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Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.
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Cisterna Magna/metabolismo , Dependovirus/genética , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Transducción Genética , Animales , Catéteres , Sistema Nervioso Central/metabolismo , Genes Reporteros , Terapia Genética , Vectores Genéticos/administración & dosificación , Humanos , Inyecciones Espinales , Imagen por Resonancia Magnética , Modelos Animales , Ovinos , Cirugía Asistida por Computador , Tomografía Computarizada por Rayos X , Transgenes , Grabación en VideoRESUMEN
BACKGROUND: Multidrug resistance-associated protein 1 (MRP1) overexpression plays a major role in chemoresistance in glioblastoma multiforme (GBM) contributing to its notorious deadly nature. Although MRP1-siRNA transfection to GBM in vitro has been shown to sensitise the cells to drug, MRP1 silencing in vivo and the phenotypic influence on the tumour and normal tissues upon MRP1 down-regulation have not been established. Here, porous silicon nanoparticles (pSiNPs) that enable high-capacity loading and delivery of siRNA are applied in vitro and in vivo. RESULT: We established pSiNPs with polyethyleneimine (PEI) capping that enables high-capacity loading of siRNA (92 µg of siRNA/mg PEI-pSiNPs), and optimised release profile (70% released between 24 and 48 h). These pSiNPs are biocompatible, and demonstrate cellular uptake and effective knockdown of MRP1 expression in GBM by 30%. Also, siRNA delivery was found to significantly reduce GBM proliferation as an associated effect. This effect is likely mediated by the attenuation of MRP1 transmembrane transport, followed by cell cycle arrest. MRP1 silencing in GBM tumour using MRP1-siRNA loaded pSiNPs was demonstrated in mice (82% reduction at the protein level 48 h post-injection), and it also produced antiproliferative effect in GBM by reducing the population of proliferative cells. These results indicate that in vitro observations are translatable in vivo. No histopathological signs of acute damage were observed in other MRP1-expressing organs despite collateral downregulations. CONCLUSIONS: This study proposes the potential of efficient MRP1-siRNA delivery by using PEI-capped pSiNPs in achieving a dual therapeutic role of directly attenuating the growth of GBM while sensitising residual tumour cells to the effects of chemotherapy post-resection.
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Silenciador del Gen , Glioblastoma/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Nanopartículas/química , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Silicio/química , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ratones Desnudos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nanopartículas/ultraestructura , Especificidad de Órganos , Fenotipo , Porosidad , Propionatos/farmacología , Quinolinas/farmacologíaRESUMEN
RATIONALE: It is unclear how septic shock causes acute kidney injury (AKI) and whether this is associated with histological change. OBJECTIVES: We aimed to determine the nature and extent of changes in renal structure and function over time in an ovine model of septic shock. METHODS: Fifteen sheep were instrumented with a renal artery flow probe and renal vein cannula. Ten were given intravenous Escherichia coli to induce septic shock, and five acted as controls. Animals were mechanically ventilated for 48 hours, while receiving protocol-guided parenteral fluids and a norepinephrine infusion to maintain mean arterial pressure. Renal biopsies were taken every 24 hours or whenever animals were oliguric for 2 hours. A renal pathologist, blinded to tissue source, systematically quantified histological appearance by light and electron microscopy for 31 prespecified structural changes. MEASUREMENTS AND MAIN RESULTS: Sheep given E. coli developed septic shock, oliguria, increased serum creatinine, and reduced creatinine clearance (AKI), but there were no changes over time in renal blood flow between groups (P > 0.30) or over time within groups (P > 0.50). Renal oxygen consumption increased only in nonseptic animals (P = 0.01), but there was no between-group difference in renal lactate flux (P > 0.50). There was little structural disturbance in all biopsies and, although some cellular appearances changed over time, the only difference between septic and nonseptic animals was mesangial expansion on electron microscopy. CONCLUSIONS: In an intensive care-supported model of gram-negative septic shock, early AKI was not associated with changes in renal blood flow, oxygen delivery, or histological appearance. Other mechanisms must contribute to septic AKI.
