Breed differences in natriuretic peptides in healthy dogs.

BACKGROUND: Measurement of plasma concentration of natriuretic peptides (NPs) is suggested to be of value in diagnosis of cardiac disease in dogs, but many factors other than cardiac status may influence their concentrations. Dog breed potentially is 1 such factor. OBJECTIVE: To investigate breed variation in plasma concentrations of pro-atrial natriuretic peptide 31-67 (proANP 31-67) and N-terminal B-type natriuretic peptide (NT-proBNP) in healthy dogs. ANIMALS: 535 healthy, privately owned dogs of 9 breeds were examined at 5 centers as part of the European Union (EU) LUPA project. METHODS: Absence of cardiovascular disease or other clinically relevant organ-related or systemic disease was ensured by thorough clinical investigation. Plasma concentrations of proANP 31-67 and NT-proBNP were measured by commercially available ELISA assays. RESULTS: Overall significant breed differences were found in proANP 31-67 (P < .0001) and NT-proBNP (P < .0001) concentrations. Pair-wise comparisons between breeds differed in approximately 50% of comparisons for proANP 31-67 as well as NT-proBNP concentrations, both when including all centers and within each center. Interquartile range was large for many breeds, especially for NT-proBNP. Among included breeds, Labrador Retrievers and Newfoundlands had highest median NT-proBNP concentrations with concentrations 3 times as high as those of Dachshunds. German Shepherds and Cavalier King Charles Spaniels had the highest median proANP 31-67 concentrations, twice the median concentration in Doberman Pinschers. CONCLUSIONS AND CLINICAL IMPORTANCE: Considerable interbreed variation in plasma NP concentrations was found in healthy dogs. Intrabreed variation was large in several breeds, especially for NT-proBNP. Additional studies are needed to establish breed-specific reference ranges.

Investigation of apoptosis in a canine model of chronic heart failure induced by tachycardia.

Participation of apoptosis during the development of pacing-induced dilated cardiomyopathy is not fully understood. After 7 weeks rapid right ventricular pacing, gene expressions of Bax, Bcl-2 and Caspase-3 were measured by RTQ-PCR from interventricular septum biopsies that were taken weekly in 21 beagle dogs during the development of heart failure. We evaluated protein levels of these genes by Western blot and DNA fragmentation by TUNEL method from autopsy samples. Gene expression of Bax remained unchanged during the pacing period; Bcl-2 mRNA expression transiently decreased in moderate heart failure and their ratio (Bcl-2/Bax) was not significantly altered. Caspase-3 gene expression increased in heart failure. Compared to the control group, expression of Bax and Bcl-2 proteins and their ratio were increased in dogs only after 4 weeks of pacing. No band of activated Caspase was found in the normal nor in the paced myocardium. In the TUNEL assay there was no significant difference between numbers of apoptotic cells in any of the groups, although a few TUNEL-positive cells were detected in the paced groups. Our results are not in favour of apoptosis in the pathogenesis of heart failure in this model and may be it could be attributed to activation of other systems.

Lack of efficacy of low-dose spironolactone as adjunct treatment to conventional congestive heart failure treatment in dogs.

Aldosterone plays an important role in the pathophysiology of heart failure. Aldosterone receptor blockade has been shown to reduce morbidity and mortality in human patients with advanced congestive left ventricular heart failure. This study was designed to assess the efficacy and tolerance of long-term low-dose spironolactone when added to conventional heart failure treatment in dogs with advanced heart failure. Eighteen client-owned dogs with advanced congestive heart failure due to either degenerative valve disease (n=11) or dilated cardiomyopathy (n=7) were included in this prospective, placebo-controlled, double-blinded, randomized clinical study. After initial stabilization including furosemide, angiotensin-converting enzyme inhibitors, pimobendan and digoxin, spironolactone at a median dose of 0.52 mg/kg (range 0.49-0.8 mg/kg) once daily (n=9) or placebo (n=9) was added to the treatment, and the dogs were reassessed 3 and 6 months later. Clinical scoring, echocardiography, electrocardiogram, systolic blood pressure measurement, thoracic radiography, sodium, potassium, urea, creatinine, alanine aminotransferase, aldosterone and aminoterminal atrial natriuretic propeptide were assessed at baseline, 3 and 6 months. Survival times were not significantly different between the two treatment groups. Spironolactone was well tolerated when combined with conventional heart failure treatment.

