RESUMEN
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. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.
Asunto(s)
Endotelio/metabolismo , Púrpura Trombocitopénica Idiopática/etiología , Púrpura Trombocitopénica Idiopática/metabolismo , Animales , Biomarcadores , Biopsia , Coagulación Sanguínea , Plaquetas/metabolismo , Plaquetas/ultraestructura , Modelos Animales de Enfermedad , Perros , Endotelio/ultraestructura , Citometría de Flujo , Lisofosfolípidos/sangre , Masculino , Activación Plaquetaria , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Esfingosina/análogos & derivados , Esfingosina/sangre , Factor de von Willebrand/metabolismoRESUMEN
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. Validation of the imaging methods is provided by preclinical studies in pig kidneys, in which ARF-induced PD values were significantly higher ( , Wilcoxon) when the transducer executing asymmetric ARF was oriented across versus along the nephrons. The ratio of these PD values obtained with the transducer oriented across versus along the nephrons strongly linearly correlated ( R2 = 0.95 ) to the ratio of shear moduli measured by shear wave elasticity imaging. On the contrary, when a symmetric ARF was implemented, no significant difference in PD was observed ( p > 0.01 ). Similar results were demonstrated in vivo in the kidney allografts of 14 patients. The symmetric ARF produced PD measures with no significant difference ( p > 0.01 ) between along versus across alignments, but the asymmetric ARF yielded PD ratios that remained constant over a six-month observation period post-transplantation, consistent with stable serum creatinine level and urine protein-to-creatinine ratio in the same patient population ( p > 0.01 ). The results of this pilot in vivo clinical study suggest the feasibility of 1) implementing symmetrical ARF to obviate mechanical anisotropy in the kidney cortex when anisotropy is a confounding factor and 2) implementing asymmetric ARF to exploit mechanical anisotropy when mechanical anisotropy is a potentially relevant biomarker.
Asunto(s)
Aloinjertos , Diagnóstico por Imagen de Elasticidad/métodos , Corteza Renal , Trasplante de Riñón , Adulto , Anciano , Aloinjertos/diagnóstico por imagen , Aloinjertos/fisiología , Animales , Anisotropía , Módulo de Elasticidad/fisiología , Femenino , Humanos , Corteza Renal/diagnóstico por imagen , Corteza Renal/fisiología , Masculino , Persona de Mediana Edad , Insuficiencia Renal Crónica/diagnóstico por imagen , Insuficiencia Renal Crónica/cirugía , PorcinosRESUMEN
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 0.8-630.0 pmol cm-2 s-1 and 2-13 d, respectively). The NO-releasing materials were implanted in the subcutis for 3, 10, or 25 d for histological and immunohistochemical evaluation of the FBR. A delayed, more severe inflammatory response to control (i.e., non-NO-releasing) implants was observed in diabetic pigs relative to healthy swine. Regardless of the animal disease state, each NO-releasing implant tested elicited reduced inflammation compared to controls at both 3 and 10 d. However, only the NO-release materials capable of releasing low NO fluxes (0.8-3.3 pmol cm-2 s-1) for 7-13 d durations mitigated the inflammatory response at 25 d. Using immunohistochemical staining for the endothelial cell surface marker CD-31, we also observed poor blood vessel development at non-NO-releasing implants in diabetic swine. Relative to controls, NO-releasing implants with the longest NO-release duration (13 d) increased blood vessel densities by 47.1 and 70.4% in the healthy and diabetic pigs, respectively. In the healthy model, tissues surrounding the long NO-release materials contained sparse amounts of collagen, whereas implants with shorter NO-release durations (2, 3, and 7 d) were characterized with a dense collagen encapsulation layer, similar to controls. Collagen deposition in diabetic swine was inhibited, and unaffected by NO. These results emphasize several key differences in the FBR in the setting of acute onset diabetes. The observation that NO release counteracts the more severe FBR in diabetic swine while simultaneously promoting tissue integration may help guide the design of medical implants (e.g., glucose sensors) with improved performance for diabetes management.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Reacción a Cuerpo Extraño/patología , Implantes Experimentales , Inflamación/patología , Neovascularización Patológica/patología , Óxido Nítrico/metabolismo , Poliuretanos/química , Animales , Colágeno/metabolismo , Femenino , Reacción a Cuerpo Extraño/metabolismo , Masculino , Neovascularización Patológica/metabolismo , Tejido Subcutáneo/metabolismo , Tejido Subcutáneo/patología , PorcinosRESUMEN
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. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude mice were experimentally inoculated with embryonated A. tetraptera ova harvested from enzootically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/mL piperazine in water for 4 or 8 wk. Regardless of the mouse strain or treatment, no A. tetraptera were recovered at necropsy, indicating the lack of resistance in the worms to anthelmintic treatment. In addition, 10 of 12 DBA/2 positive-control mice cleared the A. tetraptera infection without treatment. To evaluate the feasibility of EAD PCR for A. tetraptera, 69 cages of breeder mice enzootically infected with A. tetraptera were housed on a Tecniplast IVC rack as a field study. On day 0, 56% to 58% of the cages on this rack tested positive for A. tetraptera by PCR and fecal centrifugation flotation (FCF). PCR from EAD swabs became positive for A. tetraptera DNA within 1 wk of placing the above cages on the rack. When these mice were treated with 150 ppm fenbendazole in feed, EAD PCR reverted to pinworm-negative after 1 mo of treatment and remained negative for an additional 8 wk. The ability of EAD PCR to detect few A. tetraptera positive mice was investigated by housing only 6 infected mice on another IVC rack as a field study. The EAD PCR from this rack was positive for A. tetraptera DNA within 1 wk of placing the positive mice on it. These findings demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive assay that may be a useful diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF.
