Development Of Inclusion Bodies Research Articles

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Dopaminergic Neurons Exhibit an Age-Dependent Decline in Electrophysiological Parameters in the MitoPark Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most diagnosed neurodegenerative disorder, and the classic motor symptoms of the disease are attributed to selective loss of dopaminergic neurons of the substantia nigra. The MitoPark mouse is a genetic model of PD that mimics many of the key characteristics of the disease and enables the study of progressive neurodegeneration in parkinsonism. Here we have identified functional deficits in the ion channel physiology of dopaminergic neurons from MitoPark mice that both precede and are concurrent with the time course of behavioral symptomatology. Because PD is a progressive disease with a long asymptomatic phase, identification of early functional adaptations could lay the groundwork to test therapeutic interventions that halt or reverse disease progression.

Inclusões intracitoplasmáticas hialinas na medular da adrenal de bovinos

Cytoplasmic inclusion bodies in adrenal medullary chromaffin cells have been described in various species including humans. These inclusions are believed to be related to certain infectious, toxic and neurodegenerative diseases. No reports concerning such adrenal inclusions have been described in bovines. Adrenal glands from twenty bovines were evaluated in a retrospective study. Seven of these exhibited inclusions - three cases of rabies, two cases of chronic suppurative bronchopneumonia, one case of chronic suppurative peritonitis, and one case of gangrenous mastitis. The inclusions were present in higher numbers especially in cases of rabies and also in one case of chronic suppurative bronchopneumonia. The inclusions were intracytoplasmic, eosinophilic, rounded, single or multiple, of various sizes, strongly stained by PAS and were present in higher numbers in the external layer of the adrenal medulla. The inclusions were negative when subjected to immunohistochemistry for detection of viral antigens in the cases of rabies. Although inclusion bodies were present in adrenal glands devoid of other histological alterations, they were more abundant in cases in which the adrenal gland had other alterations. The correlation between certain diseases and the development of inclusion bodies is not known, which highlights the importance of further studies on these inclusions in adrenal glands of bovines.

890. Improvement of Behavior and Mortality Following CNS Administration of AAVrh.10hCLN2 to CLN2 −/− Mice, a Model LINCL (Battan Disease) a Fatal Childhood Neurological Disorder (Batten Disease)

Top of pageAbstract Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies, leading to diffuse neuronal cell death. We have compared CNS gene transfer of CLN2 with serotype 2, 5, 8 and rh.10 AAV vectors and observed that AAVrh.10 provided the highest levels of TPP-I protein in the CNS of rodents. In order to assess the functional consequences of AAVrh.10CLN2 CNS administration, a total of 1.6|[times]|1011 genome copies of AAVrh.10hCLN2, was administered into 4 locations per hemisphere of CLN2 |[minus]|/|[minus]| mice ages 7 to 8 wk and the mice were assessed weekly for performance in tests that combine motor and sensory skills. The treated CLN2 |[minus]|/|[minus]| mice were compared to untreated CLN2 |[minus]|/|[minus]| mice (negative controls) and wild type (CLN2 +/+) mice (positive controls). Analysis of TPP-I levels in the brain demonstrated the AAVrh.10CLN2-treated mice had levels higher than the wild type control (CLN2 +/+) in all regions of the brain. Analysis of neuronal lipofucsin-associated autofluorescence, a hallmark of the disease, demonstrated between 40 to 45 % decrease for the treated CLN2|[minus]|/|[minus]| mice in the injected areas in comparison to the untreated CLN2 |[minus]|/|[minus]| mice. In a test for gait analysis, for wild type mice, the footprints for the hindpaws are largely superimposed on those for the front paws and there was no dragging. In untreated CLN2 |[minus]|/|[minus]| mice, by the age of 21 wk, there was poor gait with dragging of feet and no clear coordination of location between the forepaw and the ipsilateral hindpaw. However, in the 21 wk CLN2 |[minus]|/|[minus]| mice treated with AAVrh.10hCLN2, there was better coordination of the paws and much less dragging. In a grip strength test, the treated mice had a better performance than untreated CLN2 |[minus]|/|[minus]| mice (p 0.05). In a balance beam test, the treated mice had a better performance than untreated CLN2 |[minus]|/|[minus]| mice (p<0.005, ANOVA with group as factor and time as covariate), although the performance of AAVrh.10hCLN2 treated CLN2 |[minus]|/|[minus]| mice was not as good as wild type mice (p<0.005). Kaplan-Meier analysis of survival curves also showed that there was a statistically significant survival advantage of AAVrh.10hCLN2 treated CLN2 |[minus]|/|[minus]| mice over the untreated CLN2 |[minus]|/|[minus]| mice. The median age of survival of the injected CLN2 |[minus]|/|[minus]| mice was 147 days compared to 118 days for the uninjected CLN2 |[minus]|/|[minus]| mice (p<0.01). Together, this data suggests the conclusion that AAVrh.10CLN2 is a suitable candidate for preclinical development for the treatment of LINCL.

