OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs.
SAMPLE Blood samples from 8 healthy adult dogs.
PROCEDURES Blood samples were incubated with calcitriol (10−7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent.
RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression.
CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.
OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs.
SAMPLE Blood samples from 6 healthy adult dogs.
PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10−7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry.
RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes.
CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.
To determine the in vitro effects of epinephrine, norepinephrine, and dobutamine on lipopolysaccharide (LPS)-stimulated production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in blood from healthy dogs.
Blood samples from 9 healthy dogs.
Blood samples were incubated with LPS from Escherichia coli O127:B8 or PBSS (control) for 1 hour. Afterward, the samples were incubated with 10μM epinephrine, norepinephrine, or dobutamine or with saline (0.9% NaCl) solution (control) for 23 hours. Leukocyte viability was assessed by use of trypan-blue exclusion in blood from 2 dogs to ensure cell viability was not altered by the catecholamines. Tumor necrosis factor-α, IL-6, and IL-10 concentrations were measured in the supernatant in duplicate with a canine-specific multiplex bead-based assay. Blood samples from 2 dogs were used to create dose-response curves to evaluate whether the observed cytokine modulation was dependent on catecholamine concentration.
Incubation of blood with epinephrine and norepinephrine significantly increased LPS-stimulated production of IL-10, compared with the control. Epinephrine and norepinephrine significantly decreased LPS-stimulated production of TNF-α, compared with the control. Epinephrine and norepinephrine did not significantly alter LPS-stimulated production of IL-6. Dobutamine did not alter catecholamine production.
CONCLUSIONS AND CLINICAL RELEVANCE
Epinephrine and norepinephrine, but not dobutamine, had immunomodulatory effects on LPS-stimulated TNF-α and IL-10 production in blood from healthy dogs in this in vitro model of sepsis. Data suggested that dobutamine may have immune system-sparing effects in dogs with sepsis.
Objective—To evaluate assessment of circulating amino terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration as a means to discriminate between congestive heart failure and primary pulmonary disease in dogs.
Design—Prospective case series.
Animals—46 dogs with signs of respiratory distress or coughing.
Procedures—All dogs underwent physical and thoracic radiographic examinations. Dogs with evidence of heart disease (eg, murmur, arrhythmia, or large cardiac silhouette detected by radiography) also underwent echocardiography. Dogs with no evidence of heart disease or failure were included if they underwent bronchoalveolar lavage (with cytologic examination and bacterial culture of the lavage fluid). Blood samples for NT-proBNP assay were obtained within 12 hours of the diagnosis of heart failure or prior to bronchoalveolar lavage in dogs with primary pulmonary disease. Circulating concentrations of NT-proBNP were compared between groups and correlated with radiographic and echocardiographic measures of cardiac size.
Results—Congestive heart failure and primary pulmonary disease were diagnosed in 25 and 21 dogs, respectively. Dogs with congestive heart failure had significantly higher median serum or plasma NT-proBNP concentration (2,554 pmol/L; interquartile [25% to 75%] range, 1,651.5 to 3,475.5 pmol/L) than dogs with primary pulmonary disease (357 pmol/L; interquartile range, 192.5 to 565.5 pmol/L). Radiographic vertebral heart score and echocardiographic left atrial-to-aortic diameter ratio were not correlated with NT-proBNP concentration. Left ventricular end-diastolic diameter (measured echocardiographically) and NT-proBNP concentration were weakly correlated.
Conclusions and Clinical Relevance—Serum or plasma NT-proBNP concentration assessment may be useful for discrimination of congestive heart failure from primary pulmonary disease in dogs with respiratory distress or cough.
Objective—To compare clinical findings and inflammatory mediator production among cats with sepsis, cats with noninfectious systemic inflammatory response syndrome (SIRS), and healthy cats.
Animals—Cats with sepsis (n = 16) or SIRS (19) and 8 healthy control cats.
Procedures—Clinical variables were recorded for each cat, and plasma tumor necrosis factor (TNF) and interleukin (IL)-1β activities and IL-6 and CXC chemokine ligand (CXCL)-8 concentrations were determined at initial evaluation.
Results—Clinicopathologic abnormalities associated with sepsis in cats included a high band neutrophil percentage, eosinopenia, hyponatremia, hypochloremia, hypoalbuminemia, hypocalcemia, and hyperbilirubinemia. When the sepsis and SIRS groups were compared, the only significant differences in the CBC and plasma biochemical findings were band neutrophil percentage and albumin concentration. Cats with sepsis had significantly greater plasma TNF activity than did healthy cats and were more likely to have detectable concentrations of IL-6 than were cats with SIRS or healthy cats. Plasma IL-1β activity did not differ among groups, and CXCL-8 was not detectable in most (32/43) cats. Mortality rate was not significantly greater for cats with sepsis (7/16) than for cats with SIRS (5/19). Plasma IL-1β activity and IL-6 and chloride concentrations were the only variables correlated with nonsurvival in the sepsis group.
