Multicenter Placebo-Controlled Randomized Study of Ethyl Pyruvate in Horses Following Surgical Treatment for ≥ 360° Large Colon Volvulus.

Identifying therapies that mitigate ischemic colonic injury and improve mucosal healing and intestinal viability are crucial to improving survival in horses with ≥360° large colon volvulus (LCV). Ethyl pyruvate is the ethyl ester of pyruvate with diverse pharmacologic effects that limit ischemic injury and hasten intestinal mucosal repair in preclinical rodents, sheep and swine models. The objective of this study was to determine the effects of ethyl pyruvate on systemic indices of colon viability, expression of inflammatory genes in whole blood, morbidity and survival after surgical correction of LCV compared to controls. Horses received either 150 mg/kg ethyl pyruvate in 1 liter lactated Ringer's solution (LRS) or 1 liter LRS intravenously (IV) every 6 h for 24 h following surgical recovery for correction of LCV. Colic duration, perioperative heart rate (HR), packed cell volume (PCV), total solids (TS), blood L-lactate concentration, surgical time, intraoperative episodes of hypoxemia and hypotension, expression of inflammatory cytokine genes, fecal consistency and survival to hospital discharge were compared between ethyl pyruvate treated horses and controls. Twenty-two horses, 12 receiving ethyl pyruvate and 10 controls, were enrolled in the study. Ethyl pyruvate was safely administered to horses following surgical correction of LCV. No significant effects of ethyl pyruvate on post-operative variables, including survival, were found. Seven of 12 ethyl pyruvate treated horses and 5/10 controls survived to hospital discharge. Higher HR, PCV and blood L-lactate concentration at the time of hospital admission, P = 0.005, 0.01, 0.04, respectively, 24 h after surgery, P = 0.001, 0.03, 0.02, respectively, were associated with death. Heart rate, P = 0.005, 48 h after surgery was associated with death. Ethyl pyruvate was safely administered to horses following correction of LCV with no apparent adverse events but was not associated with improved post-operative outcomes including survival. A larger, randomized control trial is needed to fully evaluate the effectiveness of ethyl pyruvate. A major limitation of this investigation is the small sample size, making the study underpowered and creating a high possibility of type II error.

Multiple adrenocortical steroid response to administration of exogenous adrenocorticotropic hormone to hospitalized foals.

BACKGROUND: The hypothalamic-pituitary-adrenal axis regulates the response to sepsis-associated stress. Relative adrenal insufficiency or adrenocorticotropic hormone (ACTH):cortisol imbalance, defined as a poor cortisol response to administration of ACTH, is common and associated with death in hospitalized foals. However, information on other adrenal steroid response to ACTH stimulation in sick foals is minimal. OBJECTIVE: To investigate the response of multiple adrenocortical steroids to administration of ACTH in foals. ANIMALS: Hospitalized (n = 34) and healthy (n = 13) foals. METHODS: In this prospective study, hospitalized foals were categorized into 2 groups using cluster analysis based on adrenal steroids response to ACTH stimulation: Cluster 1 (n = 11) and Cluster 2 (n = 23). After baseline blood sample collection, foals received 10 µg of ACTH with additional samples collected at 30 and 90 minutes after ACTH. Steroid and ACTH concentrations were determined by immunoassays. The area under the curve (AUC) and Delta0-30 were calculated for each hormone. RESULTS: The AUC for cortisol, aldosterone, androstenedione, pregnenolone, 17α-OH-progesterone, and progesterone were higher in critically ill (Cluster 1) compared to healthy foals (P < .01). Delta0-30 for cortisol and 17α-OH-progesterone was lower in Cluster 1 (24%, 26.7%) and Cluster 2 (16%, 11.2%) compared to healthy foals (125%, 71%), respectively (P < .05). Foals that died had increased AUC for endogenous ACTH (269 versus 76.4 pg/mL/h, P < .05) accompanied by a low AUC for cortisol (5.5 versus 15.5 µg/dL/h, P < .05), suggesting adrenocortical dysfunction. CONCLUSION AND CLINICAL IMPORTANCE: The 17α-OH-progesterone response to administration of ACTH was a good predictor of disease severity and death in hospitalized foals.

