Emilie Krafft

Serum and bronchoalveolar lavage fluid endothelin-1 concentrations as diagnostic biomarkers of canine idiopathic pulmonary fibrosis.

BACKGROUND Diagnosis of canine idiopathic pulmonary fibrosis (IPF) is challenging. Endothelin-1 (ET1) is a biomarker of IPF in humans, but whether ET1 can detect and differentiate IPF from other canine respiratory diseases is unknown. OBJECTIVE To evaluate whether measurement of the concentration of ET1 in serum and bronchoalveolar lavage fluid (BALF) can be used to distinguish canine IPF from chronic bronchitis (CB) and eosinophilic bronchopneumopathy (EBP). ANIMALS Twelve dogs with IPF, 10 dogs with CB, 6 dogs with EBP, 13 privately owned healthy West Highland White Terriers (WHWT), and 9 healthy Beagle dogs. METHODS Prospective, case control study. ET1 concentration was determined by ELISA in serum and in BALF. RESULTS No significant difference in serum ET1 concentration was detected between healthy Beagle dogs and WHWT. Serum ET1 concentration was higher in dogs with IPF (median interquartile range; 2.32 pg/mL, 2.05-3.38) than healthy Beagle dogs (1.28, 1.07-1.53; P < .001), healthy WHWT (1.56, 1.25-1.85; P < .001), dogs with EBP (0.94 0.68-1.01; P = .001), and dogs with CB (1.54 0.74-1.82; P = .005). BALF ET1 concentration was below the detection limit in healthy WHWT and in dogs with CB, whereas it was measurable in all dogs with IPF. A cut-off serum concentration of 1.8 pg/mL had a sensitivity of 100% and a specificity of 81.2% for detection of IPF, with an area under the receiver operating characteristic curve of 0.818. CONCLUSIONS AND CLINICAL IMPORTANCE Serum ET1 can differentiate dogs with IPF from dogs with EBP or CB. ET1 can be detected in BALF of dogs with IPF.

Long‐Term Outcome and Use of 6‐Minute Walk Test in West Highland White Terriers with Idiopathic Pulmonary Fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease occurring mainly in West Highland White Terriers (WHWTs). The effects of IPF on survival and on exercise tolerance in WHWTs are unknown. Objectives To evaluate survival, prognostic factors, and exercise tolerance in WHWTs with IPF. Animals Privately owned WHWTs; 15 with IPF and 11 healthy controls. Methods Prospective case‐control study conducted in 2007–2012. For survival, descriptive statistics and Kaplan–Meier (KM) survival curves with Cox proportional hazard ratios were performed. For the prognostic factor study, KM curves, Cox regression analysis, and logistic regression models were used. The 6‐minute walk test (6MWT) was used for measurement of exercise tolerance. Results The median IPF‐specific survival of deceased WHWTs (7/15) with IPF was 32 (range 2–51) months from onset of clinical signs. The risk of death from birth in WHWTs with IPF in age‐adjusted Cox model was significantly higher (hazard ratio 4.6; 95% confidence interval 1.05–19.74, P = .04) than in control WHWTs. No significant prognostic factors were identified. In 6MWT, WHWTs with IPF walked a shorter distance, median 398 m (range 273–519 m), than healthy controls, median 492 m (420–568 m), P = .05, and the partial pressure of oxygen in arterial blood in diseased dogs had a moderate positive correlation with walking distance (Kendall′s tau‐b = 0.69, P = .06). Conclusion and Clinical Importance IPF had a negative impact on life expectancy, but individual survival varied considerably. 6MWT proved to be a well‐tolerated, noninvasive test to evaluate exercise tolerance.

Transforming Growth Factor Beta 1 Activation, Storage, and Signaling Pathways in Idiopathic Pulmonary Fibrosis in Dogs

Background The pathogenesis of idiopathic pulmonary fibrosis (IPF) in dogs is poorly understood. In human, transforming growth factor β1 (TGF‐β1) is considered central in the pathogenesis. Objectives To investigate TGF‐β1 pathway in IPF. Animals Lung tissues from 12 affected and 11 control dogs. Serum from 16 affected West Highland white Terriers (WHWTs) and healthy dogs from predisposed (13 WHWTs, 12 Scottish Terriers and 13 Bichons Frise) and nonpredisposed breeds (10 Whippets, 10 Belgian shepherds, 8 Labradors). Methods In this prospective study, immunohistochemistry was used to evaluate expression and localization of TGF‐β1 protein and proteins involved in TGF‐β1 signaling (TGF‐β receptor type I and phospho‐Smad2/3). Pulmonary expression of TGF‐β1 and molecules involved in its storage (latent TGF‐β binding proteins [LTBP] 1, 2, and 4), activation (ανβ6 and ανβ8 integrins, thrombospondin‐1) and signal inhibition (Smad 7) was analyzed by quantitative reverse transcriptase PCR. Circulating TGF‐β1 concentration was measured by ELISA. Results In IPF, high level of TGF‐β1 protein was found in areas of fibrosis, epithelial cells had strong expression of TGF‐β receptor type 1 and phospho‐Smad2/3, gene expression was decreased for LTBP 4 (P = .009) and β8 integrin (P < .001) and increased for thrombospondin‐1 (P = .016); no difference was seen for Smad7, LTBP1 and 2. Serum TGF‐β1 concentration was higher in predisposed compared with nonpredisposed breeds (P < .0001). Conclusions and Clinical Importance This study identified an enhanced TGF‐β1 signaling activity in IPF. TGF‐β1 storage and activation proteins with altered expression represent potential therapeutic targets. Higher circulating TGF‐β1 concentration in predisposed breeds might partly explain their susceptibility for IPF.

