Effect of 5'-adenosine monophosphate-activated protein kinase agonists on insulin and glucose dynamics in experimentally induced insulin dysregulation in horses.

BACKGROUND: 5'-adenosine monophosphate-activated protein kinase (AMPK) agonists, particularly resveratrol (RES), have not been extensively evaluated for their effect on insulin dysregulation (ID) in horses. OBJECTIVES: Evaluate the effects of treatment with RES (10 mg/kg PO q12h), metformin (MET; 30 mg/kg PO q12h), and aspirin (ASP; 20 mg/kg PO q24h) on experimentally induced ID. ANIMALS: Thirty-three healthy, adult, light-breed horses. METHODS: Unblinded, placebo-controlled, experimental trial evaluating effects of AMPK agonists (RES, MET, and ASP) on experimentally induced ID. Horses were randomly assigned to a treatment group (RES, MET/ASP, RES/ASP, RES/MET/ASP, or placebo [CON]) after induction of ID with dexamethasone (0.08 mg/kg PO q24h for 7 days). Frequently sampled insulin-modified IV glucose tolerance tests (FSIGTT) and oral sugar tests (OST) were performed at baseline, 7 days after ID, and ID plus 7 days of treatment. Minimal model and OST variables were compared between (1-way ANOVA) and within (1-way ANOVA for repeated measures) groups over time to determine effects of treatment on ID. RESULTS: Administration of dexamethasone for 14 days resulted in significantly altered insulin and glucose dynamics (SI, DI, basal [glucose], and [insulin]) and produced clinical signs of laminitis in 5 out of 33 (15%) of horses included in the study. Combination therapy with RES, MET, and ASP did not significantly improve insulin and glucose dynamics in horses with experimentally induced ID. CONCLUSIONS AND CLINICAL IMPORTANCE: Metabolic testing before glucocorticoid administration should be considered in horses with clinical signs of metabolic syndrome.

Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing.

BACKGROUND: Supporting limb laminitis (SLL) is a complication of severe orthopedic disease in horses and is often life-limiting, yet the pathophysiology remains obscure. HYPOTHESIS/OBJECTIVES: To investigate the role of digital lamellar inflammatory signaling in the pathophysiology of SLL using a model of unilateral weight bearing, hypothesizing that there would be evidence of lamellar inflammation in limbs subjected to the model. ANIMALS: Thirteen healthy adult Standardbred horses were used for this study (11 geldings, 2 mares; mean age 6.5 ± 2.5 years; mean body weight 458.3 ± 32.8 kg). METHODS: Randomized controlled experimental study. A steel shoe with a custom insert was applied to a randomly selected front foot of 7 horses; 6 horses were unshod and served as controls. After 92 hours, all horses were humanely euthanized, and digital lamellar samples were collected. Lamellar protein and mRNA were isolated and used to perform western blot and PCR. RESULTS: Lamellar concentrations of IL-6 mRNA were higher in SL tissue than IL HIND tissue (median [25%-75%] normalized copy number 191 [111-3060] and 48 [25-74], respectively; P=.003), and lamellar concentrations of COX-2 mRNA were higher in SL tissue than CON tissue (normalized copy number 400 [168-634] and 125 [74-178], respectively; P=.007). Lamellar concentrations of IL-1B, IL-10, and COX-1 mRNA were not significantly different between groups. The concentrations of phosphorylated (activated) STAT1 and STAT3 proteins were higher in SL (0.5 [0.35-0.87] and 1.35 [1.1-1.7], respectively) compared to CON (0.24 [0.09-0.37] and 0.31 [0.16-037]) and UL HIND (0.27 [0.19-0.37] and 0.38 [0.24-0.5]); P=0.01 and P<0.001. CONCLUSIONS AND CLINICAL IMPORTANCE: Lamellar inflammatory signaling was higher in tissue from horses subjected to prolonged unilateral weight-bearing, suggesting that these pathways could be relevant to the pathophysiology of SLL.

Codon-optimization of the respiratory syncytial virus (RSV) G protein expressed in a vesicular stomatitis virus (VSV) vector improves immune responses in a cotton rat model.

Respiratory syncytial virus is an important cause of pneumonia in children, the elderly, and immunocompromised individuals. The attachment (G) protein of RSV generates neutralizing antibodies in natural RSV infection which correlate with protection against disease. The immune response to RSV is typically short-lived, which may be related to the heavy glycosylation of RSV-G. In order to improve its immunogenicity, we expressed G protein mutants in a vesicular stomatitis virus (VSV) vector system and tested their ability to protect cotton rats from RSV challenge. We found that the most protective construct was codon-optimized RSV-G, followed by wild-type G and membrane-bound G. Constructs which expressed the G protein with reduced glycosylation or the secreted G protein provided either partial or no protection. Our results demonstrate that modifications to the G protein are not advantageous in a VSV vector system, and that an intact, codon-optimized G is a superior vaccine candidate.