A-Raf associates with and regulates platelet-derived growth factor receptor signalling.

Raf kinases are important intermediates in epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) mediated activation of the mitogen-activated protein kinase (MAPK) pathway. In this report, we show that the A-Raf kinase is associated with activated EGF receptor complexes and with PDGF receptor (PDGFR) complexes independent of prior PDGF treatment. The ability of A-Raf to associate with receptor tyrosine kinases could provide a Ras-GTP-independent mechanism for the membrane localization of A-Raf. Expression of a partially activated A-Raf mutant resulted in decreased tyrosine phosphorylation of the PDGFR, specifically on Y857 (autophosphorylation site) and Y1021 (phospholipase Cgamma1 (PLCgamma1) binding site), but not the binding sites for other signalling proteins (Nck, phosphatidylinositol 3'-kinase (PI3K), RasGAP, Grb2, SHP). Activated A-Raf expression also altered the activation of PLCgamma1, and p85-associated PI3K. Thus, A-Raf can regulate PLCgamma1 signalling via a PDGFR-dependent mechanism and may also regulate PI3K signalling via a PDGFR-independent mechanism.

Identification of key residues in the A-Raf kinase important for phosphoinositide lipid binding specificity.

Raf kinases are involved in regulating cellular signal transduction pathways in response to a wide variety of external stimuli. Upstream signals generate activated Ras-GTP, important for the relocalization of Raf kinases to the membrane. Upon full activation, Raf kinases phosphorylate and activate downstream kinase in the mitogen-activated protein kinase (MAPK) signaling pathway. The Raf family of kinases has three members, Raf-1, B-Raf, and A-Raf. The ability of Raf-1 and B-Raf to bind phosphatidylserine (PS) and phosphatidic acid (PA) has been show to facilitate Raf membrane associations and regulate Raf kinase activity. We have characterized the lipid binding properties of A-Raf, as well as further characterized those of Raf-1. Both A-Raf and Raf-1 were found to bind to 3-, 4-, and 5-monophosphorylated phosphoinositides [PI(3)P, PI(4)P, and PI(5)P] as well as phosphatidylinositol 3,5-bisphosphate [PI(3,5)P(2)]. In addition, A-Raf also bound specifically to phosphatidylinositol 4,5- and 3,4-bisphosphates [PI(4,5)P(2) and PI(3,4)P(2)] and to PA. A mutational analysis of A-Raf localized the PI(4,5)P(2) binding site to two basic residues (K50 and R52) within the Ras binding domain. Additionally, an A-Raf mutant lacking the first 199 residues [i.e., the entire conserved region 1 (CR1) domain] bound the same phospholipids as full-length Raf-1. This suggests that a second region of A-Raf between amino acids 200 and 606 was responsible for interactions with the monophosphorylated PIs and PI(3,5)P(2). These results raise the possibility that Raf-1 and A-Raf bind to specific phosphoinositides as a mechanism to localize them to particular membrane microdomains rich in these phospholipids. Moreover, the differences in their lipid binding profiles could contribute to their proposed isoform-specific Raf functions.

Two phosphorylation-independent sites on the p85 SH2 domains bind A-Raf kinase.

Src homology 2 (SH2) domains mediate phosphotyrosine (pY)-dependent protein:protein interactions involved in signal transduction pathways. We have found that the SH2 domains of the 85-kDa alpha subunit (p85) of phosphatidylinositol 3-kinase (PI3 kinase) bind directly to the serine/threonine kinase A-Raf. In this report we show that the p85 SH2:A-Raf interaction is phosphorylation-independent. The affinity of the p85 C-SH2 domain for A-Raf and phosphopeptide pY751 was similar, raising the possibility that a p85:A-Raf complex may play a role in the coordinated regulation of the PI3 kinase and Raf-MAP kinase pathways. We further show that the p85 C-SH2 domain contains two distinct binding sites for A-Raf; one overlapping the phosphotyrosine-dependent binding site and the other a separate phosphorylation-independent site. This is the first evidence for a second binding site on an SH2 domain, distinct from the phosphotyrosine-binding pocket.