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Riñón/fisiopatología , Choque Séptico/fisiopatología , Lesión Renal Aguda/etiología , Animales , Biopsia , Presión Sanguínea , Gasto Cardíaco , Modelos Animales de Enfermedad , Femenino , Riñón/patología , Circulación Renal , Ovinos , Choque Séptico/complicaciones , Choque Séptico/patologíaRESUMEN
Gaucher disease arises from mutations in the ß-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the ß-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in ß-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.
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Enfermedad de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Glicoesfingolípidos/metabolismo , Lisosomas/metabolismo , Enfermedad Aguda , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Neuronas/metabolismo , Fenotipo , Ovinos , Bazo/metabolismoRESUMEN
BACKGROUND: The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. METHODS: Six normal ferrets (7 -8 weeks old) were treated with a 150 µL LPC pre-treatment, followed one hour later by a 500 µL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. RESULTS: The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. CONCLUSIONS: This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.
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Expresión Génica/efectos de los fármacos , Operón Lac/genética , Lentivirus/genética , Lisofosfatidilcolinas/farmacología , Mucosa Respiratoria/efectos de los fármacos , Transducción Genética/métodos , Animales , Femenino , Hurones , Vectores Genéticos , Proteína p24 del Núcleo del VIH/sangre , Pulmón , Masculino , Mucosa Respiratoria/patología , TráqueaRESUMEN
Triiodothyronine (T3) concentrations in plasma decrease during acute illness and it is unclear if this contributes to disease. Clinical and laboratory studies of T3 supplementation in disease have revealed little or no effect. It is uncertain if short term supplementation of T3 has any discernible effect in a healthy animals. Observational study of intravenous T3 (1 µg/kg/h) for 24 h in a healthy sheep model receiving protocol-guided intensive care supports (T3 group, n=5). A total of 45 endpoints were measured including hemodynamic, respiratory, renal, hematological, metabolic and endocrine parameters. Data were compared with previously published studies of sheep subject to the same support protocol without administered T3 (No T3 group, n=5). Plasma free T3 concentrations were elevated 8-fold by the infusion (pmol/l at 24 h; T3 group 34.9±9.9 vs. No T3 group 4.4±0.3, P<0.01, reference range 1.6 to 6.8). There was no significant physiological response to administration of T3 over the study duration. Supplementation of intravenous T3 for 24 h has no physiological effect on relevant physiological endpoints in healthy sheep. Further research is required to understand if the lack of effect of short-term T3 may be related to kinetics of T3 cellular uptake, metabolism and action, or acute counterbalancing hormone resistance. This information may be helpful in design of clinical T3 supplementation trials.
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In this study, lead (Pb) bioaccessibility was assessed in peri-urban contaminated soils using a variety of established in vitro assays. Bioaccessibility data was then used to predict Pb relative bioavailability (RBA) using published in vivo-in vitro regression models in order to compare calculated estimates and measured values. Lead bioaccessibility varied depending on the in vitro methodology employed with the relative bioavailability leaching procedure (RBALP) and in vitro gastrointestinal (IVG) assays providing more conservative Pb bioaccessibility values compared to those determined using PBET, UBM and Rel-SBRC-I assays. When Pb RBA was calculated, predicted values using PBET-G and UBM-G data were similar to measured Pb RBA values. However, Pb RBA was over-estimated by 1.6-5.5- and 2.6-6.6-fold when data and regression models from RBALP and IVG-G assays were employed.
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Monitoreo del Ambiente/métodos , Plomo/análisis , Plomo/farmacocinética , Modelos Biológicos , Contaminantes del Suelo/análisis , Contaminantes del Suelo/farmacocinética , Animales , Disponibilidad Biológica , Monitoreo del Ambiente/estadística & datos numéricos , Predicción , Mucosa Gástrica/metabolismo , Humanos , Incineración , Mucosa Intestinal/metabolismo , Modelos Lineales , Eliminación de Residuos , Porcinos , Remodelación UrbanaRESUMEN
Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. ARTICLE HIGHLIGHTS: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.
