Casey J. LeBlanc

Effects of dietary supplementation with fish oil on in vivo production of inflammatory mediators in clinically normal dogs

OBJECTIVE To evaluate the effect of diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on in vivo production of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha, prostaglandin E2 (PGE2), and platelet-activating factor (PAF) in dogs. ANIMALS 15 young healthy dogs. PROCEDURES Dogs were randomly allocated to receive an isocaloric ration supplemented with sunflower oil (n=5), fish oil (5), or fish oil plus vitamin E (5) for 12 weeks. At week 12, in vivo production of inflammatory mediators was evaluated in serum at multiple time points for 6 hours following stimulation with IV administration of lipopolysaccharide (LPS). RESULTS Serum activity or concentration (area under the curve) of IL-1, IL-6, and PGE2 significantly increased after LPS injection in all groups but to a lesser extent in dogs receiving the fish oil diet, compared with results for dogs receiving the sunflower oil diet. Serum activity of TNF-alpha and PAF concentration also increased significantly after LPS injection in all groups but did not differ significantly among groups. CONCLUSIONS AND CLINICAL RELEVANCE A fish oil-enriched diet consisting of 1.75 g of EPA/kg of diet and 2.2 g of DHA/kg of diet (dry-matter basis) with an n-6:n-3 fatty acid ratio of 3.4:1 was associated with significant reductions in serum PGE2 concentrations and IL-1 and IL-6 activities. Results supported the use of EPA- and DHA-enriched diets as part of antiinflammatory treatments for dogs with chronic inflammatory diseases. Additional studies in affected dogs are warranted to further evaluate beneficial anti-inflammatory effects of EPA- and DHA-enriched diets.

Exposure of adult mice to environmental tobacco smoke fails to enhance the immune response to inhaled antigen.

Epidemiologic evidence supports a role for environmental tobacco smoke (ETS) in the occurrence and severity of allergies/asthma. However, neither the precise combination of ETS and allergen exposure nor the mechanism (or mechanisms) by which these factors interact and contribute to asthma induction is known. Animal model studies have failed to establish a convincing relationship between ETS exposure and asthma induction, perhaps because of methodological inadequacies. Here, we tested the hypothesis that ETS inhalation would provoke an asthmatic response by overcoming normal airway tolerance to inhaled antigens. Our protocol combined daily ETS exposure with nose-only sensitization to ovalbumin. Three strains of mice were tested, each with a different level of susceptibility to airway hypersensitivity. Immunological responses were assessed by immunoglobulin production. Airway inflammation was assessed by bronchoalveolar lavage differentials and lung histopathology. Airway hyperresponsiveness was determined by methacholine challenge. The mice produced ovalbumin-specific antibodies following ovalbumin exposure in a strain-dependent manner. Only the A/J mice produced detectable levels of ovalbumin-specific immunoglobulin (Ig) E. Both A/J and BALB/c mice produced ovalbumin-specific IgG1 antibodies. The C57Bl/6 mice did not produce detectable levels of antibodies. The A/J mice also exhibited airway inflammation following ovalbumin exposure. Neither the C57Bl/6 nor the BALB/c mice exhibited signs of airway inflammation. Exposure to ETS failed to enhance ovalbumin-specific antibody production, airway inflammation, or hyperresponsiveness. Together these results indicate that ETS exposure accompanied by nose-only allergen sensitization fails to overcome aerosol tolerance in adult mice.

Comparison of aspiration and nonaspiration techniques for obtaining cytologic samples from the canine and feline spleen

BACKGROUND Ultrasound-guided fine needle aspiration of the spleen is commonly used in the diagnostic evaluation of veterinary patients. Techniques using suction delivered through a 6-20-cm(3) syringe are the most commonly described means of obtaining cytologic samples of the spleen. Comparison studies of various human lesions have shown nonaspiration techniques to produce equal or superior cytologic specimens with less blood than specimens obtained using aspiration techniques. OBJECTIVE The purpose of this study was to compare the quality of splenic cytology specimens obtained using aspiration and nonaspiration techniques. METHODS Client-owned dogs (n=24) and cats (n=7) receiving an abdominal ultrasound at the University of Tennessee College of Veterinary Medicine were enrolled in the study between January and June 2005. Samples were obtained from patients with and without sonographic splenic abnormalities. Two clinical pathologists, working independently and blinded to the method of sample collection, graded the cytologic specimens using a subjective scoring system for cellularity, amount of blood, and preservation of cellular morphology. RESULTS Agreement between the 2 independent observers was good. Direct comparison of the 2 techniques showed that samples obtained by the nonaspiration method had higher cellularity (P=.0002), less blood (P=.0023), and similar cell morphology (P=1.0000) compared with samples obtained by the aspiration method. CONCLUSION These results suggest the nonaspiration technique is a superior method for obtaining a high-quality cytologic specimen from the canine and feline spleen.

May-Hegglin anomaly in a dog

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 μM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dogs MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.

Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes

OBJECTIVE To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes. SAMPLE POPULATION Nodal lymphocytes from 21 clinically normal dogs. PROCEDURES Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors. RESULTS Expression of P-gp was (mean +/- SEM) 50.62 +/- 13.39 (n = 21) and that of MRP was 2.16 +/- 0.25 (13). Functional activity was 1.27 +/- 0.06 (n = 21) for P-gp and both inhibitors and 21.85 +/- 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated. CONCLUSIONS AND CLINICAL RELEVANCE Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.

Chronic lymphocytic leukaemia in a pot-bellied pig.

An 11-year-old, neutered male, Vietnamese pot-bellied pig was admitted for routine dental and foot care. As a part of routine geriatric evaluation, blood was submitted for a complete blood count and serum biochemical analysis. The blood count revealed a marked leucocytosis due to lymphocytosis. Further diagnostic evaluation, including abdominal and thoracic radiography, abdominal ultrasonography and blood lymphocyte immunophenotyping confirmed a diagnosis of chronic lymphocytic leukaemia of T-cell origin. Treatment was initiated with oral prednisone and cyclophosphamide. The pig did well on treatment for about 1 month after discharge but was then euthanized.