Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner.

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, ß, γ isoforms as well as cytokines, including interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-ß was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.

Effects of coadministration of corn oil and ponazuril on serum and cerebrospinal fluid concentrations of ponazuril in horses.

BACKGROUND: Ponazuril is used for the treatment of equine protozoal myeloencephalitis (EPM). Coadministration of ponazuril with oil could result in higher serum and cerebrospinal fluid (CSF) concentrations of ponazuril. HYPOTHESIS: Coadministration of corn oil will result in higher serum and CSF concentrations of ponazuril than when ponazuril is administered alone. ANIMALS: Ten resident university-owned adult horses of either sex and >2 years of age. METHODS: Cohort study. Ponazuril oral paste (5 mg/kg BW; ponazuril treatment group (PON); n = 5), or ponazuril oral paste (5 mg/kg BW; ponazuril and oil treatment group (PONOIL; n = 5) coadministered with 2 oz of corn oil q24h for 21 days. Horses were treated once daily, for 21 days. Blood was collected on days 0, 7, 14, and 21 before dosing. In addition, CSF was collected on days 1, 7, 14, and 21. The concentration of ponazuril was determined in serum and CSF and results compared using repeated measures ANOVA. RESULTS: Coadministration of ponazuril with 2 oz of corn oil resulted in higher concentrations of ponazuril in serum (at steady state) than that found in horses given ponazuril alone (6.2 ± 0.9 mg/L versus 4.5 ± 1.0 mg/L; P = .004) (mean ± 1 SD). Cerebrospinal fluid concentrations of ponazuril were also greater in horses that received ponazuril and oil (0.213 mg/L ± 0.04 versus 0.162 ± 0.04 mg/L) (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that coadministration of corn oil with ponazuril might enhance the effectiveness of treatment with ponazuril.

An investigation of the role of soluble CD14 in hospitalized, sick horses.

The objectives of this study were to (1) evaluate the effects of equine soluble CD14 (sCD14) and monoclonal antibodies (mAb) to equine CD14 on lipopolysaccharide-induced tumor necrosis factor α (TNF-α) secretion from equine peripheral blood mononuclear cells (PBMC); and to (2) determine serum concentrations of sCD14 in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia. Equine PBMC isolated from 10 healthy horses were incubated with Escherichia coli LPS plus CD14 mAb or sCD14 and assayed for TNF-α activity. Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF-α production, whereas use of sCD14 inhibited LPS-induced TNF-α production in a concentration-dependent manner. Additionally, blood samples from 55 ill and 23 healthy horses were used to determine serum concentrations of sCD14. Concentrations of sCD14 were positively correlated to respiratory rate, duration of clinical signs and band neutrophil count. Although serum sCD14 was significantly increased in the ill horses compared to healthy horses, sCD14 did not correlate with outcome. Results of this study indicate that release of sCD14 is increased in ill horses and that TNF-α production by PBMC is decreased when cells are treated with sCD14.

Evaluation of neutrophil apoptosis in horses with acute abdominal disease.

OBJECTIVE: To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery. ANIMALS: 20 client-owned adult horses. PROCEDURES: Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 hours, the proportion of neutrophils in apoptosis was detected by flow cytometric evaluation of cells stained with annexin V and 7-aminoactinomycin D. Values were compared between the colic and arthroscopy groups; among horses with colic, values were further compared between horses with and without strangulating intestinal lesions. RESULTS: Percentage recovery of neutrophils was significantly smaller in preoperative samples (median, 32.5%) and in all samples combined (35.5%) for the colic group, compared with the arthroscopy group (median, 66.5% and 58.0%, respectively). No significant differences in the percentages of apoptotic neutrophils were detected between these groups. Among horses with colic, those with strangulating intestinal lesions had a significantly lower proportion of circulating apoptotic neutrophils in postoperative samples (median, 18.0%) than did those with nonstrangulating lesions (66.3%). CONCLUSIONS AND CLINICAL RELEVANCE: The smaller proportion of apoptotic neutrophils in horses with intestinal strangulation suggested that the inflammatory response could be greater or prolonged, compared with that of horses with nonstrangulating intestinal lesions. Further investigations are needed to better understand the relationship between neutrophil apoptosis and inflammation during intestinal injury.

Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro.

Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as 'endotoxin tolerance' (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three concentrations of endotoxin, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells exposed to the 2-step endotoxin challenge. The relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMC. A marked suppression of IL-12 production is associated with ET.

Factors associated with outcome following treatment of horses with septic tenosynovitis: 51 cases (1986-2003).

OBJECTIVE: To identify factors associated with outcome (ie, survival and return to function) following treatment of horses with septic tenosynovitis. DESIGN: Retrospective case series. ANIMALS: 51 horses with septic tenosynovitis. PROCEDURES: Information was obtained from medical records and through follow-up conversations with owners. Factors analyzed for an association with outcome included affected limb, etiology, duration of clinical signs prior to examination, presence of complications, primary treatment, secondary treatments, number of surgical procedures, and hospitalization time. RESULTS: Concurrent complications were identified in 41 (80%) horses. The primary treatment consisted of through-and-through lavage in 26 (51%) horses, tenoscopy in 20 (39%), and tenosynoviotomy combined with lavage in 5 (10%). Forty (78%) horses were discharged, and 37 (73%) survived at least 1 year after surgery; 21 of the 37 (57%) returned to their previous or a higher level of performance. Percentages of horses that survived 1 year after discharge and percentages that returned to their intended use did not vary significantly among treatments. Horses with tendon rupture or sepsis of an adjacent joint were significantly less likely to survive. Horses with tendon injury or pannus were significantly less likely to return to their intended use. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that various factors were associated with outcome in horses with septic tenosynovitis. However, surgical technique was not found to be associated with survival rate or rate of return to intended use.

In vivo pretreatment with PGG-glucan fails to alter cytokine mRNA expression of equine peripheral blood mononuclear cells exposed to endotoxin ex vivo.

Twelve healthy horses were assigned to treatment or control groups. Treated horses received PGG-Glucan[ED-1] (1 mg/kg, IV) 24 hours prior to peripheral blood mononuclear cell (PBMC) isolation. PBMCs were isolated and incubated in the presence of lipopolysachharide (LPS). At 0, 6, 12, 24 and 48 hours messenger RNA (mRNA) was extracted. Reverse transcription polymerase chain reaction (PCR) was performed and cytokine mRNA expression for tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), interleukin 10 (IL-10) and interferon gamma (IFN-gamma) determined using real time PCR. A significant effect of LPS stimulation over time was seen on TNFalpha, IL-1beta, IL-10 and IFN-gamma production. No significant difference was observed between the PGG-Glucan treatment group and control cells at any time point.