Diagnostic evaluation of insulin and glucose dynamics in light-breed horses receiving dexamethasone.

Objective: Insulin dysregulation is a hallmark of equine metabolic syndrome (EMS) and increases the risk for development of laminitis. Accurate diagnosis of insulin dysregulation is crucial for implementation of preventative strategies in this population. The objective was to assess the effects of dexamethasone administration on insulin and glucose dynamics in light-breed horses and assess the agreement of various diagnostic tests for insulin dysregulation [basal [insulin] (BI), oral sugar test (OST), and combined glucose-insulin test (CGIT)]. Animal: Fourteen adult light-breed horses. Procedure: Prospective, experimental study to assess insulin and glucose dynamics by performing basal insulin, OST, and CGIT before (baseline) and post-dexamethasone administration (0.08 mg/kg, PO, q24h) for 7 d. Insulin and glucose dynamics were assessed by the BI, OST, CGIT, and insulin sensitivity proxy measurements (RISQI, QUICKI, FGIR, HOMA-IR, IG) at the baseline and post-dexamethasone time points. Results: The OST area under the insulin and glucose curves were increased following dexamethasone treatment (P < 0.001 and P < 0.01, respectively). Basal insulin, OST [insulin] at 60 min and CGIT [insulin] at 45 min were increased at the post-dexamethasone time point (P < 0.001, < 0.001, and < 0.01). Similarly, time spent in the positive glucose phase during the CGIT was longer at the post-dexamethasone time point (P < 0.001). The proxy measurements for insulin sensitivity (RISQI, QUICKI, FGIR) were decreased (P < 0.01) and the proxy measurements for insulin resistance (HOMA-IR) and ß-cell function (IG) were increased after dexamethasone administration (P < 0.01). More horses were classified with following dexamethasone administration, based on the diagnostic criteria for basal insulin (P = 0.03), OST (P = 0.01), and CGIT (P < 0.01). Kappa coefficients, measuring agreement between basal insulin, OST, and CGIT, showed none to moderate agreement at the baseline time point. Conclusion: Dexamethasone administration at 0.08 mg/kg, PO, q24h for 7 d worsened insulin dysregulation in adult light-breed horses based on findings of a basal insulin, OST, CGIT, and insulin sensitivity proxy measurements. There was none to moderate agreement between the basal insulin, OST, CGIT for the diagnosis of insulin dysregulation. Clinical relevance: Horses administered dexamethasone at a dose of 0.08 mg/kg, PO, q24h for 7 d should be considered insulin dysregulation and appropriate preventative strategies should be implemented. The variability of diagnostic performance of common tests for insulin dysregulation (basal insulin, OST, CGIT) may affect clinical decisions; therefore, performing multiple tests, including proxy measurements, may improve diagnostic accuracy of insulin dysregulation.