Early increase in pulmonary vascular reactivity with overexpression of endothelin-1 and vascular endothelial growth factor in canine experimental heart failure.

Heart failure is a cause of pulmonary vasoconstriction and remodelling, leading to pulmonary hypertension (PH) and decreased survival. The pathobiology of PH in heart failure remains incompletely understood. We investigated pulmonary vascular function and signalling molecules in early stage PH secondary to experimental heart failure. Eight beagle dogs with overpacing-induced heart failure underwent haemodynamic assessment and postmortem pulmonary arterial reactivity, morphometry and quantification of genes encoding for factors involved in vascular reactivity and remodelling: endothelin-1 (ET-1), ETA and ETB receptors, vascular endothelial growth factor (VEGF), VEGF receptors 1 and 2 (VEGFR1 and VEGFR2), endothelial nitric oxide synthase, angiopoietin-1, bone morphogenetic protein receptors (BMPR1A and BMPR2), serotonin transporter (5-HTT) and the 5-HT(2B) receptor. Overpacing was associated with a decrease in cardiac output and an increase in pulmonary vascular pressures. However, there were no changes in pulmonary vascular resistance or in arteriolar medial thickness. There were increased expressions of genes encoding for ET-1, ETB, VEGF and VEGFR2, while expression of the other genes analysed remained unchanged. In vitro, pulmonary arteries showed decreased relaxation and increased reactivity, while systemic mammary arteries were unaffected. Early PH in heart failure is characterized by altered vasoreactivity and increased ET-1/ETB and VEGF/VEGFR2 signalling.

Rhinitis/Bronchopneumonia syndrome in Irish Wolfhounds.

This study describes the clinical, immunologic, genetic, and pathologic features of Irish Wolfhounds with rhinitis/bronchopneumonia syndrome. The dogs examined were from Belgium, The Netherlands, UK, Canada, Germany, and Switzerland. Signs included transient to persistent mucoid or mucopurulent rhinorrhea, cough, and respiratory dyspnea. Radiographic, rhinoscopic, and bronchoscopic findings were variable. Analysis of ciliary ultrastructure was performed in 5 affected dogs, but no characteristic primary ciliary defects (primary ciliary dyskinesia) were detected. Serum and bronchoalveolar lavage fluid (BALF) concentrations of IgA, IgG, and IgM were determined in some affected dogs and clinically normal Irish Wolfhounds. Serum IgA concentration was below the reference range in 5 of 8 affected dogs tested, whereas BALF IgA concentration was above the normal range in 2 affected adult dogs. The CD4 to CD8 lymphocyte subset ratio (CD4:CD8) in peripheral blood was tested in 3 affected dogs and was within the normal range. BALF CD4:CD8 was tested in 1 affected dog and was higher than the normal range. Decreased neutrophil phagocytosis was observed in 1 of the 4 dogs tested. Analysis of pedigrees of the Belgian, Canadian, German, and Swiss dogs revealed common ancestry, suggesting a heritable syndrome.

Expression of the serotonin 1b receptor in experimental pulmonary hypertension.

The pathogenesis of pulmonary arterial hypertension (PAH) remains uncertain. Both the serotonin and endothelin (ET) systems are believed to be involved. Recent studies pointed to the importance of the serotonin 2B receptor as a limiting step. The current authors investigated the lung tissue expression of serotonin receptors and of the serotonin transporter (5-HTT) by real-time-quantitative polymerase chain reaction in chronic overcirculation-induced PAH in growing piglets, with and without treatment with the dual ET receptor blocker bosentan. Pulmonary haemodynamic changes were described by pulmonary arterial impedance spectra. Three months after the surgical anastomosis of the left subclavian artery to the pulmonary arterial trunk, there was a shift of the impedance spectra to higher ratios of pressure and flow moduli, with increases in both 0 Hz impedance and characteristic impedance, and these changes were completely prevented by bosentan therapy. There was an increase in the expression of the serotonin 1B receptor. There was no change in the expression of the 5-HTT, and of the serotonin 2B, 1D, and 4 receptors. The overexpression of the serotonin 1B receptor was partially prevented by bosentan therapy. The present authors conclude that this early pulmonary arterial hypertension model is characterised by an endothelin receptor-dependent increased expression of the serotonin 1B receptor.

