Journal: Scientific Reports

Article Title: Platelet activation determines the severity of thrombocytopenia in dengue infection

doi: 10.1038/srep41697

Figure Lengend Snippet: Whole blood of healthy individual was incubated with or without DENV2 (3.4 MOI) and perfused over HUVEC monolayer under shear of 2.5 dyne/cm 2 in presence of mepacrine (label platelets). (A) Platelet thrombus area and (B) platelet-VWF string number and (C) string length were measured. Data are the mean ± SEM from ten view-fields in three independent experiments. The thrombus area, and string number and length were increased in presence of DENV2, *p < 0.05. The string length (calculated as median with highest and lowest value) was increased in presence of DENV2, **p < 0.005. (D) The 40X images show the platelet thrombus (marked by dotted area) and platelet- VWF string (marked by arrow) in absence (D1) and presence (D2) DENV2.

Article Snippet: The whole blood collected from healthy individuals was perfused over the petri plate confluent with human umbilical vein endothelial cells (HUVEC) from ATCC, USA as mentioned in our recent work .

Techniques: Incubation, Shear

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: A. CLL cells from 10 patients (n. 1-10; Table ) (i) or normal B cells isolated from the peripheral blood of 10 healthy donors (ii) were added onto TNFα pretreated confluent monolayer of HUVEC (TNF-HUVEC) in transwell plates (Transendothelial Migration (TEM) assays) or to the bottom compartment of separate transwells (Suspension cultures)(5×10 5 cells/well). Suspension cultures of CLL cells were carried out in bottom wells either with or without a TNF-HUVEC onto the upper filter. The percentage of apoptotic and viable cells in each experimental condition were determined after 12 hours by flow cytometry analysis of Annexin-V-PE/7AAD double staining. Each experimental condition was compared to basal levels in control suspension cultures without HUVEC (TEM assays: non-TM, non-transmigrated; TM, transmigrated). (iii) Transwell filters of TEM assays with CLL cells performed with TNF-α activated (+TNF) or untreated HUVEC (No TNF) were fixed and fluorescently stained with TUNEL (red; apoptotic nuclei), anti-CD31 (green; HUVEC junctions) and Hoechst (blue; total nuclei). Laser confocal microscopy images were taken from the upper and underside of filters (magnification 20x; ≥ 3 microscopy fields per filter; 1-2 filters per sample). Cartoons are from a representative TEM experiment with TNF-HUVEC. Total and TUNEL stained nuclei were counted (Image J; > 200 nuclei per sample and experimental condition) and the frequency of apoptotic cells in each side of the filters calculated (Right panel). Note that although the absolute number of nuclei was higher in the upper than the undersides the percentages of apoptotic CLL cells increased in the later one. Data are mean (±SD) from ten independent samples. B. i) CLL cells were cultured onto alive or paraformaldehyde fixed (fixed) TNF-α preactivated or untreated confluent HUVEC monolayers. The percentages of viable CLL cells (Annexin-V-neg 7AAD neg) were determined at the indicated time points by flow cytometry and compared to control suspension cultures without HUVEC. ii) CLL cells were cultured for 12 hours in suspension or onto alive HUVEC with normal culture medium or conditioned medium from 5 days CLL-HUVEC cocultures. iii) CLL cells were harvested at the indicated time-points from cocultures onto untreated or TNF-activated HUVEC and left in culture alone up to 12 hours. Two-tailed Student's t-test significance values: * P<0.05; ** P<0.01; *** P<0.001; n.s. non-significant.

Article Snippet: Briefly confluent monolayers of HUVECs (PromoCell, Spain) were grown onto the filters of transwell plates (96xwell transwell plates, 5 μm pore size, Corning) and stimulated or not with TNF-α (10 ng/mL; PeProtech, Europe) for 4 hours.