Objectif: La dysrégulation de l'insuline est une caractéristique du syndrome métabolique équin (EMS) et augmente le risque de développement de la fourbure. Un diagnostic précis de la dysrégulation de l'insuline est crucial pour la mise en oeuvre de stratégies préventives dans cette population. L'objectif était d'évaluer les effets de l'administration de dexaméthasone sur la dynamique de l'insuline et du glucose chez les chevaux de race légère et d'évaluer la concordance de divers tests de diagnostic pour le dérèglement de l'insuline [insuline basale] (BI), test de sucre oral (OST) et un test glucose-insuline combiné (CGIT). Animal: Quatorze chevaux adultes de race légère. Procédure: Étude prospective et expérimentale pour évaluer la dynamique de l'insuline et du glucose en effectuant l'insuline basale, l'OST et le CGIT avant (valeur de base) et après l'administration de dexaméthasone (0,08 mg/kg, PO, q24h) pendant 7 jours. La dynamique de l'insuline et du glucose a été évaluée par les mesures indirectes de BI, de l'OST, du CGIT et de la sensibilité à l'insuline (RISQI, QUICKI, FGIR, HOMA-IR, IG) aux points temporels de base et post-dexaméthasone. Résultats: La zone OST sous les courbes d'insuline et de glucose a augmenté après le traitement à la dexaméthasone (P < 0,001 et P < 0,01, respectivement). L'insuline basale, l'OST [insuline] à 60 minutes et le CGIT [insuline] à 45 minutes ont augmenté au point temporel post-dexaméthasone (P < 0,001, < 0,001 et < 0,01). De même, le temps passé dans la phase de glucose positif pendant le CGIT était plus long au moment post-dexaméthasone (P < 0,001). Les mesures indirectes de la sensibilité à l'insuline (RISQI, QUICKI, FGIR) ont diminué (P < 0,01) et les mesures indirectes de la résistance à l'insuline (HOMA-IR) et de la fonction des cellules ß (IG) ont augmenté après l'administration de dexaméthasone (P < 0,01). Plus de chevaux ont été classés avec l'administration suivante de dexaméthasone, sur la base des critères de diagnostic de l'insuline basale (P = 0,03), OST (P = 0,01) et CGIT (P < 0,01). Les coefficients Kappa, mesurant la concordance entre l'insuline basale, l'OST et le CGIT, ont montré une concordance nulle à modérée au point de référence. Conclusion: L'administration de dexaméthasone à 0,08 mg/kg, PO, toutes les 24 h pendant 7 jours a aggravé la dysrégulation de l'insuline chez les chevaux adultes de race légère d'après les résultats d'une insuline basale, d'OST, de CGIT et de mesures indirectes de la sensibilité à l'insuline. Il n'y avait aucun accord à modéré entre l'insuline basale, l'OST, le CGIT pour le diagnostic de dysrégulation de l'insuline. Pertinence clinique: Les chevaux ayant reçu de la dexaméthasone à une dose de 0,08 mg/kg, PO, q24h pendant 7 jours doivent être considérés comme ayant un dérèglement de l'insuline et des stratégies préventives appropriées doivent être mises en oeuvre. La variabilité des performances diagnostiques des tests courants de dysrégulation de l'insuline (insuline basale, OST, CGIT) peut affecter les décisions cliniques; par conséquent, la réalisation de plusieurs tests, y compris des mesures indirectes, peut améliorer la précision du diagnostic du dérèglement de l'insuline.(Traduit par Dr Serge Messier).

Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model.

Respiratory syncytial virus (RSV) is one of the most important causes of respiratory disease in infants, immunocompromised individuals, and the elderly. Natural infection does not result in long-term immunity, and there is no licensed vaccine. Vesicular stomatitis virus (VSV) is a commonly used vaccine vector platform against infectious diseases, and has been used as a vector for a licensed Ebola vaccine. In this study, we expressed the RSV fusion (F) protein, the RSV F protein stabilized in either a pre-fusion or a post-fusion configuration, the attachment glycoprotein (G), or the G and F proteins of RSV in combination in a VSV vector. Cotton rats were immunized with these recombinants intranasally or subcutaneously to test immunogenicity. RSV F stabilized in either a pre-fusion or a post-fusion configuration proved to be poorly immunogenic and protective when compared to unmodified F. RSV G provided partial protection and moderate levels of neutralizing antibody production, both of which improved with intranasal administration compared to subcutaneous inoculation. The most successful vaccine vector was VSV expressing both the G and F proteins after intranasal inoculation. Immunization with this recombinant induced neutralizing antibodies and provided protection from RSV challenge in the upper and lower respiratory tract for at least 80 days. Our results demonstrate that co-expression of F and G proteins in a VSV vector provides synergistic effects in inducing RSV-specific neutralizing antibodies and protection against RSV infection.

The selective inhibitor of nuclear export (SINE) verdinexor exhibits biologic activity against canine osteosarcoma cell lines.

Verdinexor (KPT-335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose-dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21 to 74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50  = 21 µM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.

Lamellar perfusion and energy metabolism in a preferential weight bearing model.

BACKGROUND: Supporting limb laminitis (SLL) is suspected to be caused by lamellar ischaemia as a consequence of increased mechanical load. OBJECTIVES: Examine the effects of prolonged preferential weight bearing (PWB) on lamellar perfusion and metabolism. STUDY DESIGN: In vivo experiment. METHODS: Microdialysis probes were inserted in the lamellar and sublamellar dermis of one forelimb in 13 Standardbred horses. In six horses, a platform shoe (contralateral forelimb) was used to induce increased load on the microdialysis-instrumented forelimb (PWB). The remaining seven horses were controls (CON). All horses were housed in stocks with limb weight distribution logged continuously for 92 hours. Microdialysate was collected and analysed every 4 hours for glucose, lactate, pyruvate, and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Median [IQR] load on the microdialysis-instrumented limb was equivalent to 38.7% bwt. [37.3-40.3] in PWB and 27.3% bwt. [26.6-28] in CON. Limb offloading frequency increased in CON (P < .001) but not PWB (P = .2). Lamellar microdialysate glucose decreased in PWB (P < .001) and CON (P = .004), however, the rate of decrease was higher in PWB (P = .007). Lamellar L:P increased in PWB (P < .001) and peaked at 196 [79-656], whereas L:P did not change over time in CON (P = .6) and peaked at 42 [41-49]. Lamellar urea clearance decreased in PWB (P < .001) but not in CON (P = .3). Sublamellar L:P and urea clearance did not change over time in either group (P > .05). MAIN LIMITATIONS: The PWB model may not be representative of naturally occurring SLL. CONCLUSIONS: Evidence of lamellar ischaemia (increased L:P and decreased urea clearance) was detected exclusively in the lamellar dermis of PWB feet subjected to persistently increased load. Lamellar ischaemia is a consequence of increased mechanical load and likely contributes to the development of SLL.

Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. HYPOTHESIS/OBJECTIVES: To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. ANIMALS: Eight Standardbred horses. METHODS: Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL-11, cyclooxygenase 1 and 2, tumour necrosis factor-alpha [TNF-α], E-selectin, and intercellular adhesion molecule-1 [ICAM-1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3). RESULTS: Compared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL-6 (P = .008), IL-1ß (P = .008), IL-11 (P = .008), and cyclooxygenase-2 (P = .008) were decreased in CDH. Cyclooxygenase-1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP-1 (P = .05), MCP-2 (P = .46), TNF-α (P = .05), E-selectin (P = .15), and ICAM-1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P-STAT3 [S727] P = .19; P-STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P-STAT1 [S727], P = .25; P-STAT1 [Y701], P = .64). CONCLUSIONS AND CLINICAL IMPORTANCE: These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light-breed horses.

BACKGROUND: Hyperinsulinemia is associated with equine laminitis, and digital lamellar inflammation in equine metabolic syndrome-associated laminitis (EMSAL) is modest when compared with sepsis-associated laminitis. OBJECTIVES: To characterize digital lamellar inflammation in horses in a euglycemic-hyperinsulinemic clamp (EHC) model of laminitis. ANIMALS: Sixteen healthy adult Standardbred horses. METHODS: Prospective experimental study. Horses underwent EHC or saline infusion (CON) for 48 hours or until the onset of Obel grade 1 laminitis. Horses were euthanized, and digital lamellar tissue was collected and analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL11, cyclooxygenases 1 and 2, tumor necrosis factor alpha [TNF-α], E-selectin, and ICAM-1), immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1], STAT3, and p38MAPK), and immunohistochemistry (markers of leukocyte infiltration: CD163, MAC387). RESULTS: Lamellar mRNA concentrations of IL-1ß, IL-6, IL-11, COX-2, and E-selectin were increased; the concentration of COX-1 was decreased; and concentrations of CXCL1, CXCL6, MCP-1, MCP-2, IL-8, TNF-α and ICAM-1 were not significantly different in the EHC group compared to the CON group (P ≤ .003). Lamellar concentrations of phosphorylated STAT proteins (P-STAT1 [S727], P-STAT1 [Y701], P-STAT3 [S727], and P-STAT3 [Y705]) were increased in the EHC group compared to the CON group, with phosphorylated STAT3 localizing to nuclei of lamellar basal epithelial cells. There was no change in the lamellar concentration of P-p38 MAPK (T180/Y182), but the concentration of total p38 MAPK was decreased in the EHC samples. There was no evidence of notable lamellar leukocyte emigration. CONCLUSIONS AND CLINICAL IMPORTANCE: These results establish a role for lamellar inflammatory signaling under conditions associated with EMSAL.

The effect of hypothermia on influx of leukocytes in the digital lamellae of horses with oligofructose-induced laminitis.

Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia ("cryotherapy"), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10g/kg, n=14 horses). Lamellae were harvested at 24h post-oligofructose administration (DEV, n=7) or at the onset of OG1 laminitis (OG1, n=7). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n=10 (40× fields/digit for MAC387 and 20x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p<0.05. MAC387-positive cells were present in low numbers in the lamellar tissue and were decreased in the hypothermic limbs (vs. AMB limbs, p<0.05) in the OG1 group; no change in CD163-positive cell numbers was noted across the conditions of the model. This study demonstrated that hypothermia of the distal limbs instituted early in the disease process in the horse at risk of SRL significantly attenuates the increase of MAC387-positive leukocytes in the digital lamellae, but has minimal effect on increases in lamellar concentrations of the major leukocyte cell type present in that tissue, CD163-positive mononuclear cells.

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis.

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.