Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy

Kummerfeld, Maren; Meens, Jochen; Haas, Ludwig; Baumgärtner, Wolfgang; Beineke, Andreas

doi:10.1016/j.jviromet.2009.04.030

Cited by 42 publications

(44 citation statements)

References 23 publications

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“…In order to detect viral antigen (TMEV), axonal proteins (p-NF, n-NF, β-APP) and cell surface antigens (CD3, CD107b), a standard avidin-biotin-peroxidase complex (ABC; Vector Laboratories, Burlingame, Calif., USA) method was used as described [8,22]. Briefly, sections were dewaxed and hydrated through graded alcohols.…”

Section: Methodsmentioning

confidence: 99%

“…Quantification of macrophages/microglia and T cells was performed using a biotinylated CD107b-specific antibody (monoclonal rat anti-mouse, clone M3/84, diluted 1:200; AbD Serotec., Oxford, UK) and a CD3-specific antibody (polyclonal rabbit anti-human, diluted 1:300; DakoCytomation GmbH, Hamburg, Germany), respectively. Virus protein detection was performed using a polyclonal rabbit anti-TMEV capsid protein VP1-specific antibody as described before [22]. Control sections were incubated with isotype-matched control antibodies or with nonimmune sera.…”

Section: Methodsmentioning

confidence: 99%

“…Methanol-fixed frozen sections of preselected tissues were rinsed in 0.1% Triton X-100 in PBS for 30 min. Nonspecific binding was blocked with 20% goat serum diluted in PBS/0.1% Triton X-100/1% BSA (bovine serum albumin) for 30 min, before incubation with the primary polyclonal antibody against TMEV at 4° overnight (polyclonal anti-TMEV BeAn VP1 antibody, diluted 1:2,000) [22]. After washing with 0.1% Triton X-100 in PBS, incubation with a Cy3-conjugated goat anti-rabbit IgG antibody (H+L, diluted 1:200; Jackson ImmunoResearch Europe, Newmarket, UK) was performed for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Controls were incubated with rabbit preimmune serum and/or a mouse or rat IgG1 anti-isotype antibody (Millipore GmbH, Schwalbach/Ts., Germany) instead of primary antibodies. Expression of cell-type specific markers was studied by a Leica TCS SP5 laser confocal microscope (Leica, Wetzlar, Germany) equipped with a 63-fold water immersion objective as described [22,25]. For excitation of Cy2-conjugated secondary antibody and Alexa Fluor 488, a 488-nm Argon laser was used.…”

Section: Methodsmentioning

confidence: 99%

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Periventricular Demyelination and Axonal Pathology Is Associated with Subependymal Virus Spread in a Murine Model for Multiple Sclerosis

et al. 2012

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Objectives: Theiler’s murine encephalomyelitis virus (TMEV) infection of mice is a widely used animal model for demyelinating disorders, such as multiple sclerosis (MS). The aim of the present study was to identify topographical differences of TMEV spread and demyelination in the brain of experimentally infected susceptible SJL/J mice and resistant C57BL/6 mice. Methods: Demyelination was confirmed by Luxol fast blue and cresyl violet staining and axonal damage by neurofilament-specific and β-amyloid precursor protein-specific immunohistochemistry. Viral dissemination within the central nervous system (CNS) was quantified by immunohistochemistry and in situ hybridization. Further, the phenotype of infected cells was determined by confocal laser scanning microscopy. Results: An early transient infection of periventricular cells followed by demyelination and axonopathies around the fourth ventricle in SJL/J mice was noticed. Periventricular and brain stem demyelination was associated with a predominant infection of microglia/macrophages and oligodendrocytes. Conclusions: Summarized, the demonstration of ependymal infection and subjacent spread into the brain parenchyma as well as regional virus clearance despite ongoing demyelination and axonal damage in other CNS compartments allows new insights into TME pathogenesis. This novel aspect of TMEV CNS interaction will enhance the understanding of region-specific susceptibilities to injury and regenerative capacities of the brain in this MS model.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Periventricular Demyelination and Axonal Pathology Is Associated with Subependymal Virus Spread in a Murine Model for Multiple Sclerosis

et al. 2012

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“…To investigate the in diluted in sodium cacodylate buffer (0.1M, pH 7.3). vivo behavior of BO1-cells in the nondemyelinated Osmium-fixed samples were dehydrated and embedded environment, animals (n = 4) received 2 µl DMEM conin epon resin as described previously (38,69). Ultrathin taining 10 5 BO-1 cells.…”

