Background Pituitary pars intermedia dysfunction (PPID) is a common endocrinopathy of horses diagnosed with a thyrotropin‐releasing hormone (TRH) stimulation test. Hypothesis/Objectives Describe the repeatability of TRH stimulation in horses with and without PPID in winter and autumn. Animals Twenty adult horses; 6 controls and 6 with PPID tested in autumn, 8 controls and 6 with PPID tested in winter with 3 controls and 3 with PPID tested in both seasons. Methods Thyrotropin‐releasing hormone stimulation was performed on 2 consecutive occasions, 1 week before and 1 week after the winter solstice and the autumn equinox. Blood was collected before and 30 minutes after IV injection of 1 mg of TRH. ACTH concentration was determined by a chemiluminescent assay. Repeatability and test‐retest reliability were assessed by repeated measures analysis of variance, intraclass correlation coefficient and within‐horse coefficients of variation (CV). Bland‐Altman plots were generated to visualize agreement between repetitions. Results In winter, no week effect was detected on the results of the TRH simulation and the test had an excellent test‐retest reliability. In autumn, after‐TRH ACTH concentrations were significantly lower on week 2 (P = .02) and the test only had a good test‐retest reliability. There were significantly larger within‐horse CV during autumn (P = .04) and after TRH stimulation (P = .04). There were 2 misclassifications in winter and 4 in autumn. Conclusions and Clinical Importance The TRH stimulation test was repeatable when performed 2 weeks apart in winter; however, in autumn, more variability in after‐TRH ACTH concentrations resulted in decreased repeatability.

Recent studies have demonstrated that innate immune cells, i.e. neutrophils and monocytes, provide the initiating stimulus for venous thrombus development. Gas6 was found to promote inflammation by inducing interactions between the endothelium and innate immune cells. In addition, we recently showed that Gas6 was involved in venous thrombosis by inducing tissue factor expression in the endothelium. Since Gas6 is expressed in monocytes, we hypothesize that Gas6 may be involved in monocyte recruitment during venous thrombosis. Venous thrombosis was induced in the inferior vena cava of wild type (WT) and Gas6 deficient (-/-) mice using 5% FeCl3. Using ultrasonography, we found that global monocyte depletion by clodronate resulted in the formation of smaller thrombi. Selective depletion of the pro-inflammatory (Ly6Chi) monocyte subset, using an anti-CCR2 antibody, also induced the formation of smaller clots. In addition, MOMA-2 (monocyte-macrophage marker) staining showed a reduced number of monocytes in thrombi from Gas6-/- mice. More importantly, immunofluorescent staining revealed that fewer Ly6Chi monocytes were recruited to the thrombi of Gas6-/- mice compared to WT. However, Ly6Clow (patrolling) monocytes were equivalently recruited between Gas6-/- and WT mice. In vitro, mRNA expression of CCR2 was increased by thrombin in WT but not in Gas6-/- monocytes. The mRNA and protein expression of the CCR2 ligand, CCL2, was also increased by thrombin in WT but not in Gas6-/- endothelial cells. CCL2 secretion, as demonstrate by ELISA, was induced by thrombin treatment in WT but not in Gas6-/- endothelial cells. Conditioned media from WT or Gas6-/- endothelial treated by thrombin was used for monocyte migration experiments. The conditioned media from WT endothelial cells treated with thrombin increased migration of WT monocytes compared to media from untreated or Gas6-/- endothelial cells. Our results demonstrate an important role for Ly6Chi monocytes in thrombus formation and that Gas6 is specifically involved in the recruitment of these monocytes through the expression of CCL2 and CCR2. Disclosures No relevant conflicts of interest to declare.

Background: Endocrinopathic laminitis develops in association with insulin dysregulation, but the role of insulin in the pathogenesis remains unclear.Hyperinsulinemia can cause hypoaminoacidemia, which is associated with integumentary lesions in other species and therefore warrants investigation as a potential mechanism in laminitis.Objective: Evaluate plasma amino acid concentrations in the euglycemichyperinsulinemic clamp (EHC) and prolonged glucose infusion (PGI) laminitis models.

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