Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated in vitro and ex vivo

Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material

Soy Phosphatidylinositol–Containing Lipid Nanoparticle Prolongs the Plasma Survival and Hemostatic Efficacy of B-domain–Deleted Recombinant Canine Factor VIII in Hemophilia A Dogs

Soy phosphatidylinositol (PI) containing lipid nanoparticles prolong plasma survival, improve hemostatic efficacy, and decrease immunogenicity of human B-domain deleted Factor VIII (BDD FVIII) in Hemophilia A (HA) mice. We hypothesize that PI associated BDD FVIII is more potent than the free protein, and using mathematical modeling, have projected that PI associated BDD FVIII could be used for once-weekly prophylactic dosing in patients. To facilitate translation to the clinic, comparative plasma survival and ex vivo efficacy of PI associated recombinant canine FVIII (PI-rcFVIII) were evaluated in HA dogs. 2 HA dogs were administered a 50 U/kg iv dose of free or PI-rcFVIII. rcFVIII activity measurements and ex vivo efficacy analyses like whole blood clotting time (WBCT) and thromboelastography (TEG) were conducted on recovered plasma and whole blood samples. PI association decreased clearance (~25%) and increased plasma exposure (~1.4 fold) of rcFVIII. PI-rcFVIII treated animals had prolonged improvements in WBCTs and TEG parameters compared to free rcFVIII treated animals. Since rcFVIII is a BDD form of FVIII, these studies provide proof-of-principle that observations with human BDD FVIII in mice translate to higher animal species. Additionally, PI-rcFVIII has potential applications in canine HA management and as a bypass therapy in inhibitor-positive HA patients

Experimental Validation of ARFI Surveillance of Subcutaneous Hemorrhage (ASSH) Using Calibrated Infusions in a Tissue-Mimicking Model and Dogs

Acoustic radiation force impulse (ARFI) Surveillance of Subcutaneous Hemorrhage (ASSH) has been previously demonstrated to differentiate bleeding phenotype and responses to therapy in dogs and humans, but to date, the method has lacked experimental validation. This work explores experimental validation of ASSH in a poroelastic tissue-mimic and in vivo in dogs. The experimental design exploits calibrated flow rates and infusion durations of evaporated milk in tofu or heparinized autologous blood in dogs. The validation approach enables controlled comparisons of ASSH-derived bleeding rate (BR) and time to hemostasis (TTH) metrics. In tissue-mimicking experiments, halving the calibrated flow rate yielded ASSH-derived BRs that decreased by 44% to 48%. Furthermore, for calibrated flow durations of 5.0 minutes and 7.0 minutes, average ASSH-derived TTH was 5.2 minutes and 7.0 minutes, respectively, with ASSH predicting the correct TTH in 78% of trials. In dogs undergoing calibrated autologous blood infusion, ASSH measured a 3-minute increase in TTH, corresponding to the same increase in the calibrated flow duration. For a measured 5% decrease in autologous infusion flow rate, ASSH detected a 7% decrease in BR. These tissue-mimicking and in vivo preclinical experimental validation studies suggest the ASSH BR and TTH measures reflect bleeding dynamics

Porcine and Canine von Willebrand Factor and von Willebrand Disease: Hemostasis, Thrombosis, and Atherosclerosis Studies

Use of animal models of inherited and induced von Willebrand factor (VWF) deficiency continues to advance the knowledge of VWF-related diseases: von Willebrand disease (VWD), thrombotic thrombocytopenic purpura (TTP), and coronary artery thrombosis. First, in humans, pigs, and dogs, VWF is essential for normal hemostasis; without VWF bleeding events are severe and can be fatal. Second, the ADAMTS13 cleavage site is preserved in all three species suggesting all use this mechanism for normal VWF multimer processing and that all are susceptible to TTP when ADAMTS13 function is reduced. Third, while the role of VWF in atherogenesis is debated, arterial thrombosis complicating atherosclerosis appears to be VWF-dependent. The differences in the VWF gene and protein between humans, pigs, and dogs are relatively few but important to consider in the design of VWF-focused experiments. These homologies and differences are reviewed in detail and their implications for research projects are discussed. The current status of porcine and canine VWD are also reviewed as well as their potential role in future studies of VWF-related disorders of hemostasis and thrombosis

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals

Acoustic radiation force beam sequence performance for detection and material characterization of atherosclerotic plaques: preclinical, ex vivo results

