human brain vascular endothelial cells  (BioWhittaker Molecular Applications)

Journal: PLoS ONE

Article Title: Pharmacologic Inhibition of CXCL10 in Combination with Anti-malarial Therapy Eliminates Mortality Associated with Murine Model of Cerebral Malaria

doi: 10.1371/journal.pone.0060898

Figure Lengend Snippet: The table displays biological processes or functions enhanced by Atorvastatin treatment identified by Ingenuity Pathway Analysis as significantly (p <0.05) associated with the dataset.

Article Snippet: Human brain vascular endothelial cells (HBVEC; Biowhittaker, Walkersville, MD) were cultured at 37°C with 5% CO 2 in Complete Serum-free Medium Kit (Cell Systems, Kirkland, WA) supplemented with 2% fetal bovine serum (FBS; ATCC, Manassas, VA) and 100 U/ml of streptomycin, 100 U/ml of penicillin (Gibco, Grand Island, NY) and were harvested and passaged at about 70–90% confluence.

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Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: A, after reaching confluence, hCMEC/D3 cells were treated with 50 nm and 100 nm hydrocortisone (HC) for 48 h and GR mRNA expression was assessed. Treatment was repeated every 24 h. A down-regulation of GR transcript to 0.81 ± 0.06-fold after 48 h of treatment with 50 nm HC, and to 0.63 ± 0.1-fold after 48 h of treatment with 100 nm HC was observed in hCMEC/D3 cells. B, confluent monolayers of hCMEC/D3 cells were grown in collagen-IV coated cell culture flasks in the presence of 100 nm HC as indicated. Cell lysates were analysed by Western blot for GR protein contents. After 48 h of HC treatment of hCMEC/D3, a down-regulation of GR protein to an estimated protein content of 83 ± 0.6% of that in untreated cells occurred (n = 3). C, immunocytochemistry visualizing the cellular localization of GR protein in hCMEC/D3 endothelial cells maintained in serum-reduced medium (0.25% FCS) ‘control’ as compared to cells maintained in differentiation medium (0.25% FCS, 110 nm HC) ‘HC’. GR stain (FITC = green), propidium iodide nuclear counterstain (red), and merged images (GR/PI) of GR immunofluorescence (green) and nuclei counterstained by propium iodide (red). After 48 h of HC treatment a nuclear concentration of GR (green) in hCMEC/D3 cells was observed, visualized by propidium iodide nuclear counterstaining (red). The nuclear concentration of GR could be confirmed for HC treated hCMEC/D3 cells by the use of computer imaging software to merge the individual images for FITC-GR and propidium iodide counterstain to assess similarityof staining pattern. The slides were analysed using a Zeiss Axioscop2 microscope. All pictures within each experiment were captured and manipulated identically with SpotAdvanced software and Adobe Photoshop. Bar in the lower panels indicates 20 μm for all panels.

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: Expressing, Cell Culture, Western Blot, Immunocytochemistry, Control, Staining, Immunofluorescence, Concentration Assay, Imaging, Software, Microscopy

Journal:

Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: Influence of the addition of HC on the electrical barrier properties (TER) of hCMEC/D3 monolayers. Growth medium (2.5% FCS) was changed after 5 days in culture to differentiation medium (0.25% FCS, ±additions) and analysis of the TER was performed after an additional 48 h in vitro, while treatment was repeated every 24 h. Incubation medium: control: with 0.25% (v/v) FCS, without hydrocortisone; HC: with 0.25% (v/v) FCS, 100 nm HC; Data are given as means ±s.d. (n = 6).

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: In Vitro, Incubation, Control

Journal:

Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: Permeability coefficients (Pe) for fluorescein and FITC-dextrans (4, 10, 70 and 150 kDa) of control and HC-treated hCMEC/D3 cells

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: Permeability, Control

Journal:

Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: Modulation of TJ gene expression in hCMEC/D3 cells by the GC HC

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: Gene Expression

Journal:

Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: A, GC treatment prevents a compromise of BBB function in response to TNFα administration for 8 h: TER drops from 74 ± 12 Ω cm2 in control cells to 42 ± 7 Ω cm2 in TNFα-treated cells, while treatment with HC increased TER values to 324 ± 33 Ω cm2. Simultaneous administration of TNFα with HC effectively prevented barrier breakdown; TER values amounted to 157 ± 27 Ω cm2. B, hCMEC/D3 cells were grown in collagen IV-coated cell culture flasks to confluence for 5 days and thereafter maintained in differentiation medium containing various additions for an additional 8 h: 0.25% FCS; 0.25% FCS + 100 nm HC; 0.25% FCS + 10 nm TNFα; 0.25% FCS + 100 nm HC + 10 nm TNFα. After 8 h, cell lysates were prepared. Cell lysates were analysed by Western blot for occludin, claudin-5, and VE-cadherin. Eight hours of TNFα-treatment decreased occludin protein to 75 ± 1% of untreated cells. Pretreatment with HC before TNFα administration increased occludin to 110 ± 1.5% of untreated cells and prevented occludin loss. When treated with HC alone, occludin was significantly increased to 139 ± 4% of control values. In contrast, 8 h of TNFα-treatment decreased claudin-5 protein to 57 ± 1% of untreated cells. Simultaneous administration of TNFα and HC yielded reduced levels of claudin-5 to 79 ± 2% of untreated cells. When treated with HC alone, claudin-5 increased to 110 ± 1.5% of control (Fig. 4B). Levels of the adherens junction protein VE-cadherin did not show significant changes under most treatment regimes. However, HC treatment concomitant with TNFα treatment led to an increase in detectable VE-cadherin protein (118 ± 5% of control) 8 h after treatment. C, densitometric evaluation of B.

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: Control, Cell Culture, Western Blot

Journal:

Article Title: Differential effects of hydrocortisone and TNF? on tight junction proteins in an in vitro model of the human blood-brain barrier

doi: 10.1113/jphysiol.2007.146852

Figure Lengend Snippet: Modulation of TJ gene expression in hCMEC/D3 cells by the inflammatory mediator TNFα and HC

Article Snippet: Isolation and culture of cerebral endothelial cells The immortalized human brain micro-vascular endothelial cell line hCMEC was generated as described previously ( Weksler et al. 2005 ).

Techniques: Gene Expression