Charlotte Sandersen

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.

Effects of moderate amounts of barley in late pregnancy on growth, glucose metabolism and osteoarticular status of pre-weaning horses

In stud management, broodmares are commonly fed concentrates in late pregnancy. This practice, however, was shown to correlate with an increased incidence of osteochondrosis in foals, which may be related to insulin sensitivity. We hypothesized that supplementation of the mare with barley in the last trimester of pregnancy alters the pre-weaning foal growth, glucose metabolism and osteoarticular status. Here, pregnant multiparous saddlebred mares were fed forage only (group F, n=13) or both forage and cracked barley (group B, n=12) from the 7th month of pregnancy until term, as calculated to cover nutritional needs of broodmares. Diets were given in two daily meals. All mares and foals returned to pasture after parturition. Post-natal growth, glucose metabolism and osteoarticular status were investigated in pre-weaning foals. B mares maintained an optimal body condition score (>3.5), whereas that of F mares decreased and remained low (<2.5) up to 3 months of lactation, with a significantly lower bodyweight (-7%) than B mares throughout the last 2 months of pregnancy. B mares had increased plasma glucose and insulin after the first meal and after the second meal to a lesser extent, which was not observed in F mares. B mares also had increased insulin secretion during an intravenous glucose tolerance test (IVGTT). Plasma NEFA and leptin were only temporarily affected by diet in mares during pregnancy or in early lactation. Neonatal B foals had increased serum osteocalcin and slightly increased glucose increments and clearance after glucose injection, but these effects had vanished at weaning. Body measurements, plasma IGF-1, T4, T3, NEFA and leptin concentrations, insulin secretion during IVGTT, as well as glucose metabolism rate during euglycemic hyperinsulinemic clamps after weaning, did not differ between groups. Radiographic examination of joints indicated increased osteochondrosis relative risk in B foals, but this was not significant. These data demonstrate that B or F maternal nutrition has very few effects on foal growth, endocrinology and glucose homeostasis until weaning, but may induce cartilage lesions.

Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses

In equids, placentation is diffuse and nutrient supply to the fetus is determined by uterine size. This correlates with maternal size and affects intra-uterine development and subsequent post-natal growth, as well as insulin sensitivity in the newborn. Long-term effects remain to be described. In this study, fetal growth was enhanced or restricted through ET using pony (P), saddlebred (S) and draft (D) horses. Control P-P (n = 21) and S-S (n = 28) pregnancies were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P or S embryos into D mares (P-D, n = 6 and S-D, n = 8) or S embryos into P mares (S-P, n = 6), respectively. Control and experimental foals were raised by their dams and recipient mothers, respectively. Weight gain, growth hormones and glucose homeostasis were investigated in the foals from birth to weaning. Fetal growth was enhanced in P-D and these foals remained consistently heavier, with reduced T3 concentrations until weaning compared to P-P. P-D had lower fasting glucose from days 30 to 200 and higher insulin secretion than P-P after IVGTT on day 3. Euglycemic clamps in the immediate post-weaning period revealed no difference in insulin sensitivity between P-D and P-P. Fetal growth was restricted in S-P and these foals remained consistently lighter until weaning compared to S-D, with elevated T3 concentrations in the newborn compared to S-S. S-P exhibited higher fasting glycemia than S-S and S-D from days 30 to 200. They had higher maximum increment in plasma glucose than S-D after IVGTT on day 3 and clamps on day 200 demonstrated higher insulin sensitivity compared to S-D. Neither the restricted nor the enhanced fetal environment affected IGF-1 concentrations. Thus, enhanced and restricted fetal and post-natal environments had combined effects that persisted until weaning. They induced different adaptive responses in post-natal glucose metabolism: an early insulin-resistance was induced in enhanced P-D, while S-P developed increased insulin sensitivity.

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.

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.

Gene expression profiling from leukocytes of horses affected by osteochondrosis.

Osteochondrosis (OC) is a developmental disease that affects growing horses and that severely affects their ability to perform. The genetic basis of its pathogenesis is poorly understood. The aim of the study was to analyze the transcript profile of leukocytes from horses affected with OC. Two transcriptome libraries were constructed from leukocytes of OC‐affected and non–OC‐affected horses using digital gene expression analysis (DGE) and real‐time PCR. Statistical analysis allowed selection of 1,008 tags upregulated in the non–OC‐affected group and 1,545 tags upregulated in the OC‐affected group. Among these genes, 16 regulated genes and 5 housekeeping genes were selected. Metabolic pathways analysis showed an obvious dysregulation of several signaling pathways related to cartilage formation or cartilage repair, including Wnt, Indian hedgehog, and TGF‐beta signaling. Other genes, including ISG, ApoB, MGAT4, and TBC1D9, showed a significantly different expression between groups. These genes may play a role in high carbohydrate diet, abnormal insulin metabolism, or inflammation, mechanisms suspected to be involved in OC. This DGE analysis of the transcript profile of leukocytes from OC‐affected horses demonstrated significant differences in comparison to the control library. These results open new perspectives for the understanding of equine OC.

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 sports monitoring of horses presenting predisposing factors.

Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.

Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4h), was obtained with plasma from EDTA anticoagulated blood. The mean value (+/-SEM) measured in 37 healthy horses was 32.53+/-4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids.

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.

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.