This work presents preclinical data demonstrating performance of acoustic radiation force (ARF) based elasticity imaging with five different beam sequences for atherosclerotic plaque detection and material characterization. Twelve trained, blinded readers evaluated parametric images taken ex vivo under simulated in vivo conditions of 22 porcine femoral arterial segments. Receiver operating characteristic (ROC) curve analysis was carried out to quantify reader performance using spatially-matched immunohistochemistry for validation. The beam sequences employed had high sensitivity and specificity for detecting Type III+ plaques (Sens: 85%, Spec: 79%), lipid pools (Sens: 80%, Spec: 86%), fibrous caps (Sens: 86%, spec: 82%), calcium (Sens: 96%, Spec: 85%), collagen (Sens: 78%, Spec: 77%), and disrupted internal elastic lamina (Sens: 92%, Spec: 75%). 1:1 single-receive tracking yielded the highest median areas under the ROC curve (AUC), but was not statistically significantly higher than 4:1 parallel-receive tracking. Excitation focal configuration did not result in statistically different AUCs. Overall, these results suggest ARF-based imaging is relevant to detecting and characterizing plaques and support its use for diagnosing and monitoring atherosclerosis

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD

Common Security of the Czech Republic and Slovakia Airspace by JAS-39 GRIPEN Fighter Aircrafts

Diplomová práce se zabývá problematikou ochrany vzdušného prostoru České republiky a Slovenska nadzvukovými letouny JAS-39 Gripen. První část práce je zaměřena na teoretickou rovinu obrany jako služby v obecném zájmu nehospodářské povahy a na vybrané pojmy z oblasti obrany a bezpečnosti státu. Druhá část se zabývá komparací Armády České republiky a Ozbrojených sil Slovenské republiky se zaměřením na vzdušné síly a možnosti ochrany vzdušného prostoru pomoci nadzvukových bojových letounů obou zemí. Třetí částí je zhodnocení možnosti pořízení a provozování nadzvukových letounů JAS-39 Gripen Vzdušnými silami Ozbrojených sil Slovenské republiky, které vychází z předchozího zkoumání a zároveň přibližuje možnosti zabezpečení společného vzdušného prostoru obou zemí. V poslední části jsou doporučení pro společné zabezpečení vzdušného prostoru obou zemí, vycházející z jednotlivých možností, ale i se stávající bezpečnostní a ekonomické situace.This thesis deals with the airspace protection of Czech Republic and Slovakia by JAS-39 Gripen supersonic aircrafts. The first part is focused on the theoretical level of defense as a service of general non-economic interest and certain terms of defense and national security. The second part deals with the comparsion of the Czech Army and Slovak Army with focuse on options of airspace protection by supersonic fighter aircrafts of both countries. The third part evaluates the possibility of acquisition and usage JAS-39 supersonic aircraft by the Slovak Air Force which is based on previous research and shows how to secure the common airspace of both countries. In the final chapter are recommendations for common airspace security of both countries based on each option, but also on current security and economic situation.153 - Katedra veřejné ekonomikyvelmi dobř

Academic Senate - Meeting Minutes, 4/18/2017

<p>All values are presented with SD. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: *p<0.05, **p<0.01.</p

A Monoclonal Antibody Against V 3 Integrin Inhibits Development of Atherosclerotic Lesions in Diabetic Pigs

Atherosclerotic lesions develop and progress more rapidly in diabetic patients than in nondiabetic individuals. This may be caused by accelerated lesion formation in the high-glucose environment of diabetes. Smooth muscle cells (SMCs) cultured in high glucose are more responsive to growth factors such as insulin-like growth factor–1 (IGF-1). This enhanced response to IGF-1 is due in part to increased activation of the αVβ3 integrin. We tested whether αVβ3 integrin activation was increased in diabetic animals and whether an antibody to β3 would inhibit IGF-1 action and development of atherosclerosis. Eight male pigs were made diabetic with streptozotocin and fed a high-fat diet. A F(ab)2 antibody fragment directed at β3 was infused into one femoral artery, whereas the other artery received control F(ab)2 for 3.5 months. There was a 65 ± 8% reduction in atherosclerotic lesion area in the arteries treated with F(ab)2 antibody to β3. Phosphorylation of β3 was reduced by 75 ± 18% in vessels treated with the antibody. Shc and mitogen-activated protein kinase phosphorylation, which are required for IGF-1–stimulated SMC proliferation, were also significantly reduced. We conclude that activation of IGF-1 receptor and αVβ3-linked signaling pathways accelerates atherosclerosis in diabetes and that administration of an antibody to β3 to diabetic pigs inhibits αVβ3 activation, IGF-1–stimulated signaling, and atherosclerotic lesion development. This approach offers a potential therapeutic approach to the treatment of this disorder