Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression. We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway. PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors. PEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

Objective—To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses Animals—Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses. Procedures—Whole-body protein synthesis was measured with a 2-hour primed constant infusion of 13C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-13C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis. Results—Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples. Conclusions and Clinical Relevance—The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

Objective-To determine the effect of biopsy collection depth on the postprandial activation of mammalian target of rapamycin (mTOR) signaling factors, particularly protein kinase B, ribosomal protein S6 kinase, ribosomal protein S6, and eukaryotic initiation factor 4E binding protein 1 in middle-aged horses. Animals-6 healthy Thoroughbred mares (mean +/- SD age, 13.4 +/- 3.4 years). Procedures-Horses were fed a high-protein feed at 3 g/kg. Sixty minutes after horses were fed, the percutaneous needle biopsy technique was used to collect biopsy specimens from the gluteus medius muscle at 6, 8, and 10 cm below the surface of the skin. Muscle specimens were analyzed for the activation of upstream and downstream mTOR signaling factors, myosin heavy chain (MHC) isoform composition, and amino acid concentrations. Results-A 21% increase in MHC IIA isoform expression and a 21% decrease in MHC IIX isoform expression were identified as biopsy depth increased from 8 to 10 cm below the surface of the skin; however, no significant change was evident in the degree of MHC I expression with muscle depth. Biopsy depth had no significant effect on the phosphorylation of any of the mTOR signaling factors evaluated. Conclusions and Clinical Relevance-Postprandial mTOR signaling could be compared between middle-aged horses when biopsy specimens were collected between 6 and 10 cm below the surface of the skin. Optimization of muscle biopsy techniques for evaluating mTOR signaling in horses will facilitate the design of future investigations into the factors that regulate muscle mass in horses.

Objective-To use in vitro assays to evaluate the effects of a novel immunosuppressive agent, FTY720, on biological functions (migration, phagocytosis, and production of reactive-oxygen species ROS) of feline peripheral neutrophils and determine the cytotoxic effects of FTY720 on feline peripheral neutrophils. Sample Population-Peripheral neutrophils obtained from 8 healthy cats. Procedure-Peripheral neutrophils were isolated from blood samples obtained from the 8 cats and exposed to the phosphorylated form of FTY720 (FTY720-P). A fluorescence-based in vitro evaluation of migration was performed. Phagocytosis of microbes and production of ROS were evaluated by use of a 2-color flow cytometry system. Samples of whole blood obtained from the cats were incubated with various concentrations of FTY720-P, fluorescein-labeled Staphylococcus aureus, and dihydroethidium. Cytotoxic effects were evaluated by use of propidium iodide staining. Results-Addition of FTY720-P caused a slight nonsignificant decrease in phagocytosis and production of ROS by feline peripheral neutrophils. Migration activity of feline peripheral neutrophils was significantly increased by the addition of FTY720-P. Addition of FTY720-P at concentrations considered for clinical use did not increase the death rate of feline peripheral neutrophils. Conclusions and Clinical Relevance-FTY720 does not inhibit critical functions of feline peripheral neutrophils in vitro.

Objective-To study the antiviral activity of genistein, a soya isoflavone, on in vitro replication of bovine herpesvirus type 1 (BHV-1). Sample Population-Madin-Darby bovine kidney (MDBK) cells. Procedure-Effects of genistein on the magnitude and kinetics of inhibition of BHV-1 phosphorylation of glycoprotein E (gE) and in vitro replication of BHV-1 in MDBK cells were evaluated. Antiviral activity of genistein was compared with 2 compounds, estradiol-17β (EST) and tamoxifen (TAM), that have estrogenic and antiestrogenic activity, respectively. High-performance liquid chromatography (HPLC) was used to determine the concentration of genistein in medium from infected and uninfected MDBK cultures. Results-Genistein reduced BHV-1, but not gE-deleted BHV-1 (BHV-1gEΔ3.1), replication by 90% at 18 hours after inoculation. This inhibition was not sustained through 24 hours after inoculation. The genistein concentration in media from MDBK cells was decreased by 40% during BHV-1 infection, compared with 16% for uninfected cells, at 24 hours after inoculation. Genistein inhibited gE phosphorylation and BHV- 1 replication in a dose-dependent manner. Dosing with 25 µMgenistein at 0 and 12 hours after inoculation of BHV-1 was optimal for decreasing BHV-1 replication. Estradiol-17β EST and TAM did not affect BHV-1 replication. Conclusions and Clinical Relevance-The decrease in genistein concentration was a viral infection-dependent event. Genistein is an inhibitor of BHV-1 replication because of its ability to inhibit tyrosine kinase activity. A possible application may be for the control of BHV-1 infection in cattle by feeding soya products rich in genistein prior to or during periods of stress.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.