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Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Porcinos , Humanos , Animales , Ratones , Trasplante de Islotes Pancreáticos/métodos , Supervivencia de Injerto , Islotes Pancreáticos/irrigación sanguínea , Diabetes Mellitus Tipo 1/cirugía , ColágenoRESUMEN
BACKGROUND: Huntington's disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1â:â20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. OBJECTIVE AND METHODS: Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. RESULTS: Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. CONCLUSION: This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.
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Enfermedad de Huntington , Sustancia Blanca , Animales , Humanos , Femenino , Ovinos , Anciano , Enfermedad de Huntington/metabolismo , Distribución Tisular , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Sustancia Blanca/diagnóstico por imagen , Imagen por Resonancia Magnética , Proteínas Mutantes/metabolismoRESUMEN
Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the luciferase (Luc), LacZ, and hCFTR transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline [PBS]) conditioning. At 1 month, bioluminescence was detected in vivo in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined ex vivo there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected in vivo in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged ex vivo. LacZ expression was predominantly observed in the alveolar regions of the lung. hCFTR was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.
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Fibrosis Quística , Animales , Callithrix , Fibrosis Quística/genética , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genes Reporteros , Terapia Genética , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Pulmón , Ratones , Proyectos Piloto , Transducción GenéticaRESUMEN
The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.
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Tejido Adiposo , Músculo Esquelético , Tejido Adiposo/metabolismo , Animales , Feto , Glucólisis , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , OvinosRESUMEN
Preclinical imaging studies of fetal hemodynamics require anesthesia to immobilize the animal. This may induce cardiovascular depression and confound measures under investigation. We compared the impact of four anesthetic regimes upon maternal and fetal blood gas and hemodynamics during baseline periods of normoxia, and in response to an acute hypoxic challenge in pregnant sheep. Merino ewes were surgically prepared with maternal and fetal vascular catheters and a fetal femoral artery flow probe at 105-109 days gestation. At 110-120 days gestation, ewes were anesthetized with either isoflurane (1.6%), isoflurane (0.8%) plus ketamine (3.6 mg·kg-1 ·h-1 ), ketamine (12.6 mg·kg-1 ·h-1 ) plus midazolam (0.78 mg·kg-1 ·h-1 ), propofol (30 mg·kg-1 ·h-1 ), or remained conscious. Following 60 min of baseline recording, nitrogen was administered directly into the maternal trachea to displace oxygen and induce maternal and thus fetal hypoxemia. During normoxia, maternal PaO2 was ~30 mmHg lower in anesthetized ewes compared to conscious controls, regardless of the type of anesthesia (p < .001). There was no effect of anesthesia on fetal mean arterial blood pressure (MAP; p > .05), but heart rate was 32 ± 8 bpm lower in fetuses from ewes administered isoflurane (p = .044). During maternal hypoxia, fetal MAP increased, and peripheral blood flow decreased in all fetuses except those administered propofol (p < .05). Unexpectedly, hypoxemia also induced fetal tachycardia regardless of the anesthetic regime (p < .05). These results indicate that despite maternal anesthesia, the fetus can mount a cardiovascular response to acute hypoxia by increasing blood pressure and reducing peripheral blood flow, although the heart rate response may differ from when no anesthesia is present.
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Anestesia/efectos adversos , Hipoxia Fetal/fisiopatología , Anestesia/métodos , Animales , Presión Sanguínea , Femenino , Hipoxia Fetal/etiología , Hipnóticos y Sedantes/administración & dosificación , Hipnóticos y Sedantes/efectos adversos , Isoflurano/administración & dosificación , Isoflurano/efectos adversos , Ketamina/administración & dosificación , Ketamina/efectos adversos , Midazolam/administración & dosificación , Midazolam/efectos adversos , Nitrógeno/administración & dosificación , Nitrógeno/efectos adversos , Oxígeno/metabolismo , Propofol/administración & dosificación , Propofol/efectos adversos , Flujo Sanguíneo Regional , OvinosRESUMEN
The assessment of arsenic (As) bioavailability from contaminated matrices is a crucial parameter for reducing the uncertainty when estimating exposure for human health risk assessment. In vivo assessment of As utilising swine is considered an appropriate model for human health risk assessment applications as swine are remarkably similar to humans in terms of physiology and As metabolism. While limited in vivo As bioavailability data is available in the literature, few details have been provided regarding technical considerations for performing in vivo assays. This paper describes, with examples, surgical, experimental design and analytical issues associated with performing chronic and acute in vivo swine assays to determine As bioavailability in contaminated soil and food.