Use of ciliogenesis in the diagnosis of primary ciliary dyskinesia in a dog.

Primary ciliary dyskinesia is a congenital condition that may cause chronic rhinitis and bronchopneumonia. Primary ciliary dyskinesia may be diagnosed by induction of ciliogenesis by use of in vitro cell culture. Induction of ciliogenesis allows for differentiation between primary and secondary ciliary dyskinesia.

Eosinophilic bronchopneumopathy in dogs.

Eosinophilic bronchopneumopathy was diagnosed in 23 young dogs. Clinical signs included cough, gagging, and retching in all dogs, dyspnea in 21 dogs (91%), and nasal discharge in 12 dogs (52%). The most common radiographic findings were a moderate to severe bronchointerstitial pattern (68%, 13 of 19 dogs). Bronchoscopic findings included the presence of abundant yellow-green mucus or mucopurulent material (70%, 16 of 23 dogs) and severe mucosal thickening with an irregular or polypoid appearance (52%, 12 of 23 dogs), with partial airway closure during expiration in 3 dogs (13%). Peripheral blood eosinophilia was noted in 14 of 23 dogs (61%). Inflammatory cells in brush or bronchoalveolar lavage fluid cytologic preparations comprised more than 50% eosinophils in 14 of 23 dogs (61%), and 20-50% eosinophils in 6 dogs (26%). Eosinophilic infiltration of the bronchial mucosa was observed in biopsies from 19 dogs and was graded as mild (37%, 7 dogs), moderate (32%, 6 dogs), or severe (32%, 6 dogs). The mean serum immunoglobulin A concentration was almost double that of a population of 20 healthy dogs of various breeds. Oral glucocorticoids were administered on alternate days with progressive tapering of the dose; the dosage at maintenance varied between 0.1 and 1.0 mg/kg every other day. No relationship was found between the duration of clinical signs and the maintenance dosage or the cytologic and histopathologic grades.

Genetic analysis of the influence of pertussis toxin on experimental allergic encephalomyelitis susceptibility: an environmental agent can override genetic checkpoints.

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.

Doppler echocardiographic study of left and right ventricular function during dobutamine stress testing in conscious healthy dogs.

OBJECTIVE: To evaluate left and right ventricular filling and ejection performances by use of Doppler echocardiography in healthy, conscious dogs submitted to dobutamine stress testing. ANIMALS: 10 unsedated, healthy adult Beagles. PROCEDURE: Doppler echocardiography was performed during cardiac stress testing on each dog twice at 24-hour intervals. Dobutamine was infused in 10 micrograms/kg of body weight/min incremental dosages, from 12.5 to 42.5 micrograms/kg/min. Duration of each step was 15 minutes. Doppler measurements were recorded at baseline and at each stage of dobutamine infusion, whereas aortic diameter was measured at baseline and at peak dosage by use of two-dimensional echocardiography. RESULTS: Dobutamine infusion induced a significant increase in velocity time integrals and in peak flow velocities at the aortic, pulmonic, mitral, and tricuspid valves. Acceleration time-to-deceleration time ratio at the aortic wave also was increased significantly. On the other hand, ejection time, acceleration time, and deceleration time at the aortic and pulmonic valves and peak flow velocity of the E wave-to-peak flow velocity of the A wave ratio at the mitral and tricuspid valves decreased significantly during the test. The acceleration time-to-deceleration time ratio at the pulmonic wave was unchanged. A significant, progressive increase in cardiac index also was observed during dobutamine infusion, with a maximal increase of 104% from baseline. This was mediated initially by an increase in stroke index and, at higher dosages, by an increase in heart rate. CONCLUSIONS AND CLINICAL RELEVANCE: Doppler echocardiography performed during dobutamine stress testing may be a reliable method of assessing myocardial function in dogs with cardiovascular disease.

Nonresponsive generalized bacterial infection associated with systemic lupus erythematosus in a Beauceron.