Asunto(s)
Monitoreo Epidemiológico/veterinaria , Oxiuriasis/veterinaria , Oxyuroidea/aislamiento & purificación , Animales , Antihelmínticos/farmacología , Brotes de Enfermedades , Polvo/análisis , Heces/parasitología , Femenino , Fenbendazol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Oxiuriasis/epidemiología , Oxiuriasis/parasitología , Oxyuroidea/clasificación , Oxyuroidea/efectos de los fármacos , Oxyuroidea/crecimiento & desarrollo , Reacción en Cadena de la PolimerasaRESUMEN
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). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a CâT transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies ('inhibitors') to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia.
Asunto(s)
Enfermedades de los Perros/genética , Perros/genética , Factor VIII/genética , Hemofilia A/veterinaria , Mutación Puntual , Animales , Codón de Terminación , Perros/sangre , Hemofilia A/genética , Análisis de Secuencia de ADN/veterinariaRESUMEN
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. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency.
Asunto(s)
Deficiencia del Factor VII/genética , Deficiencia del Factor VII/terapia , Factor VII/genética , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Precursores de Proteínas/genética , Adenoviridae/genética , Animales , Perros , Deficiencia del Factor VII/sangre , Expresión Génica , Vectores Genéticos/administración & dosificación , Células HEK293 , Humanos , Mutación Puntual , TransgenesRESUMEN
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. PRINCIPAL FINDINGS: Twenty pigs developed severe and diffuse distal coronary artery atherosclerosis (i.e., severe = intimal area as a percent medial area > 200% in at least 2 coronary artery cross sections and diffuse distal = intimal area as a percent medial area ≥ 150% over 3 sections separated by 2 cm in the distal half of the coronary artery). The other 17 pigs had substantially less coronary artery atherosclerosis. All 37 pigs had blood pressure in a range that would be considered hypertensive in humans and developed elevations in total and LDL and HDL cholesterol, weight gain, increased backfat, and increased insulin resistance (Bergman Si) without overt diabetes. Insulin resistance was not associated with atherosclerosis severity. Five additional pigs fed regular pig chow also developed increased insulin resistance but essentially no change in the other variables and little to no detectible coronary atherosclerosis. Most importantly, the 20 high fat/high NaCl diet-fed pigs with severe and diffuse distal coronary artery atherosclerosis had substantially greater increases (p< 0.05) in oxidized LDL (oxLDL) and fructosamine consistent with increased protein glycation. CONCLUSION: In pigs fed a high fat/high NaCl diet, glycated proteins are induced in the absence of overt diabetes and this degree of increase is associated with the development of severe and diffuse distal coronary artery atherosclerosis.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Enfermedad de la Arteria Coronaria , Fructosamina/sangre , Resistencia a la Insulina , Lipoproteínas LDL/sangre , Cloruro de Sodio Dietético/farmacología , Animales , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/fisiopatología , Modelos Animales de Enfermedad , Índice de Severidad de la Enfermedad , Cloruro de Sodio Dietético/efectos adversos , Sus scrofaRESUMEN
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. Treatment options for Ulcerative Dermatitis are largely palliative and ineffective. We have proposed mouse Ulcerative Dermatitis as a model for human Skin Picking Disorder based on similar epidemiology, behavior, and its comorbidity and mechanistic overlap with hair pulling (trichotillomania). We predicted that mouse Ulcerative Dermatitis would be treated by N-Acetylcysteine, as this compound is highly effective in treating both Skin Picking Disorder and Trichotillomania. Furthermore, we hypothesized that N-Acetylcysteine's mode of action is as a precursor to the production of the endogenous antioxidant glutathione in the brain, and therefore intranasal glutathione would also treat Ulcerative Dermatitis. Accordingly, we show in a heterogenous prospective trial, the significant reduction in Ulcerative Dermatitis lesion severity in mice receiving either N-acetylcysteine (oral administration) or glutathione (intranasal). The majority of mice treated with N-acetylcysteine improved slowly throughout the course of the study. Roughly half of the mice treated with glutathione showed complete resolution of lesion within 2-4 weeks, while the remainder did not respond. These findings are the first to show that the use of N-acetylcysteine and Glutathione can be curative for mouse Ulcerative Dermatitis. These findings lend additional support for mouse Ulcerative Dermatitis as a model of Skin Picking Disorder and also support oxidative stress and glutathione synthesis as the mechanism of action for these compounds. As N-Acetylcysteine is poorly tolerated by many patients, intranasal glutathione warrants further study as potential therapy in Skin Picking, trichotillomania and other body-focused repetitive behavior disorders.