Conclusions and Clinical Relevance—Cats with sepsis may have various clinicopathologic abnormalities but are more likely to have a high band neutrophil percentage and hypoalbuminemia than cats with noninfectious SIRS. Plasma interleukin-1β activity and plasma IL-6 and chloride concentrations may be useful prognostic biomarkers for septic cats.
Objective—To determine the effects of ketamine hydrochloride on hemodynamic and immunologic alterations associated with experimentally induced endotoxemia in dogs.
Animals—9 mixed-breed dogs.
Procedures—In a crossover study, dogs were randomly allocated to receive ketamine (0.5 mg/kg, IV, followed by IV infusion at a rate of 0.12 mg/kg/h for 2.5 hours) or control solution (saline [0.9% NaCl] solution, 0.25 mL, IV, followed by IV infusion at a rate of 0.5 mL/h for 2.5 hours). Onset of infusion was time 0. At 30 minutes, lipopolysaccharide (LPS; 1 μg/kg, IV) was administered. Heart rate (HR), systolic arterial blood pressure (SAP), plasma tumor necrosis factor (TNF)-α activity, and a CBC were evaluated.
Results—Mean SAP was significantly reduced in dogs administered ketamine or saline solution at 2 and 2.5 hours, compared with values at time 0. However, there was no significant difference between treatments. At 1, 2, and 2.5 hours, dogs administered ketamine had a significantly lower HR than dogs administered saline solution. Although plasma TNF-α activity significantly increased, compared with values at time 0 for both groups, ketamine-treated dogs had significantly lower peak plasma TNF-α activity 1.5 hours after LPS administration. All dogs had significant leukopenia and neutropenia after LPS administration, with no differences detected between ketamine and saline solution treatments.
Conclusions and Clinical Relevance—Administration of a subanesthetic dose of ketamine had immunomodulating effects in dogs with experimentally induced endotoxemia (namely, blunting of plasma TNF-α activity). However, it had little effect on hemodynamic stability and no effect on WBC counts.
OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples.
SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats.
PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups.
RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection.
CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.
Objective—To compare concentrations of interleukin (IL)-4, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and total nitric oxide (NO) metabolites in bronchoalveolar lavage fluid (BALF) for discrimination between asthma and chronic bronchitis in cats.
Procedures—Cats screened with cytologic examination of BALF included 13 client-owned cats with naturally developing asthma, 8 client-owned cats with chronic bronchitis, 23 research cats with experimentally induced asthma, 33 research cats with experimentally induced nonseptic suppurative inflammation of the airways, and 20 healthy control cats. Banked unconcentrated BALF supernatant samples were assayed for concentrations of IL-4, IFN-γ, TNF-α, and total NO metabolites.
Results—Concentrations of IL-4 and IFN-γ in BALF were less than the limits of detection for most cats, precluding statistical analysis. No significant differences were detected among groups for TNF-α concentrations. Concentrations of total NO metabolites were significantly higher in cats with clinical chronic bronchitis, compared with research cats with nonseptic suppurative inflammation or research cats with asthma.
Conclusions and Clinical Relevance—There were no significant differences in tested biomarkers between cats with asthma and healthy control cats. None of the measured cytokines or NO metabolites were useful for discriminating between cats with naturally developing asthma and those with chronic bronchitis.
Objective—To determine the lowest dose of cosyntropin on a per body weight basis that would produce maximal cortisol and aldosterone secretion and the ideal timing of blood sample collection after ACTH stimulation in healthy cats.
Design—Randomized crossover trial.
Animals—7 adult sexually intact male purpose-bred cats.
Procedures—Each cat received saline (0.9% NaCl) solution (control) and 5 doses (125 μg/cat and 10, 5, 2.5, and 1 μg/kg [4.54, 2.27, 1.14, and 0.45 μg/lb]) of cosyntropin IV with a 2-week washout period between treatments. Blood samples were obtained before (baseline) and at 15, 30, 45, 60, 75, and 90 minutes after administration of saline solution or cosyntropin.
Results—Serum cortisol and aldosterone concentration increased significantly, compared with baseline values, after administration of all cosyntropin doses. Lower doses of cosyntropin resulted in an adrenocortical response equivalent to the traditional dose of 125 μg/cat. The lowest doses of cosyntropin that stimulated a maximal cortisol and aldosterone response were 5 and 2.5 μg/kg, respectively. Lower doses of cosyntropin resulted in a shorter interval between IV administration of cosyntropin and peak serum cortisol and aldosterone concentrations.
Conclusions and Clinical Relevance—Low-dose ACTH stimulation testing with IV administration of cosyntropin at 5 μg/kg followed by blood sample collection at 60 to 75 minutes resulted in concurrent peak serum cortisol and aldosterone concentrations that were equivalent to those achieved following administration of cosyntropin at 125 μg/cat, the standard dose currently used.