A case of concurrent canine systemic lupus erythematosus (SLE) and generalized bacterial infection in a six-year-old female Beauceron is reported. The dog presented with purulent nasal and ocular discharges, skin lesions (including seborrhea, hyperkeratotic areas, and papules as well as ecchymoses around the eyes, on both sides of the pinnae, and on the vulva), generalized lymph node enlargement, a mitral murmur, and lameness. Later, facial swelling, a retrobulbar abscess, and a cough also developed. Occurrence of a generalized bacterial infection was established by culture of group-C, beta-hemolytic Streptococcus from the throat, the mouth, a biopsy site (popliteal lymph node area), the retrobulbar abscess, and the lung. The diagnosis of SLE was based on the clinical signs and particularly on the occurrence of antinuclear antibody (ANA) and antidoublestranded-desoxyribonucleic acid (ds-DNA) antibody. Interestingly, the latter type of antibodies were also detected in two young female puppies whelped by this dog. Salient histological findings included an extreme cell depletion of the lymph nodes and spleen and severe pneumonitis and peribronchiolitis. The results of this case indicate that a definite diagnosis of canine SLE can, at times, be made on the basis of the presence of serum ANA and ds-DNA antibodies.

Multiple independent regulatory pathways control UBI4 expression after heat shock in Saccharomyces cerevisiae.

Transcription of the polyubiquitin gene UBI4 of Saccharomyces cerevisiae is strongly induced by a variety of environmental stresses, such as heat shock, nutrient depletion and exposure to DNA-damaging agents. This transcriptional response of UBI4 is likely to be the primary mechanism for increasing the pool of ubiquitin for degradation of stress-damaged proteins. Deletion and promoter fusion studies of the 5' regulatory sequences indicated that two different elements, heat shock elements (HSEs) and stress response element (STREs), contributed independently to heat shock regulation of the UBI4 gene. In the absence of HSEs, STRE sequences localized to the intervals -264 to -238 and -215 to -183 were needed for stress control of transcription after heat shock. Site-directed mutagenesis of the STRE (AG4) at -252 to -248 abolished heat shock induction of UBI4 transcription. Northern analysis demonstrated that cells containing either a temperature-sensitive HSF or non-functional Msn2p/Msn4p transcription factors induced high levels of UBI4 transcripts after heat shock. In cells deficient in both heat stress pathways, heat-induced UBI4 transcript levels were considerably lower but not abolished, suggesting a role for another factor(s) in stress control of its expression.

Transcriptional factor mutations reveal regulatory complexities of heat shock and newly identified stress genes in Saccharomyces cerevisiae.

A computer-aided pattern search of the entire yeast genome was designed and used to identify 186 putative stress response element-regulated genes in Saccharomyces cerevisiae. Transcript levels of eight of these candidate genes were examined, and three (37%) were shown to be heat shock- and DNA damage-inducible and to require the Msn2p and Msn4p transcriptional activators for stress regulation. Significantly, several heat shock protein (HSP) genes were identified in this computer search. Using a series of single and multiple regulatory mutants, we demonstrate unexpected regulatory complexities among the HSP genes from S. cerevisiae following heat shock.

Laryngeal rhabdomyoma in a golden retriever.

A three-year-old male golden retriever had had progressive dyspnoea, exercise intolerance, stridor, and a modified bark for five months. A mass 2 cm in diameter was present dorsal to the right side of the larynx. Histological examination revealed cross-striations in some elongated cells, consistent with a diagnosis of rhabdomyoma, a diagnosis which was confirmed by positive immunohistochemical staining for myoglobin and desmin. The mass could not be removed without total laryngectomy and a permanent tracheostomy and the dog was euthanased.

Physiologic response to dobutamine infusion during cardiac stress testing of dogs.

