ABSTRACT. We investigated the anti-inflammatory effects of ethyl pyruvate (EP) on LPS-stimulated canine PBMCs in vitro. We found that EP treatment inhibited the mRNA expressions of proinflammatory cytokines (TNF- and IL-6), but induced mRNA expression of anti-inflammatory cytokines (IL-10). ELISA measurements revealed that EP also effectively downregulates the LPS-induced increase in proinflammatory cytokine release, while upregulating anti-inflammatory cytokine release. These data indicate that EP could be an effective anti-inflammatory agent in dogs.KEY WORDS: canine peripheral blood mononuclear cells, cytokine, ethyl pyruvate, mRNA.J. Vet. Med. Sci. 72(10): 1379-1381, 2010 Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite pyruvic acid and has been shown to exhibit antioxidant [5] and anti-inflammatory [1] properties. While EP is known to inhibit activation of p28 mitogen-activated protein kinase and NF-B in murine macrophages [10], its anti-inflammatory mechanism remains unclear.We hypothesized that lipopolysaccharide (LPS) treatment of canine PBMCs would induce proinflammatory cytokine expression and that EP would attenuate this increase at either the mRNA or protein level. To address this hypothesis, we selected TNF- and IL-6 as representative proinflammatory cytokines that are upregulated after LPS administration. In addition, we selected IL-10 as an anti-inflammatory cytokine.Blood was collected from six healthy beagles and peripheral blood mononuclear cells (PBMCs) were separated using Histopaque ) and 100 U/ml penicillin G and 10 g/ml streptomycin (Sigma) at 37C in 5% CO 2 and incubated overnight. The next day, the mononuclear cells were exposed to 100 ng/ml of LPS (Escherichia coli serotype O111: B4, Sigma) and simultaneously treated with various concentrations of EP (Sigma) (5 d 10 mM each) based on previous in vitro studies [9]. The cell pellets were used for RNA isolation, and the cell-free supernatants were stored at -80C until they were analyzed. Samples were harvested after 3, 6, 12 and 24 hr of cultivation. Unstimulated cells and cells in 10 mM ethyl pyruvate were used as controls. All procedures used in this study were approved by local animal welfare authorities (CBU2008-021).For the quantification of cytokine gene expression, SYBR ® green real-time PCR was used. Total RNA was extracted using an RNeasy The primers utilized in this study are summarized in Table 1. The optimal annealing temperature (T a ) for all assays ranged from 60C for GAPDH and IL-6 to 62C for TNF- and IL-10. Primer efficiency calculations [E (%) = (10 (1/-S) -1) 100, S=slope] for all of the standard lines were between 95.7 and 102.4%. Relative quantification was analyzed using the -2 CT method as previously described [4]. Tests were performed in duplicate.Our quantification of cytokine mRNA abundance demonstrated that EP inhibits TNF- and IL-6 mRNA synthesis and promotes IL-10 mRNA expression on LPS stimulation,
A 3-month-old intact male, Labrador retriever was presented with the history of coagulopathy and anemia. The results of initial screening tests of the hemostatic system yielded a tentative diagnosis of hemophilia. Activated partial thromboplastin time (APTT) was distinctly prolonged (106 seconds) and prothrombin time (PT) was not detected due to markedly prolonged test time. Whole blood transfusions (20 me l/kg body weight) were carried out prior to assays of coagulation factor. After transfusion, the patient recovered well and hemorrhage ceased. Blood samples were assessed for coagulation factor activity. The patient showed markedly low factor IX coagulation activity (5%, reference range: 7∼140%) and was diagnosed with hemophilia B. After recovery, the patient was discharged from the hospital. However, 4 months later the patient was re-hospitalized for recurrence of the initial symptoms. The owner did not want to pursue further treatment and the patient died of respiratory distress two days later.
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