OBJECTIVE To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). ANIMALS 8 healthy adult horses. PROCEDURES Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays. RESULTS Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone. CONCLUSIONS AND CLINICAL RELEVANCE Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.

In horses, hyperinsulinemia and insulin resistance (insulin dysregulation) are associated with the development of laminitis. Although obesity is associated with insulin dysregulation, the mechanism of obesity-associated insulin dysregulation remains to be established. We hypothesized that oxidative stress in skeletal muscle is associated with obesity-associated hyperinsulinemia in horses. Thirty-five light breed horses with body condition scores (BCS) of 3/9 to 9/9 were studied, including 7 obese, normoinsulinemic (BCS ≥ 7, resting serum insulin < 30 μIU/mL) and 6 obese, hyperinsulinemic (resting serum insulin ≥ 30 μIU/mL) horses. Markers of oxidative stress (oxidative damage, mitochondrial function, and antioxidant capacity) were evaluated in skeletal muscle biopsies. A Spearman’s rank correlation coefficient was used to determine relationships between markers of oxidative stress and BCS. Furthermore, to assess the role of oxidative stress in obesity-related hyperinsulinemia, markers of antioxidant capacity and oxidative damage were compared among lean, normoinsulinemic (L-NI); obese, normoinsulinemic (O-NI); and obese, hyperinsulinemic (O-HI) horses. Increasing BCS was associated with an increase in gene expression of a mitochondrial protein responsible for mitochondrial biogenesis (estrogen-related receptor alpha, ERRα) and with increased antioxidant enzyme total superoxide dismutase (TotSOD) activity. When groups (L-NI, O-NI, and O-HI) were compared, TotSOD activity was increased and protein carbonyls, a marker of oxidative damage, decreased in the O-HI compared to the L-NI horses. These findings suggest that a protective antioxidant response occurred in the muscle of obese animals and that obesity-associated oxidative damage in skeletal muscle is not central to the pathogenesis of equine hyperinsulinemia.

The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10(3) MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine.

OBJECTIVE To evaluate the effects of dietary nonphytate phosphorus (NPP) content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of hens that were or were not inoculated with Salmonella Typhimurium. ANIMALS 64 Salmonella-free hens. PROCEDURES Hens were fed a diet with 0.22% (control; n = 32) or 0.42% (high-P; 32) NPP for 6 weeks and then orally inoculated with S Typhimurium (5 × 107 CFUs) or PBSS. Tissues were obtained from 8 S Typhimurium–inoculated and 8 PBSS-inoculated hens from each group at 2 and 7 days postinoculation (DPI). Percentages of ileal CD4+ and CD8+ lymphocytes were determined by flow cytometry. Cytokine mRNA expression was determined by quantitative real-time PCR assays. RESULTS For S Typhimurium–inoculated hens, plasma parathyroid hormone concentration was significantly increased and 1,25-dihydroxyvitamin D3 concentration was decreased in hens fed the high-P diet, compared with values in hens fed the control diet. Salmonella Typhimurium inoculation caused an increase in the percentage of ileal CD8+ lymphocytes and the expression of interleukin (IL)-1β, IL-6, IL-8, interferon-γ, IL-12, and IL-18 in the cecal tonsils and spleen and a decrease in the expression of IL-4 and IL-10 in the cecal tonsils. Hens fed the high-P diet had significantly increased splenic expression of interferon-γ at 2 DPI and IL-1β, IL-6, IL-12, and IL-18 at 7 DPI, compared with hens fed the control diet. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested there was a T-helper 1 cytokine reaction in the cecal tonsils and spleen of S Typhimurium–inoculated hens, and dietary NPP content altered calcium regulation hormone concentrations and affected splenic cytokine expression.