Techniques: Isolation, Migration, Flow Cytometry, Double Staining, Staining, TUNEL Assay, Confocal Microscopy, Microscopy, Cell Culture, Two Tailed Test

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: CLL cells of the 12 patients used in EphA2Fc assays having (LApos) or not (LAneg) lymphadenopathy (Figure ), were nucleofected with ephrinA4 specific or negative control siRNA duplexes or left untreated. A. EphrinA4 silencing was determined by flow cytometry 48 hours after nucleofection. B. Untreated or nucleofected cells (siRNA neg or ephrinA4) were left transmigrating for 12 hours through TNF-HUVEC. i) TEM rate of samples measured by flow cytometry. ii) Transwell filters from TEM assays were stained with anti CD31 mAb (green, HUVEC junctions) and Hoechst (blue, nuclei) and examined by confocal microscopy (40x oil immersion; Leica TCS-SP2). CD31 junctions were not altered in the HUVEC monolayer of ephrinA4 knock-down samples. iii) Absolute counts of CLL cells adhered to TNF-HUVEC were measured by image analysis (ImageJ; > 200 nuclei counted per filter). C. Analysis of CLL cells viability in TEM assays after ephrinA4 knock-down. i) Absolute levels of viable CLL cells in suspension cultures or in TEM assays (non-TM and TM cells) were compared between ephrinA4-specific or neg control siRNA nucleofected to control untreated CLL cells. ii) Relative viability of TM cells as percentage of basal levels in suspension cultures or of non-TM cells. Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: Briefly confluent monolayers of HUVECs (PromoCell, Spain) were grown onto the filters of transwell plates (96xwell transwell plates, 5 μm pore size, Corning) and stimulated or not with TNF-α (10 ng/mL; PeProtech, Europe) for 4 hours.

Techniques: Negative Control, Flow Cytometry, Staining, Confocal Microscopy, Two Tailed Test

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: A. Soluble ephrinA4 levels in the serum of CLL patients were determined by indirect ELISA (ng/mL) and possible associations with clinical parameters examined. B. CLL cells from 12 patients having (LApos) or not (LAneg) lymphadenopathy, used in the EphA2Fc or siRNA assays (Figures and ), were left transmigrating 12 hours through TNF-α preactivated HUVEC monolayers preincubated (30 min) with saturating amounts of recombinant human ephrinA4 (HUVEC+ephrinA4Fc), purified human Fc (HUVEC + hFc) or untreated before addition of CLL cells. i) Absolute number of transmigrated cells (TM) was counted by flow cytometry and expressed as percentage of total input cells (TEM rate). ii) Absolute levels of viable CLL cells in suspension cultures or in TEM assays (non-TM and TM cells) in the ephrinA4Fc or hFc treated TEM assays were compared to untreated conditions. iii) Relative viability of TM cells normalized to basal levels (suspension cultures) or to the corresponding non-TM cells in the ephrinA4Fc or hFc treatments compared to untreated TEM assays. C. i) Transwell filters were stained with anti VE-Cadherin (green, HUVEC junctions) and Hoechst (blue, nuclei) and examined by confocal microscopy. Adhered CLL cells were located at VE-Cadherin interendothelial junctions or separated from them. ii-iii) Caveolin-1 and ICAM-1 immunofluorescence staining of TEM assays done in chamber slides 2 hours after coculture with LApos CLL cells. ii) CLL cells inside of HUVEC were surrounded by caveolin-1 endothelial caveola (asterisks, > 1μm from VE-Cadherin junction) and ICAM-1. Insets show Z- views of caveolin and ICAM-1 staining for a a transcellular (TC) and a paracellular (PC) TEM CLL nuclei. iii) Measurement of CLL cells located within caveolin-1, at VE-Cadherin junctions or unclassified in ephrinA4Fc treated compared to untreated TNF-HUVEC after 2 hours coculture (at least 200 nuclei were counted per condition). Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: Briefly confluent monolayers of HUVECs (PromoCell, Spain) were grown onto the filters of transwell plates (96xwell transwell plates, 5 μm pore size, Corning) and stimulated or not with TNF-α (10 ng/mL; PeProtech, Europe) for 4 hours.