Asunto(s)
Acetilcisteína/uso terapéutico , Antioxidantes/uso terapéutico , Dermatitis/tratamiento farmacológico , Úlcera Cutánea/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Glutatión/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Conducta Autodestructiva/complicaciones , Conducta Autodestructiva/tratamiento farmacológicoRESUMEN
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. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.
Asunto(s)
Terapia Genética , Hemofilia B/terapia , Lentivirus/genética , Hígado/patología , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Perros , Femenino , Vectores Genéticos/metabolismo , Ratones Endogámicos C57BL , Mutágenos/toxicidad , Factores de Tiempo , Transducción Genética , TransgenesRESUMEN
Hyperglycemia stimulates secretion of αVß3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVß3 integrin. This study determined whether blocking ligand occupancy of αVß3 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 ß3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 ± 50 µg of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 ± 57 µg/mg), whereas diabetic animals treated with the anti-ß3 F(ab)2 excreted 119 ± 55 µg/mg (P < .05). Mesangial volume/glomerular volume increased to 21 ± 2.4% in control-treated diabetic animals compared with 14 ± 2.8% (P < .01) in animals treated with active antibody. Diabetic animals treated with control F(ab)2 had significantly less glomerular podocin staining compared with nondiabetic animals, and this decrease was attenuated by treatment with anti-ß3 F(ab)2. Glomerular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals (212 ± 14 nm) compared with nondiabetic animals (170 ± 8.8 nm), but it was unchanged (159.9 ± 16.4 nm) in animals receiving anti-ß3 F(ab)2. Podocyte foot process width was greater in control, F(ab)2-treated, animals (502 ± 34 nm) compared with animals treated with the anti-ß3 F(ab)2 (357 ± 47 nm, P < .05). Renal ß3 tyrosine phosphorylation decreased from 13 934 ± 6437 to 6730 ± 1524 (P < .01) scanning units in the anti-ß3-treated group. We conclude that administration of an antibody that inhibits activation of the ß3-subunit of αVß3 that is induced by hyperglycemia attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it may have utility in preventing the progression of this disease complication.
Asunto(s)
Nefropatías Diabéticas/etiología , Integrina alfaVbeta3/metabolismo , Animales , Anticuerpos Monoclonales , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Membrana Basal Glomerular/patología , Integrina alfaVbeta3/antagonistas & inhibidores , Masculino , Ratones Endogámicos BALB C , Podocitos/patología , Proteinuria/etiología , PorcinosRESUMEN
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. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP.
Asunto(s)
Modelos Animales de Enfermedad , Púrpura Trombocitopénica Idiopática/diagnóstico , Púrpura Trombocitopénica Idiopática/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Plaquetas/inmunología , Plaquetas/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Perros , Hemorragia/inmunología , Fenotipo , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangreRESUMEN
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. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/- pigs were generated. Subsequent breeding of heterozygotes produced LDLR-/- pigs. When fed a standard swine diet (low fat, no cholesterol), LDLR+/- pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR-/- pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.
