Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Background Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. Results In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. Conclusion Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated

A Potential Role for Pro-Inflammatory Cytokines in the Development of Insulin Resistance in Horses

Understanding the mechanisms involved in the development of insulin resistance in horses should enable development of effective treatment and prevention strategies. Current knowledge of these mechanisms is based upon research in obese humans and rodents, in which there is evidence that the increased production of pro-inflammatory cytokines by adipose tissue negatively influences insulin signaling in insulin-responsive tissues. In horses, plasma concentrations of the cytokine, tumor necrosis factor-α, have been positively correlated with body fatness and insulin resistance, leading to the hypothesis that inflammation may reduce insulin sensitivity in horses. However, little evidence has documented a tissue site of production and a direct link between inflammation and induction of insulin resistance has not been established. Several mechanisms are reviewed in this article, including the potential for macrophage infiltration, hyperinsulinemia, hypoxia, and lipopolysaccharide to increase pro-inflammatory cytokine production by adipose tissue of obese horses. Clearly defining the role of cytokines in reduced insulin sensitivity of horses will be a very important step in determining how obesity and insulin resistance are related

De novo fatty acid synthesis and NADPH generation in equine adipose and liver tissue

The lipogenic capacities of equine liver and adipose tissue explants were investigated in vitro. Preference for glucose or acetate as the primary carbon source for de novo fatty acid synthesis was determined using (14)C labeled substrates. Additional aims included determining the relative contribution of NADPH generating pathways to reducing equivalent generation and comparing the lipogenic activity of two adipose depots, mesenteric and subcutaneous harvested from the crest region of the neck. Mesenteric adipose tissue had greater lipogenic activity than subcutaneous adipose tissue, and liver tissue showed minimal (14)C incorporation into fatty acids, indicating a low hepatic lipogenic capacity. Acetate was found to be the primary carbon source for fatty acid synthesis due to both the appearance of the (14)C label in the lipid fraction and the low activity of ATP-citrate lyase. Finally, the pentose phosphate and isocitrate dehydrogenase enzymes contributed to NADPH production in equine adipose tissue

Effects of Crude Rice Bran Oil and a Flaxseed Oil Blend in Young Horses Engaged in a Training Program

Rice bran oil and flaxseed oil contain omega-3 fatty acids with the potential to reduce post-exercise inflammation and muscle damage. This study measures plasma interleukin-1β and creatine kinase and fatty acid profiles in lightly worked, young horses (Equus caballus) undergoing an exercise test after 60 days (d) of oil consumption, where the oil replaced 25% of concentrate calories. Treatments consisted of CON (no oil), FLAX (flaxseed oil blend), and RICE (crude rice bran oil). Blood was collected pre-exercise, and again at 1 min, 30 min, 24 h, 48 h, and 72 h post-IET. Data were analyzed by repeated measures ANOVA. Plasma creatine kinase activity was not different in CON during the study, greater (p < 0.05) in RICE from pre-exercise to 30 min post-exercise across all exercise tests, and lesser (p < 0.05) in FLAX at 30 min post-exercise on d 30 compared to d 0. Plasma interleukin-1β was greater (p < 0.01) in CON on d 60, but no differences were observed in FLAX and RICE throughout the study. Plasma alpha-linolenic and linoleic acids were greatest (p < 0.05) in FLAX after 30 d of inclusion, while CON horses had greater (p < 0.05) EPA across all exercise tests and DHA after 60 d. These results indicate that 60 d of inclusion of crude rice bran oil or a flaxseed oil blend may benefit lightly worked, young horses by reducing training-program-related increases in interleukin-1β, while a flaxseed oil blend may reduce exercise-induced increases in creatine kinase. Additionally, the flaxseed oil blend has the potential to increase plasma omega-3 and omega-6 fatty acids. Replacing 25% of concentrate calories with flaxseed or rice bran oil has potential benefits for young horses in training

Effects of the insulin sensitizing drug, pioglitazone, and lipopolysaccharide administration on markers of systemic inflammation and clinical parameters in horses

Equine metabolic syndrome (EMS) is a condition of obese horses characterized by insulin resistance, systemic inflammation, and an increased risk of laminitis. The pathogenesis of EMS is thought, in part, to be due to inflammatory proteins produced by adipose tissue. Reducing inflammation may decrease the incidence of laminitis in horses with EMS. Pioglitazone hydrochloride, a thiazolidinedione, has efficacy to reduce obesity associated inflammation in humans. Eight normal, adult, horses were administered 1mg/kg pioglitazone for 14 days, and eight horses served as controls. Physical examination and hematologic variables, transcript abundance of pro-inflammatory cytokines in skeletal muscle and adipose tissue, and circulating concentrations of the acute phase protein, serum amyloid A and pro-inflammatory cytokine, TNF-α were assessed prior to, and following, an LPS infusion (35 ng/kg). The objective was to determine if pre-treatment with pioglitazone would mitigate the development of inflammation and associated clinical markers of inflammation following LPS administration. Lipopolysaccharide administration induced systemic inflammation, as assessed by clinical and hematological aberrations, increased TNF-α, SAA and adipose tissue IL-6 mRNA abundance, however no mitigating effects of pioglitazone were detected. A longer treatment period or higher dose might be indicated for future experiments