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Arsénico/farmacocinética , Contaminación de Alimentos , Contaminantes del Suelo/farmacocinética , Animales , Disponibilidad Biológica , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Humanos , Modelos Animales , Medición de Riesgo/métodos , Especificidad de la Especie , PorcinosRESUMEN
Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics.
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Arsenic (As) bioavailability in spiked soils aged for up to 12 months was assessed using in vitro and in vivo methodologies. Ageing (natural attenuation) of spiked soils resulted in a decline in in vivo As bioavailability (swine assay) of over 75% in soil A (Red Ferrosol) but had no significant effect on in vivo As bioavailability even after 12 months of ageing in soil B (Brown Chromosol). Sequential fractionation, however, indicated that there was repartitioning of As within the soil fractions extracted during the time course investigated. In soil A, the As fraction associated with the more weakly bound soil fractions decreased while the residual fraction increased from 12% to 35%. In contrast, little repartitioning of As was observed in soil B indicating that natural attenuation may be only applicable for As in soils containing specific mineralogical properties.
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Arsénico/metabolismo , Arsénico/farmacocinética , Suelo/análisis , Animales , Disponibilidad Biológica , Porcinos , Factores de TiempoRESUMEN
Considerable information is available in the literature regarding the uptake of arsenic (As) from contaminated soil and irrigation water by vegetables. However, few studies have investigated As speciation in these crops while a dearth of information is available on As bioavailability following their consumption. In this study, the concentration and speciation of As in chard, radish, lettuce and mung beans was determined following hydroponic growth of the vegetables using As-contaminated water. In addition, As bioavailability was assessed using an in vivo swine feeding assay. While As concentrations ranged from 3.0 to 84.2mg As kg(-1) (dry weight), only inorganic As (arsenite and arsenate) was detected in the edible portions of the vegetables. When As bioavailability was assessed through monitoring blood plasma As concentrations following swine consumption of As-contaminated vegetables, between 50% and 100% of the administered As dose was absorbed and entered systemic circulation. Arsenic bioavailability decreased in the order mung beans>radish>lettuce=chard.
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Alimentación Animal/análisis , Arsénico/farmacocinética , Contaminantes del Suelo/farmacocinética , Porcinos , Verduras/metabolismo , Animales , Arsénico/sangre , Arsénico/metabolismo , Disponibilidad Biológica , Modelos Animales , Contaminantes del Suelo/sangre , Contaminantes del Suelo/metabolismoRESUMEN
The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth, there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. In this study, we have employed 2-photon excitation fluorescence microscopy to define the relationship between two critical metabolic cofactors nicotinamide adenine dinucleotide(P)H and flavin adenine dinucleotide, effectively utilizing a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two-photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Two-photon excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualize cardiac and other muscle pathologies.
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Colágeno/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica , Miocardio/metabolismo , Animales , Femenino , Flavina-Adenina Dinucleótido/metabolismo , NAD/metabolismo , Oxidación-Reducción , OvinosRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0125694.].
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Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential interference of NAb and to enroll serologically compatible study subjects. In this study, we report a large AAV NAb dataset comprising multiple large animal species and AAV serotypes and compare two NAb assays based on in vitro or in vivo transduction inhibition, respectively. Together with previously published AAV seroepidemiology studies, these data can serve as a reference for selecting suitable serotypes, study subjects of large animal species, and potentially human patients for rAAV treatment. In addition, we modeled the intrathalamus rAAV9 delivery in the presence of circulating anti-AAV9 NAb generated by either pre-immunization or passive transfer of NAb-positive large animal serum to mice. The data showed that circulating NAb may not be the sole determinant to inhibit brain transduction. Other aspects of pre-existing AAV immunity following natural infection or rAAV administration may be further studied to establish a more accurate inclusion criterion for clinical studies employing intraparenchymal rAAV9 injections.
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Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CßA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.