19 research outputs found

    A Study On A Velogenic Viscerotropic Newscastle Disease Virus In Vitro And In Vivo

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    The velogenic viscerotropic Newcastle disease virus or the Asiatic strain has been considered the most virulent strain of Newcastle disease virus. It is commonly found in Southeast Asia and it has been known to cause 100\ mortality in susceptible flocks. In spite of this, very little research has been conducted on it as many countries prohibit the handling of this strain of virus. In view of this, a project has been undertaken at Universiti Pertanian Malaysia to study the biological properties, cytopathogenicity and morphogenesis of a locally isolated velogenic viscerotropic Newcastle disease virus and to determine its effects on the trachea of nonvaccinated and vaccinated chickens. The in vitro study has shown that this virus has a mean death time of 66 hours, and an intracerebral pathogenicity index of 1.90 . Polykaryocytosis is the principal form of cytopathic effect it produces in chick embryo fibroblasts and cells infected by it haemadsorp red blood cells. This virus plaques in cell culture. Negatively stained virus particles have diameters ranging from 100 to 600 nanometers. Electron microscopy demonstrated that the virus replicates in the cytoplasm of infected cells and aggregates of nucleocapsids are found in the cytoplasm. The virus matures at the cell membrane and is released by budding

    Early Vascular and Neuronal Changes in a VEGF Transgenic Mouse Model of Retinal Neovascularization

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    PURPOSE. To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). METHODS. Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. RESULTS. Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. CONCLUSIONS. Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease. (Invest Ophthalmol Vis Sci. 2006;47:4638 -4645

    rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

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    PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45 ϩ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye. (Invest Ophthalmol Vis Sci. 2009;50:4279 -4287

    A case of parvovirus infection in a dog

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    A typical case of parvovirus infection in a 4 month old male German Shepherd is reported. The disease is characterised by depression, pyrexia, vomiting and diarrhoea. Electron microscopic examination of faeces from the sick dog revealed the presence of particles resembling paroooirus

    Biomarkers for Diabetic Retinopathy – Could Endothelin 2 Be Part of the Answer? - Fig 1

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    <p><b>Edn2 (A) and Ednrb (B) fold changes in mRNA expression in young and mature retinae of wt, Akita, Kimba and Akimba mice</b>. Data represented are fold changes in expression normalised against Ppia as the housekeeping gene. Differential expression was determined using delta delta Ct according to Livak and Schmittgen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160442#pone.0160442.ref023" target="_blank">23</a>]. Fold changes in expression in Akita, Kimba and Akimba retinae represent increases or decreases in mRNA expression levels compared to wt retinae. N = 4 per group; *p<0.05 compared to wt and Akita</p

    Evidence of Müller cell gliosis in mature Akimba mice with a severe phenotype.

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    <p>In the mature wt retina GFAP expression was localised and restricted to the astrocytes that reside on the GCL (arrowheads, C-D). In Akimba mice, GFAP expression was observed as thick processes in the GCL and long radial processes in the INL/OPL, indicating Müller cell gliosis (G-H). Edn2 only co-localised with GFAP in Müller cell processes in the INL/OPL (arrows, H) but not in the astrocytes of the GCL. Sections were counterstained with DAPI (B, F). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PIS/POS: photoreceptor inner and outer segments. Scale bar: 200μm</p

    Cellular localisation of Edn2 in the mature retina.

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    <p>Following 20 weeks of chronic hyperglycaemia in the mature Akita retina there was increased Edn2 staining in the IPL compared to young Akita retinae (F). Particularly in the mature Akimba retina GS staining was much reduced compared to the young retina, suggesting not only loss of photoreceptors (L) but also loss of Müller cells (I, arrowheads). Müller cell loss was also evident in mature Kimba retinae (M, arrowheads; O, arrows) compared to young Kimba retinae, but the loss was less pronounced. Scale bar: 100μm</p

    Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue

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    Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10-15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.rAAV.RPE65 was injected into the subretinal space of knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.This work demonstrated the potential benefits of using the mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina

    Serum Edn2 concentration in young and mature wt, Akita, Kimba and Akimba mice.

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    <p>Serum concentrations did not differ between young wt, Akita, Kimba and Akimba mice (A). In wt mice, Edn2 serum concentration decreased with age. In mature mice (B), there was no significant difference in serum Edn2 concentration between wt, Akita and Kimba mice. However, circulating levels of Edn2 were significantly higher in mature Akimba mice (6.9pg/mg total retina protein) compared to wt (3.9pg/mg; p<0.01), Akita (3.8pg/mg; p<0.01) and Kimba (4.6pg/mg total retina protein; p<0.05) mice.</p
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