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Abstract

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.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro.

SAMPLE Venous blood samples from 40 adult horses.

PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities.

RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis.

CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE: To investigate the distribution of T-cell markers (CD4 and CD8α) in lymphoid organs of newborn, juvenile, and adult yaks.

ANIMALS: 15 healthy male yaks of various ages from highland plateaus.

PROCEDURES: Yaks were allocated to groups on the basis of age (newborn [1 to 7 days old; n = 5], juvenile [5 to 7 months old; 5], and adult [3 to 4 years old; 5]). The thymus, spleen, 5 mesenteric lymph nodes, and 5 hemal nodes were harvested from each yak within 10 minutes after euthanasia. Morphological characteristics of those lymphoid organs were assessed by histologic examination; expression of CD4 and CD8α mRNAs and proteins were measured by quantitative real-time PCR assay and immunohistochemical staining.

RESULTS: Among the lymphoid organs evaluated, expressions of CD4 and CD8α mRNAs were highest in the thymus in all age groups. In newborn lymphoid organs, CD4 mRNA expression and CD4+ cell distribution were more predominant, whereas in juvenile and adult lymphoid organs, CD8α mRNA expression and CD8α+ cell distribution were more predominant. The CD4+ and CD8α+ cells were mainly located in the cortex and medulla of the thymus, the medulla of the hemal nodes and mesenteric lymph nodes, the periarteriolar lymphoid sheaths, and the red pulp of the spleen.

CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the CD4 mRNA expression and CD4+ T-cell distribution in yak lymphoid organs decreased and CD8α mRNA expression and CD8α+ T-cell distribution increased with age. Moreover, CD8α+ cells were present in the follicles of yaks’ secondary lymphoid organs, which differs from findings for other mammals.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To measure immunologic responses of snakes after experimentally induced infection with ferlaviruses.

ANIMALS 42 adult corn snakes (Pantherophis guttatus) of both sexes.

PROCEDURES Snakes were inoculated intratracheally with genogroup A (n = 12), B (12), or C (12) ferlavirus (infected groups) or cell-culture supernatant (6; control group) on day 0. Three snakes from each infected group were euthanized on days 4, 16, 28, and 49, and 3 snakes from the control group were euthanized on day 49. Blood samples were collected from live snakes on days −6 (baseline), 4, 16, 28, and 49. Hematologic tests were performed and humoral responses assessed via hemagglutination-inhibition assays and ELISAs. Following euthanasia, gross pathological and histologic evaluations and virus detection were performed.

RESULTS Severity of clinical signs of and immunologic responses to ferlavirus infection differed among snake groups. Hematologic values, particularly WBC and monocyte counts, increased between days 4 and 16 after infection. A humoral response was identified between days 16 and 28. Serum IgM concentrations increased from baseline earlier than IgY concentrations, but the IgY relative increase was higher at the end of the study. The hemagglutination-inhibition assay revealed that the strongest reactions in all infected groups were against the strain with which they had been infected. Snakes infected with genogroup A ferlavirus had the strongest immune response, whereas those infected with genogroup B had the weakest responses.

CONCLUSIONS AND CLINICAL RELEVANCE Results of this experimental study suggested that the ferlavirus strain with the highest virulence induced the weakest immune response in snakes.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis.

SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram.

PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti–human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed.

RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C.

CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To characterize platelet-activating factor (PAF)–induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions.

ANIMALS 6 healthy Beagles.

PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 μg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each).

RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema.

CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood.

ANIMALS 10 multiparous sows.

PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin β-2.

RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells.

CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To determine the prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7–negative dogs from Spain and Italy.

ANIMALS 252 DEA 7–negative dogs from a population of 312 dogs that were previously tested for DEA 1, DEA 4, and DEA 7.

PROCEDURES A plasma sample was obtained from each dog and evaluated for anti-DEA 7 antibodies by the use of gel column agglutination. Each plasma sample underwent major crossmatching with RBCs from DEA 7-positive dogs. Samples that resulted in agglutination were then crossmatched with RBCs from DEA 1-negative, DEA 4-positive, and DEA 7–negative dogs to confirm the presence of anti-DEA 7 antibodies. Results were then used to calculate the risk for a delayed transfusion reaction in a DEA 7–negative dog with anti-DEA 7 antibodies after a transfusion with blood that was not crossmatched or typed for DEA 7.

RESULTS 96 of 252 (38.1%) plasma samples contained anti-DEA 7 antibodies. A DEA 7–negative dog with anti-DEA 7 antibodies had a 5.9% chance of developing a delayed hemolytic reaction after transfusion with blood not crossmatched or typed for DEA 7.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that canine blood used for transfusion should be crossmatched with the blood or plasma of the intended recipient prior to transfusion to minimize the likelihood that the recipient will develop a hemolytic reaction associated with anti-DEA 7 antibodies. Ideal canine blood donors should be negative for both DEA 1 and DEA 7.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells.

SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes.

PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow–differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay.

RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system.

CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Full access
in American Journal of Veterinary Research

Abstract

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.

Full access
in American Journal of Veterinary Research