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.
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.