660. Quantitative Comparison of AAV Serotypes AAV2, AAV5, and AAVrh.10 Efficiency for CNS Gene Therapy Following Intracranial Gene Delivery

Top of pageAbstract Late infantile neuronal ceroid lipofuscinosis (Batten disease) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies which lead to neuronal cell death throughout the brain. We have previously shown that AAV2 based vectors expressing human CLN2 cDNA driven by the CMV-enhanced chicken-b-actin hybrid promoter can mediate long term expression of TPP-I in rat brain. In the present study, we have compared the ability of an AAV2-based vector to mediate distribution of TPP-I production in the rat CNS to that of recombinant AAV2-based vectors pseudotyped with capsids derived from human AAV5 and rh.10, a rhesus macaque derived AAV. Vectors (2.5|[times]|109 genome copies) were injected into the striatum of 8 wk old male F344 rats. Fluorometric enzyme assay for TPP-I in the injected region after 4 wk revealed that while AAV2- and AAV5-mediated gene delivery produced TPP-I enzyme levels that were 1.2 and 1.8 fold of endogenous (p<0.01 compared to naive) respectively, AAVrh.10-mediated delivery provided TPP-I levels that approached 17-fold above endogenous levels (p<0.01 compared to either naive or AAV5-treated animals). Immunohistochemical staining showed vector-derived TPP-I in the striatum of rats injected with each of the three vectors, with AAV5 and AAVrh.10 producing the greatest striatal filling. Volumetric analysis showed that while infection by AAV2-derived vector lead to TPP-I detection in 5.1|[plusmn]|0.4% of the striatum, AAV5 and AAVrh.10 resulted in TPP-I expression in 46.9|[plusmn]|2.9% and 51.2|[plusmn]|6.1% of the striatum respectively. Co-immunofluorescence studies demonstrated that virtually all neurons in the region of AAVrh.10 injection were TPP-I positive but no glia stained TPP-I positive. When the whole brain was examined, AAVrh.10 mediated more extensive TPP-I expression, with TPP-I being detected in the globus pallidus and basal forebrain. There was also prominent substantia nigra staining at 4 wk with AAVrh.10 delivery, suggesting retrograde distribution. In addition TPP-I positive cells were readily detected in the frontal cortex and the thalamus, both of which project to the location of the injection in the striatum. When non-striatal staining was quantitated, AAV2 administration resulted in TPP-I positive cells in 0.4|[plusmn]|0.1% of the hemisphere compared to 4.8|[plusmn]|0.6% for AAV5 and 8.6|[plusmn]|1.4% for AAVrh.10. The data show that capsids from other AAV serotypes give more widespread and higher levels of gene transfer than AAV2 vectors and suggests that high transgene expression levels may correlate with a wider distribution of transgene product. In particular, AAV vectors pseudotyped with the capsid from AAVrh.10 may be applicable to gene therapy for diffuse neurological diseases.

Development of Inclusion Bodies in Type II Pneumocyte of Human Fetus

Development of Inclusion Bodies in Type II Pneumocyte of Human Fetus

The toxicity of 2-hydroxystilbamidine: Probable fatal toxic reaction during treatment of blastomycosis.

Excerpt Following the original report of Schoenbach et al.1of the successful therapy of a case of systemic North American blastomycosis treated with the aromatic diamidine, stilbamidine, numerous p...