Techniques: Indirect ELISA, Recombinant, Purification, Flow Cytometry, Staining, Confocal Microscopy, Immunofluorescence, Two Tailed Test

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Characterization of cultured brain microvascular endothelial cells and pericytes. Confluent monolayers of brain microvascular endothelial cells (A) and pericytes (B) were observed under a light microscope. Scale bar = 50 μm. The dissociated brain microvascular endothelial cells (C) and pericytes (D) were subjected to immunofluorescence staining with isotype IgG and IgG against CD31 or α-SMA. Characterization of antibody-labeled cells was performed on a BD FACScalibur flow cytometer.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Cell Culture, Light Microscopy, Immunofluorescence, Staining, Labeling, Flow Cytometry

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Effects of JEV infection on TEER and transendothelial permeability. (A) Confluent monolayers of brain microvascular endothelial cells were mock infected or infected with JEV (MOI, 20) over time. The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured at the indicated times. The coculture of brain microvascular endothelial cells and pericytes seeded together (B) and separated by Transwell filter insert (C) was mock infected or infected with JEV (MOI, 20) over time. The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured at the indicated times. (D) Pericytes were mock infected or infected with JEV (MOI, 20) over time. The supernatants were collected at the indicated times after infection and mixed with an equal volume of fresh medium. The manipulated media were added to brain microvascular endothelial cells for 24 h. The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured. (E) Pericytes were mock infected or infected with JEV (MOI, 20) for 48 h. The supernatants were collected and mixed with an equal volume of fresh medium. The manipulated media were added to brain microvascular endothelial cells over time. The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured at the indicated times. The values of TEER were given in ohm · cm2 and the relative levels of dextran-FITC were expressed as arbitrary units. *, P < 0.05, and **, P < 0.01, versus each mock control; n = 4.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Infection, Permeability, Control

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Effects on tight junction protein expression. Pericytes were mock infected (Mock CM) or infected with JEV (MOI, 20; JEV CM) for 48 h. The supernatants were collected and mixed with an equal volume of fresh medium. The manipulated media were added to brain microvascular endothelial cells over time. (A) Total cellular proteins were isolated and subjected to Western blotting with antibodies against ZO-1, ZO-2, claudin-1, claudin-5, occludin, and β-tubulin. One representative blot of four independent experiments is shown. The content in Mock CM at each time point was defined as 100%. (B) Total RNAs were isolated and subjected to quantitative real-time RT-PCR for the measurement of ZO-1 and β-actin. Relative gene expression was determined by the ΔΔCT method, and the level in Mock CM at 4 h was defined as 1. n = 4. (C) The manipulated media (Mock CM and JEV CM) were added to brain microvascular endothelial cells for 24 h. The cells were subjected to immunofluorescence staining with antibodies against occludin (FITC) or ZO-1 (rhodamine) and counterstained with Hoechst 33342. The manipulated media (Mock CM and JEV CM) were added to brain microvascular endothelial cells in the absence or presence of MG132 (5 μM), lactacystin (50 μM), GM6001 (10 μM), or Z-DEVD (20 μM) for 24 h. Untreated cells were used as the control. (D) Total cellular proteins were isolated and subjected to Western blotting with antibodies against ZO-1 and β-tubulin. One representative blot of four independent experiments is shown. The content in control was defined as 100%. (E) The TEER (left graph) and transendothelial permeability to dextran-FITC (right graph) were measured. n = 4. Brain microvascular endothelial cells were mock infected or infected with JEV (MOI, 20) or exposed to the manipulated media (Mock CM and JEV CM) for 24 h. (F) Cellular proteins were isolated and subjected to fluorogenic assay for the determinations of trypsin-like and chemotrypsin-like proteasome activities. n = 4. (G) Cellular proteins were isolated and subjected to fluorogenic assay for the determination of caspase-3 activity. n = 4. (H) The supernatants were collected and subjected to zymography for determination of MMP-2 and MMP-9 activities. One representative blot of four independent experiments is shown. **, P < 0.01 versus Mock CM; ##, P < 0.01 versus JEV CM.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Expressing, Infection, Isolation, Western Blot, Quantitative RT-PCR, Gene Expression, Immunofluorescence, Staining, Control, Permeability, Activity Assay, Zymography