Assessment of CCL2 and CXCL8 chemokines in serum, bronchoalveolar lavage fluid and lung tissue samples from dogs affected with canine idiopathic pulmonary fibrosis

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive disease of the lung parenchyma that is more prevalent in dogs of the West Highland white terrier (WHWT) breed. Since the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 8 (CXCL8) have been implicated in pulmonary fibrosis in humans, the aim of the present study was to investigate whether these same chemokines are involved in the pathogenesis of CIPF. CCL2 and CXCL8 concentrations were measured by ELISA in serum and bronchoalveolar lavage fluid (BALF) from healthy dogs and WHWTs affected with CIPF. Expression of the genes encoding CCL2 and CXCL8 and their respective receptors, namely (C-C motif) receptor 2 (CCR2) and (C-X-C motif) receptor 2 (CXCR2), was compared in unaffected lung tissue and biopsies from dogs affected with CIPF by quantitative PCR and localisation of CCL2 and CXCL8 proteins were determined by immunohistochemistry. Significantly greater CCL2 and CXCL8 concentrations were found in the BALF from WHWTs affected with CIPF, compared with healthy dogs. Significantly greater serum concentrations of CCL2, but not CXCL8, were found in CIPF-affected dogs compared with healthy WHWTs. No differences in relative gene expression for CCL2, CXCL8, CCR2 or CXCR2 were observed when comparing lung biopsies from control dogs and those affected with CIPF. In affected lung tissues, immunolabelling for CCL2 and CXCL8 was observed in bronchial airway epithelial cells in dogs affected with CIPF. The study findings suggest that both CCL2 and CXCL8 are involved in the pathogenesis of CIPF. Further studies are required to determine whether these chemokines might have a clinical use as biomarkers of fibrosis or as targets for therapeutic intervention.

Procollagen type III amino terminal propeptide concentrations in dogs with idiopathic pulmonary fibrosis compared with chronic bronchitis and eosinophilic bronchopneumopathy

Idiopathic pulmonary fibrosis (IPF) is characterised by an abnormal accumulation of collagen type III in the pulmonary interstitium. Procollagen type III amino terminal propeptide (PIIINP) is used as a marker of collagen type III synthesis. In this study, the concentrations of PIIINP were investigated in dogs with IPF (n=15), dogs with chronic bronchitis (CB, n=19), dogs with eosinophilic bronchopneumopathy (EBP, n=13) and healthy dogs (n=25). PIIINP concentrations in serum and bronchoalveolar lavage fluid (BALF) were analysed by radioimmunoassay. Serum PIIINP values did not differ between groups, indicating that serum PIIINP is not useful in evaluating respiratory diseases in dogs. BALF PIIINP was significantly elevated in dogs with IPF compared with healthy dogs (P=0.002) and dogs with CB (P<0.001). BALF PIIINP was significantly higher in dogs with EBP than in dogs with CB (P=0.003) or healthy dogs (P=0.022). There were no differences in BALF PIIINP concentrations between dogs with IPF and dogs with EBP or between dogs with CB and healthy dogs. These results indicate that IPF is associated with elevated BALF PIIINP concentrations. BALF PIIINP concentrations also are elevated in EBP, possibly due to secondary fibrotic changes.

Evaluation of chemokines CXCL8 and CCL2, serotonin, and vascular endothelial growth factor serum concentrations in healthy dogs from seven breeds with variable predisposition for canine idiopathic pulmonary fibrosis.

The West Highland white terrier (WHWT) is particularly prone to canine idiopathic pulmonary fibrosis (CIPF). We hypothesized that higher circulating concentrations of chemokines CXCL8, CCL2, serotonin (5-HT), or vascular endothelial growth factor (VEGF) could serve as predisposing factors for CIPF development in the WHWT breed. Serum samples from 103 healthy dogs of seven different breeds variably predisposed to CIPF were collected. Serum CXCL8 concentrations were higher in healthy WHWT compared with each of the other groups of healthy dogs. Serum CCL2 concentrations were higher in healthy WHWT and Maltese compared with King Charles spaniels and Malinois Belgian shepherds. No relevant inter-breed differences were observed for serum 5-HT concentrations regarding CIPF predisposition. VEGF values from 89.3% of samples tested were below the kit detection limit. In conclusion, high CXCL8 blood concentrations and possibly CCL2 concentrations might be related to the breed predisposition of the WHWT for CIPF and warrants further investigations.