OBJECTIVE To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS 6 healthy adult horses. PROCEDURES Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography–tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS Compared with results for preadministration samples, platelet aggregation in response to 12.5μM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25μM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20μM) and prostaglandin E1 (3.3μM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition.

The cause of species difference in the susceptibility of erythrocytes to L-sorbose, and the difference in the hemolytic effect of sorbose on high potassium-containing (HK) and low potassium-containing (LK) canine erythrocytes were examined. L-sorbose was phosphorylated in canine erythrocytes, but not in human erythrocytes. Furthermore, sorbose-1-phosphate, a metabolite of L-sorbose, strongly inhibited the hexokinase of LK canine erythrocytes, but not that of HK canine erythrocytes. These results strongly indicated that inhibition of hexokinase by sorbose-1-phosphate in LK erythrocytes induced severe glycolytic limitation in these cells, resulting in hemolysis, and that HK erythrocytes are resistant to sorbose-induced hemolysis because these cells have a high hexokinase activity.

Accumulation of D-xylose by jejunal mucosa from healthy horses and rabbits was studied in Vitro. When tissue sheets were incubated with 1 mM D-xylose for 60 minutes, mucosa from horses and rabbits accumulated D-xylose against a concentration gradient. There was no accumulation when equine specimens were incubated with 5 mM D-xylose. By comparison, equine jejunum accumulated D-glucose against a concentration gradient when incubated in 5 mM D-xylose glucose. In equine and rabbit jejunum, 13.3 +/- 7.0% and 36 +/- 11.0%, respectively, of accumulated D-xylose was phosphorylated when sheets were incubated in 1 mM D-xylose. Short-circuit current and potential difference were lower in equine jejunum than in rabbit jejunum, possibly because of differences in tissue thickness. None of the transmucosal electrical measurements increased after addition of D-xylose (1 mM and 5 mM) or D-glucose (5 mM). The active transport system for D-xylose has a low affinity for this sugar and becomes saturated at low intraluminal concentrations. Therefore, abnormal D-xylose absorption test results in horses are more likely caused by abnormalities in mucosal surface area and mucosal permeability than by abnormalities of nutrient carbohydrate absorption.

The use of dietary supplements as an alternative treatment for joint-related pathologies such as osteoarthritis (OA) is increasing. However, there is little scientific evidence to support the intended use. The aim of this study was to evaluate the anti-inflammatory effects of creatine- and amino acid-based supplements in primary cultured canine chondrocytes (CnCs) as an in-vitro model of OA and compare the effects to more commonly used agents, such as the non-steroidal anti-inflammatory drug (NSAID), carprofen, and the joint supplement, glucosamine (GS). CnCs were stimulated with interleukin-1β (IL-1β) and the subsequent release of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNFα) was measured using an enzyme-linked immunosorbent assay (ELISA). Changes in oxylipins were also assessed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). All compounds examined were able to significantly reduce the release of PGE2 and TNFα and were associated with reductions in cyclooxygenase-2 (COX-2) expression and nuclear factor-kappaB (NF-κB) phosphorylation. The creatine- and amino acids-based supplements also altered the profile of oxylipins produced. All compounds examined were less effective at reducing the release of PGE2 than carprofen. Carprofen significantly increased release of TNFα from CnCs, however, while the other agents reduced TNFα release. This study suggests that creatine- and amino acid-based supplements may have a beneficial role in preventing inflammation within the joint and that further studies are warranted.