Mechanical Anisotropy Assessment in Kidney Cortex Using ARFI Peak Displacement: Preclinical Validation and Pilot In Vivo Clinical Results in Kidney Allografts.

The kidney is an anisotropic organ, with higher elasticity along versus across nephrons. The degree of mechanical anisotropy in the kidney may be diagnostically relevant if properly exploited; however, if improperly controlled, anisotropy may confound stiffness measurements. The purpose of this study is to demonstrate the clinical feasibility of acoustic radiation force (ARF)-induced peak displacement (PD) measures for both exploiting and obviating mechanical anisotropy in the cortex of human kidney allografts, in vivo.

Influence of diabetes on the foreign body response to nitric oxide-releasing implants.

The foreign body response (FBR) to nitric oxide (NO)-releasing subcutaneous implants was compared between healthy and streptozotocin-induced diabetic swine by evaluating inflammation, collagen capsule formation, and angiogenesis. Steel wire substrates were first modified with polyurethane membranes capable of diverse NO-release kinetics (NO fluxes and release durations of 0.8-630.

Endothelial alterations in a canine model of immune thrombocytopenia.

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored.

Evaluation of Anthelmintic Resistance and Exhaust Air Dust PCR as a Diagnostic Tool in Mice Enzootically Infected with .

The entry of infectious agents in rodent colonies occurs despite robust sentinel monitoring programs, strict quarantine measures, and stringent biosecurity practices. In light of several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for early detection of A. tetraptera infection.

Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII.

Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal).

Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII.

Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity.

Antioxidant Therapies for Ulcerative Dermatitis: A Potential Model for Skin Picking Disorder.

Skin Picking Disorder affects 4% of the general population, with serious quality of life impacts, and potentially life threatening complications. Standard psychoactive medications do not help most patients. Similarly, Mouse Ulcerative Dermatitis (skin lesions caused by excessive abnormal grooming behavior) is very common in widely used inbred strains of mice, and represents a serious animal welfare issue and cause of mortality.

Oxidized LDL and Fructosamine Associated with Severity of Coronary Artery Atherosclerosis in Insulin Resistant Pigs Fed a High Fat/High NaCl Diet.

Background: Insulin-resistant subjects develop more severe and diffuse coronary artery atherosclerosis than insulin-sensitive controls but the mechanisms that mediate this atherosclerosis phenotype are unknown.

Research Objective: To determine the metabolic parameters that associate with the severity of coronary atherosclerosis in insulin resistant pigs fed a high fat/high NaCl diet.

Key Methods: The primary endpoint was severity of coronary atherosclerosis in adult pigs (Sus scrofa, n = 37) fed a high fat diet that also contained high NaCl (56% above recommended levels) for 1 year.

Liver-directed lentiviral gene therapy in a dog model of hemophilia B.

We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors.

Blocking ligand occupancy of the αVβ3 integrin inhibits the development of nephropathy in diabetic pigs.

Hyperglycemia stimulates secretion of αVβ3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVβ3 integrin. This study determined whether blocking ligand occupancy of αVβ3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs received an F(ab)2 fragment of an antibody directed against the extracellular domain of the β3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks.

A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia.

Targeted disruption of LDLR causes hypercholesterolemia and atherosclerosis in Yucatan miniature pigs.

Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size.

Portal vein delivery of viral vectors for gene therapy for hemophilia.

The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.

Animal models of hemophilia.

The X-linked bleeding disorder hemophilia is caused by mutations in coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). Unless prophylactic treatment is provided, patients with severe disease (less than 1% clotting activity) typically experience frequent spontaneous bleeds. Current treatment is largely based on intravenous infusion of recombinant or plasma-derived coagulation factor concentrate.

Safety of AAV factor IX peripheral transvenular gene delivery to muscle in hemophilia B dogs.

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector-mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.

A monoclonal antibody against alphaVbeta3 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).

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs.

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.

ARFI ultrasound for in vivo hemostasis assessment postcardiac catheterization, part I: preclinical studies.

The world wide prevalence of cardiovascular disease leads to over seven million annual percutaneous coronary catheterization procedures, the majority of which exploit femoral artery access. Femoral puncture sites ('arteriotomies') can be associated with severe vessel complications after sheath removal if hemostasis is not properly achieved. Hemostasis onset is routinely determined by examination for bleeding at the skin puncture; however, clotting along the puncture path can obscure subcutaneous bleeding, and therefore hemostasis is blindly assessed.

Protein replacement therapy and gene transfer in canine models of hemophilia A, hemophilia B, von willebrand disease, and factor VII deficiency.

Dogs with hemophilia A, hemophilia B, von Willebrand disease (VWD), and factor VII deficiency faithfully recapitulate the severe bleeding phenotype that occurs in humans with these disorders. The first rational approach to diagnosing these bleeding disorders became possible with the development of reliable assays in the 1940s through research that used these dogs. For the next 60 years, treatment consisted of replacement of the associated missing or dysfunctional protein, first with plasma-derived products and subsequently with recombinant products.

Effects of cage size and enrichment on reproductive performance and behavior in C57BL/6Tac mice.

The authors examined the effects of cage size and enrichment on mouse breeding performance and behavior. Breeding trios of C57BL/6Tac mice were housed in cages of two different sizes ('standard' and 'large' cages with 82 in(2) and 124 in(2) floor space, respectively). Half of the cages of each size contained four enrichment items (Nestlet, plastic tunnel, nylon rings and running wheel), whereas the remaining cages had no enrichment.

The effect of cage size on reproductive performance and behavior of C57BL/6 mice.

Scientific research has yet to conclusively determine the optimal cage size for mice. The authors examined the effect of cage size on mouse breeding performance and on offspring behavior, which can serve as indications of overall well-being. They housed breeding trios of C57BL/6Tac mice in standard or large individually ventilated cages and measured four reproductive parameters: litter size; litter survival to weaning age; average pup weight at 7, 14 and 21 days; and the number of days between litter births.

In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice.

Introduction: Recently, in vitro models of coagulation have called into question the traditional conception of Factor IX as an intrinsic pathway protein, essential to propagation of coagulation but not central to the initiation of hemostatic plug, which has been thought instead to involve TF/FVIIa interactions with factor X and platelets. We hypothesized that the activation of factor IX, and its role in a factor IXa/FVIIa "tenase" complex leading to thrombin generation, plays a more important role than that of TF/FVIIa complex activation of factor X in the early hemostatic response to vascular injury. In vivo modeling is possible because of the generation of factor IX(-/-) mice.

Immune response after neonatal transfer of a human factor IX-expressing retroviral vector in dogs, cats, and mice.

Introduction: Gene therapy could prevent bleeding in hemophilia. However, antibodies could inhibit coagulation, while cytotoxic T lymphocytes could destroy modified cells. The immaturity of the newborn immune system might prevent these immune responses from occurring after neonatal gene therapy.