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Role of IL-6. Pericytes were mock infected (Mock CM) or infected with JEV (MOI, 20; JEV CM) for 48 h. The supernatants were collected and mixed with an equal volume of fresh medium. Brain microvascular endothelial cells were exposed to the manipulated media (Mock CM and JEV CM) or treated with IL-6 (20 ng/ml) in the absence or presence of AG490 (50 μM) for 24 h. One set of manipulated medium (JEV CM) was modified by neutralization with IL-6 neutralizing antibody (10 μg/ml) for 30 min before being subjected to exposure. Untreated cells were used as the control. (A) Cellular proteins were isolated and subjected to fluorogenic assay for the determinations of trypsin-like (left graph) and chemotrypsin-like (right graph) proteasome activity. n = 4. (B) Total cellular proteins were isolated and subjected to Western blotting with antibodies against ZO-1 and β-tubulin. One representative blot of four independent experiments is shown. The content in control was defined as 100%. (C) The TEER (left graph) and transendothelial permeability to dextran-FITC (right graph) were measured. n = 4. **, P < 0.01 versus medium control; ##, P < 0.01 versus JEV CM; &&, P < 0.01 versus IL-6 control.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Infection, Modification, Neutralization, Control, Isolation, Activity Assay, Western Blot, Permeability

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Effects of JEV infection on IL-6 expression. Pericytes were mock infected or infected with JEV (MOIs, 1, 5, 10, 20, and 40) for 48 h. (A) Supernatants isolated from infected cells were subjected to ELISA for the measurement of IL-6. n = 4. (B) The supernatants were collected and mixed with an equal volume of fresh medium. The manipulated media were added to brain microvascular endothelial cells for 24 h. The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured. n = 4. **, P < 0.01 versus mock.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Infection, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Permeability

Journal: Journal of Virology

Article Title: Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

doi: 10.1128/JVI.02738-13

Figure Lengend Snippet: Role of Ubr 1. Pericytes were mock infected (Mock CM) or infected with JEV (MOI, 20; JEV CM) for 48 h. The supernatants were collected and mixed with an equal volume of fresh medium. (A) The manipulated media were added to brain microvascular endothelial cells over time. Total cellular proteins were isolated and subjected to Western blotting with antibodies against Ubr 1 and β-tubulin. One representative blot of four independent experiments is shown. (B) Brain microvascular endothelial cells were exposed to the manipulated media (Mock CM and JEV CM) in the absence or presence of AG490 (50 μM) for 24 h. One set of manipulated medium (JEV CM) was modified by neutralization with IL-6 neutralizing antibody (10 μg/ml) for 30 min before being subjected to exposure. Total cellular proteins were isolated and subjected to Western blotting with antibodies against Ubr 1 and β-tubulin. One representative blot of four independent experiments is shown. The content in Mock CM was defined as 100%. Brain microvascular endothelial cells were transfected with control siRNA (1 nM) (mock transfection) or Ubr 1 siRNA (1 nM) for 4 h. The resultant cells were treated with IL-6 (20 ng/ml) or exposed to JEV CM for 24 h. Untreated cells were used as a control. (C) Total cellular proteins were isolated and subjected to Western blotting with antibodies against Ubr 1, ZO-1, and β-tubulin. One representative blot of four independent experiments is shown. The content in control was defined as 100%. (D) Cellular proteins were isolated and subjected to fluorogenic assay for determination of trypsin-like (upper graph) and chemotrypsin-like (lower graph) proteasome activities. n = 4. (E) The TEER (upper graph) and transendothelial permeability to dextran-FITC (lower graph) were measured. n = 4. **, P < 0.01 versus medium control; ##, P < 0.01 versus IL-6 control; &&, P < 0.01 versus JEV CM control.

Article Snippet: Confluent monolayers of brain microvascular endothelial cells ( ) and pericytes ( ) obtained from adult Sprague-Dawley rats were examined under a light microscope.

Techniques: Infection, Isolation, Western Blot, Modification, Neutralization, Transfection, Control, Permeability