM-CSF dependent increase in lung granulocyte survival. Moreover, because GM-CSF mediates its effects through activation of signal transducer and activator of transcription 5 (STAT5), we also investigated the potential role of STAT5 in allergic inflammation. METHODS: Blood granulocytes were recovered from six healthy and six heaves affected horses, a model of asthma. Lung granulocytes were obtained by bronchoalveolar lavage (BAL) from the same horses. Granulocytes were cultured in the presence or absence of anti-GM-CSF receptor antibodies for different times and apoptosis was determined using the Annexin-V/propidium iodide detection method. Nuclear protein extracts from cultured granulocytes were analysed for STAT5 binding activity by electrophoretic mobility shift assay. RESULTS: BAL fluid granulocytes from heaves affected horses demonstrated a significant delay in apoptosis compared with blood granulocytes from the same horses and blood and BAL fluid granulocytes from healthy horses. Conversely, the rate of apoptosis in blood granulocytes from healthy and heaves affected horses was comparable. The enhanced survival of BAL fluid granulocytes from affected horses was suppressed in the presence of antibodies directed against GM-CSF receptors. Increased levels of active STAT5 were found in BAL fluid granulocytes from heaves affected horses and were markedly reduced after treatment with anti-GM-CSF receptor antibodies. CONCLUSIONS: These data indicate that granulocyte survival is enhanced in the lung of heaves affected horses and suggest a role for a GM-CSF activated STAT5 pathway in delaying apoptosis of lung granulocytes in this model of asthma.

p65 Homodimer activity in distal airway cells determines lung dysfunction in equine heaves.

Nuclear factor-kappaB (NF-kappaB) activity, which is a key regulator of inflammatory gene expression, is increased in bronchial epithelial cells from horses suffering from heaves (a hypersensitivity-associated inflammatory condition of the lung). To determine whether this increased activity extends to distal airways and to other pulmonary cells, cells recovered by broncho-alveolar lavage (BAL) in healthy and heaves-affected horses were assessed for NF-kappaB activity. NF-kappaB activity was much higher in BAL cells from heaves-affected horses, especially during crisis (disease exacerbation), than in cells from healthy horses. Moreover, the level of NF-kappaB activity found in BAL cells was positively correlated to total lung resistance and to the proportion of neutrophils present in BAL fluid. Finally, prototypical p65-p50 NF-kappaB heterodimers were absent from BAL cells, which mostly contained p65 homodimers. These results (1) show that increased NF-kappaB activity is a general feature of heaves lung; (2) demonstrate the importance of p65 homodimers in neutrophilic inflammation; and (3) suggest that the use of specific NF-kappaB inhibitors could improve lung function in heaves-affected horses.