OBJECTIVE: To evaluate response of various cardiovascular variables after administration of incremental doses of dobutamine in healthy conscious dogs, using standardized dobutamine stress echocardiography (DSE). ANIMALS: 8 healthy dogs. PROCEDURE: A DSE was performed twice on each dog within 24 hours. Dobutamine was infused at a rate of 12.5 to 42.5 microg/kg/min, using incremental increases of 10 microg/kg/min. Doppler sphygmomanometry, electrocardiography, and echocardiography were performed. Left ventricular size, global ventricular performance, and left ventricular systolic myocardial function were measured by means of echocardiography. RESULTS: At the highest dosage, dobutamine induced an increase of 20+/-3% and 109+/-12% in systolic blood pressure and cardiac index, respectively. The latter was associated with a significant increase in heart rate and stroke index. Fractional shortening of the left ventricle, fractional thickening of the left ventricular free wall and interventricular septum, ejection fraction, and mean velocity of fiber shortening had a progressive and significant increase during dobutamine infusion. Preejection period and left ventricular ejection time had a progressive and significative decrease during the stress test. CONCLUSIONS: The technique used was feasable, safe, and repeatable in healthy conscious dogs. Control values were determined. CLINICAL RELEVANCE: Data for these healthy dogs might be useful for comparison with results obtained from dogs with known or suspected cardiovascular disease.

Effects of dobutamine on isovolumic and ejection phase indices of cardiac contractility in conscious healthy dogs.

The aim of this study was to determine cardiac contractility using indices derived from cardiac catheterisation in conscious healthy dogs during dobutamine infusion. Eight dogs were studied. An ECG was recorded together with left ventricular pressure and volume which were measured using a conductance catheter with an integrated microtip pressure sensor. Eight indices of left ventricular systolic performance were derived from these records. Measurements were realised under basal conditions and during an incremental dobutamine challenge. The maximal rate of rise in ventricular pressure (max dP/dt), max dP/dt divided by the developed pressure and the mean systolic ejection rate were the most sensitive indices to detect dobutamine induced changes in contractility with maximal percentage changes of 122+/-11 per cent, 130+/-7 per cent and 102+/-24 per cent respectively. Ejection fraction increased significantly during dobutamine infusion (maximal percentage change of 43+/-9 per cent) whereas the pre-ejection period (PEP) and the left ventricular ejection time (LVET) decreased significantly (maximal percentage change of -41+/-2 per cent and -28+/-3 per cent respectively). All these six indices were significantly correlated with each other. Conversely, the ratio PEP/LVET and the LVET corrected for heart rate dependency showed a maximal percentage change of only -10+/-1 per cent and -16+/-7 per cent, respectively, during the dobutamine infusion and were not significantly correlated with the other contractility indices. This study demonstrated the feasibility of the conductance method to determine cardiac contractility in conscious healthy dogs submitted to a pharmacological stress testing and provides control values for eight indices of left ventricular contractility during dobutamine infusion at increasing dosages.

Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae.

The DDR2 gene of Saccharomyces cerevisiae is a multistress response gene whose transcription is rapidly and strongly induced by a diverse array of xenobiotic agents, and environmental and physiological conditions. The multistress response of this gene requires the pentanucleotide, 5' CCCCT, (C4T;STRE (STress Response Element)) and the zinc-finger transcription factors, Msn2p and Msn4p. A 51bp oligonucleotide (oligo 31/32) containing two STREs from the DDR2 promoter region was previously shown to direct heat shock activation of a lacZ reporter gene. In this work we demonstrate that the same element conferred a complete multistress response to an E. coli galK reporter gene introduced into yeast cells. A variant oligonucleotide in which both the STRE spacing and neighboring sequences were altered responded to the same spectrum of stresses, while substitution of nucleotides within the pentanucleotide completely abolished the multistress response. These results directly demonstrate that STREs are not only necessary but are sufficient for mediating a transcriptional response to a surprisingly diverse set of environmental and physiological conditions.

Structure and functional analysis of the multistress response gene DDR2 from Saccharomyces cerevisiae.

The DDR2 gene is a multistress response gene in Saccharomyces cerevisiae that is transcriptionally activated by more than thirteen xenobiotic agents and environmental or physiological stresses. The DDR2 gene encodes a small hydrophobic 61 amino acid polypeptide located on chromosome XV adjacent to the SPE2 locus. Disruption alleles of the DDR2 gene have been constructed and these ddr2 delta mutants show no defect in heat shock recovery or thermotolerance and appear normal for DNA damage sensitivity and mutagenesis.