Analysis of gene expression in canine idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) in dogs is a rare disease of unknown aetiology, seen in terrier breeds, particularly the West Highland white terrier (WHWT). The aim of this study was to determine pulmonary gene expression in canine IPF in order to gain insights into the pathogenesis of the disease and to identify possible biomarkers. Microarray analyses were conducted to determine gene expression profiles in the lungs of dogs with IPF and control dogs of various breeds. More than 700 genes were identified as having greater than two-fold difference in expression between the two groups. The significant biological functions associated with these genes were related to cellular growth and proliferation, developmental processes, cellular movement, cell to cell signalling and interaction, and antigen presentation. Altered levels of expression were confirmed by quantitative reverse transcriptase PCR for genes encoding chemokine (C-C) ligand (CCL) 2 (+4.9 times), CCL7 (+6.8 times), interleukin 8 (+4.32 times), chemokine (C-X-C) ligand 14 (+3.4 times), fibroblast activation protein (+4.7 times) and the palate, lung and nasal associated protein (PLUNC, -25 times). Serum CCL2 concentrations were significantly higher in WHWTs with IPF (mean 628.1 pg/mL, interquartile range 460.3-652.7 pg/mL) than unaffected WHWTs (mean 344.0 pg/mL, interquartile range 254.5-415.5 pg/mL; P=0.001). The results support CCL2 as a candidate biomarker for IPF in dogs.

Pulmonary Vein-to-Pulmonary Artery Ratio is an Echocardiographic Index of Congestive Heart Failure in Dogs with Degenerative Mitral Valve Disease.

Background Early recognition of left‐sided congestive heart failure (CHF) in dogs with degenerative mitral valve disease (DMVD) is important because it influences medical therapy, timing of follow‐up, and outcome. Hypothesis Pulmonary vein diameter‐to‐pulmonary artery diameter ratio (PV/PA) measured by echocardiography can predict CHF. Animals Ninety‐eight client‐owned dogs, 37 controls, and 61 dogs with DMVD. Methods Prospective clinical cohort study. History, physical examination and Doppler‐echocardiography were performed. Dogs were classified as International Small Animal Cardiac Health Council class I, II or III. Congestive heart failure was identified in a subset of 56 dogs based on radiographic findings. The PV/PA was measured in bidimensional (2D) and M‐mode by 2 investigators blinded to the radiologists’ conclusions. Results Interobserver coefficients of variation for PV/PA acquisition and measurement were <10%. The PV/PA in control dogs was approximately 1 and increased with class of heart failure. The presence of CHF could be best predicted by measuring PV/PA in 2D echocardiography (cut‐off, 1.7; area under the curve, 0.98; CI, 0.97–0.98; P < .001) with a sensitivity of 96% and a specificity of 91%. Conclusion and clinical importance The PV/PA is a simple and reproducible echocardiographic variable that increases with class of heart failure and may help discriminate dogs in CHF from asymptomatic dogs with DMVD. Additional studies are required to determine whether PV/PA might provide additional information in the integrated interpretation of Doppler‐echocardiographic indices of left ventricular filling pressures and could be used for rapid assessment of CHF in dogs in a critical care setting.

Effect of physiological determinants and cardiac disease on plasma adiponectin concentrations in dogs.

Background In humans, a high concentration of adiponectin is associated with a favorable cardiovascular risk profile whereas, in patients with heart failure (HF), a high concentration of adiponectin is associated with a less favorable prognosis. Hypothesis/Objectives To evaluate the physiological determinants of plasma adiponectin concentration in dogs and the influence of heart disease, myxomatous mitral valve disease (MMVD), and dilated cardiomyopathy (DCM). Animals One hundred and fourteen client‐owned dogs and 9 Beagles from the research colony of the Clinical Veterinary Unit of the University of Liège. Methods We prospectively measured circulating adiponectin concentration in healthy control dogs (n = 77), dogs with MMVD (n = 22) and dogs with DCM (n = 15) of various degrees of severity. Diagnosis was confirmed by Doppler echocardiography. Plasma adiponectin concentration was measured by a canine‐specific sandwich ELISA kit. Results An analysis of covariance showed an association between adiponectin concentration and age, neuter status, and heart disease. No association between adiponectin concentration and class of HF, sex, body condition score, body weight, circadian rhythm, or feeding was found. Plasma adiponectin concentration was negatively correlated with age (P = .001). Adiponectin was lower in neutered (P = .008) compared to intact dogs. Circulating adiponectin concentration was increased in dogs with DCM compared to healthy dogs (P = .018) and to dogs with MMVD (P = .014). Conclusions and Clinical Importance Age and neutering negatively influence circulating adiponectin concentration. Plasma adiponectin concentration increased in dogs with DCM. Additional research is required to investigate if this hormone is implicated in the pathophysiology of DCM and associated with clinical outcome.