Section: Stereotaxic Implantation Of Bo-1 Cells Intomentioning

confidence: 99%

Highly Malignant Behavior of a Murine Oligodendrocyte Precursor Cell Line following Transplantation into the Demyelinated and Nondemyelinated Central Nervous System

Hansmann¹,

Pringproa²,

Ulrich³

et al. 2012

Cell Transplant

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Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromideinduced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFRα, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.Key words: Caudal cerebellar peduncle; Murine giant cell glioblastoma; Oligodendrocyte precursor cell; Signal transducer and activator of transcription-3; Transplantation INTRODUCTIONformation in the CNS (1,9,45,60). Lacking or limited production of myelin under pathophysiological conditions has been ascribed to either a loss of OPCs or a Transplantation of myelin-producing cells into the normal and demyelinated brain and spinal cord has been reduced differentiation of OPCs into mature oligodendrocytes (22,23,37,68,69). frequently used to study both the viability of the grafted tissue (16) as well as the potency of these cells to proExperimental approaches to study demyelination and remyelination include transplantation of OPCs into the duce myelin (4,10,19,25,31,58,71,73). The analysis of the basic mechanisms that control central nervous sysdemyelinated CNS as well as the analysis of naturally occurring and genetically induced mouse mutants (3,15, tem (CNS) remyelination is of particular clinical relevance since it may prompt the development of novel 17,24,35,41,49). These studies were almost exclusively performed in rats. To fully exploit the potential of this therapeutic strategies to cure demyelinating disorders, such as multiple sclerosis (22,23 (20,74). Historically, this is most Establishment and maintenance of the ...

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Transcriptional profiling predicts overwhelming homology of schwann cells, olfactory ensheathing cells, and schwann cell‐like glia

Ulrich¹,

Imbschweiler²,

Kalkuhl

et al. 2014

Glia

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Schwann cells (SCs), olfactory ensheathing cells (OECs), and central nervous system Schwann cell-like glia (SG) represent a group of nerve growth factor receptor p75 (NGFR)-positive cells, originating from different tissues. Because of their pro-regenerative capacities, these cells are subjects in experimental transplantation-based therapies of spinal cord trauma. The objective of this study was to compare the transcriptomes of uninfected and canine distemper virus-infected OECs, SCs, SG and fibroblasts (FBs) derived from four beagle dogs and cultured under identical conditions in vitro, employing canine genome 2.0 arrays (Affymetrix). Here, we observed a complete lack of transcriptional differerences between OECs and SG, a high similarity of OECs/SG to SCs, and a marked difference of SCs and OECs/SG towards FBs. Differentially expressed genes possibly involved in the maintenance of cell type-specific identity included an up-regulation of HOXD8 and HOXC4 in SCs, and an up-regulation of CNTNAP2 and EFEMP1 in OECs/SG. We identified cell type-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection. Thereby AQP1 and SCRG1 were predicted to be the most powerful biomarkers distinguishing SCs from OECs/SG. Immunofluorescence confirmed a higher expression of SCRG1 in OECs and SG, and conversely a higher expression of AQP1 in SCs in vitro. Furthermore, canine and murine olfactory nerves showed SCRG1-positive, AQP1-negative OECs and/or axons, whereas sciatic nerves displayed multifocal non-myelinated, AQP1-positive, SCRG1-negative cells. Conclusively, OECs/SG are suggested to be a uniform cell type differing only in the tissue of origin and highly related to SCs.

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Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy

Cited by 42 publications

References 23 publications

Periventricular Demyelination and Axonal Pathology Is Associated with Subependymal Virus Spread in a Murine Model for Multiple Sclerosis

Periventricular Demyelination and Axonal Pathology Is Associated with Subependymal Virus Spread in a Murine Model for Multiple Sclerosis

Highly Malignant Behavior of a Murine Oligodendrocyte Precursor Cell Line following Transplantation into the Demyelinated and Nondemyelinated Central Nervous System

Transcriptional profiling predicts overwhelming homology of schwann cells, olfactory ensheathing cells, and schwann cell‐like glia

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