Asunto(s)
Aterosclerosis/genética , Marcación de Gen , Hipercolesterolemia/genética , Receptores de LDL/genética , Animales , Animales Modificados Genéticamente , Aorta/metabolismo , Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Dieta , Modelos Animales de Enfermedad , Femenino , Orden Génico , Sitios Genéticos , Genotipo , Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos , Lípidos/sangre , Masculino , Receptores de LDL/metabolismo , Porcinos , Porcinos Enanos , Factores de TiempoRESUMEN
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. Although there are multiple delivery routes for both viral and non-viral vectors in animals, portal vein administration delivers vectors directly and efficiently into the liver. Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Terapia Genética , Vectores Genéticos/genética , Hemofilia B/genética , Hemofilia B/terapia , Administración Intravenosa , Animales , Perros , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Ratones , Vena Porta/cirugíaRESUMEN
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 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.
Asunto(s)
Plaquetas/fisiología , Enfermedades de los Perros/terapia , Factor VIII/genética , Terapia Genética/veterinaria , Hemofilia A/veterinaria , Hemostasis , Integrina alfa2/metabolismo , Animales , Enfermedades de los Perros/genética , Perros , Regulación de la Expresión Génica/fisiología , Terapia Genética/métodos , Hemofilia A/terapia , Humanos , Integrina alfa2/genéticaRESUMEN
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. More effective factor products are being developed. Moreover, gene therapies for sustained correction of hemophilia are showing much promise in preclinical studies and in clinical trials. These advances in molecular medicine heavily depend on availability of well-characterized small and large animal models of hemophilia, primarily hemophilia mice and dogs. Experiments in these animals represent important early and intermediate steps of translational research aimed at development of better and safer treatments for hemophilia, such a protein and gene therapies or immune tolerance protocols. While murine models are excellent for studies of large groups of animals using genetically defined strains, canine models are important for testing scale-up and for long-term follow-up as well as for studies that require larger blood volumes.
Asunto(s)
Hemofilia A/patología , Animales , Investigación Biomédica , Terapias Complementarias , Modelos Animales de Enfermedad , Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Humanos , Donantes de TejidosRESUMEN
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 alpha(V)beta(3) integrin. We tested whether alpha(V)beta(3) integrin activation was increased in diabetic animals and whether an antibody to beta(3) 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 beta(3) 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 beta(3). Phosphorylation of beta(3) 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 alpha(V)beta(3)-linked signaling pathways accelerates atherosclerosis in diabetes and that administration of an antibody to beta(3) to diabetic pigs inhibits alpha(V)beta(3) activation, IGF-1-stimulated signaling, and atherosclerotic lesion development. This approach offers a potential therapeutic approach to the treatment of this disorder.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Aterosclerosis/complicaciones , Aterosclerosis/prevención & control , Diabetes Mellitus Experimental/complicaciones , Integrina alfaVbeta3/inmunología , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Arteria Femoral/metabolismo , Arteria Femoral/patología , Fragmentos Fab de Inmunoglobulinas , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Miocitos del Músculo Liso/metabolismo , Fosforilación , Transducción de Señal , Porcinos , Factores de TiempoRESUMEN
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.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4(+)FoxP3(+)IL-10(+) T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Hemofilia B/terapia , Músculo Esquelético/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Perros , Factor IX/metabolismo , Citometría de Flujo , Hemofilia B/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Músculo Esquelético/patologíaRESUMEN
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. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.
Asunto(s)
Dependovirus , Factor IX/biosíntesis , Terapia Genética , Vectores Genéticos , Hemofilia B/terapia , Terapia de Inmunosupresión , Músculo Esquelético , Animales , Anticuerpos Antivirales/sangre , Inhibidores de Factor de Coagulación Sanguínea/sangre , Perros , Factor IX/genética , Hemofilia B/sangre , Hemofilia B/genética , Hemorragia/sangre , Hemorragia/genética , Hemorragia/terapia , Humanos , Inyecciones Intramusculares , Transducción GenéticaRESUMEN
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.
RESUMEN
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. We hypothesize that hemostasis assessment can be un-blinded by Acoustic Radiation Force Impulse (ARFI) ultrasound. In this first of a two-part series, we present in vivo ARFI hemostasis imaging data obtained in relevant canine models of femoral artery puncture. Above arteriotomies, ARFI-induced displacements were large (3.5 to >5.0 microm) relative to surrounding soft tissue soon after needle removal, which was consistent with our expectation for pooled extravasated blood. ARFI-induced displacements above arteriotomies decreased in magnitude (to approximately 2 microm) some time after needle removal and suggested the onset of hemostasis. This preclinical investigation served as proof of concept and justification for a pilot human study, which is presented in part two of this series.