Journal: Vaccine

Article Title: Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response

doi: 10.1016/j.vaccine.2018.07.056

Figure Lengend Snippet: Trimeric HCMV gB elicited markedly higher titers of serum HCMV neutralizing activities for fibroblasts. HCMV neutralization titers (IC50), using the MRC-5 fibroblast cell line and HCMV strain AD169wt131, of heat-inactivated (A) and non-heat-inactivated (B) day 63 sera from mice immunized in days 0, 21, and 42 with 1, 5, or 25 μg of trimeric HCMV gB versus Sino gB. HCMV neutralization titers (IC50) of heat-inactivated (C) and non-heat-inactivated (D) day 63 sera from mice immunized with trimeric HCMV gB versus Sino gB using MRC-5 and HCMV strain AD169 (*p < 0.05 compared to the sera from mice immunized with Sino gB in equal amount. **p < 0.05 compared to the sera from mice immunized with 25 μg trimeric HCMV gB).

Article Snippet: HCMV strain AD169 WT131 was provided by Drs. Xiao Wang and Haruhiko Murata (Food and Drug Administration) and strain AD169 was purchased from ATCC.

Techniques: Neutralization

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Prolonged HCMV infection induced endothelial apoptosis. A, HCMV infection resulted in substantial cytopathicity in endothelial cells. HAECs were infected with VHL/E strain of HCMV for 9 days and changes in morphology and viability were observed under light microscope. B, Detection of endothelial apoptosis in HCMV-infected HAECs. VHL/E-infected HAECs (day 9 pi) were stained with anti-IE (HCMV immediate early antigen) antibody to indicate infected cells (red), TUNEL (green) for apoptosis, and DAPI (blue) for nuclei. Merged image shows colocalization. C, Time-dependent apoptosis of HCMV-infected HAECs. HAECs were infected with VHL/E for indicated time periods; cells were stained with anti-IE antibody (red), TUNEL (green), and DAPI (blue). Merged images show colocalization. D, Time-dependent death curves of VHL/E-infected HAECs. TUNEL-positive cells and total cells were counted in 5 fields per coverslip. Results were expressed as the average percentage of TUNEL-positive cells over total cells.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Light Microscopy, Staining, TUNEL Assay

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Activation of the mitochondrial apoptosis pathway in HCMV-infected endothelial cells. A, Increased cleavage of caspases-3 and 9 in HCMV-infected endothelial cells. Total protein from mock, AD169, VHL/E, and Towne strains of infected cells (day 9) was extracted and blotted with anticaspase-9 and anticaspase-3 antibodies. β-Actin was used as an internal sample control. B, Time-dependent increases of expression and cleavage of caspase-3 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibodies against total and cleaved caspase-3. C, Cleavage of caspase-3 in HCMV-infected cells. VHL/ E-Infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-cleaved caspase-3 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, Cleavage of caspase-9 in HCMV-infected cells. VHL/ E-infected HAECs (day 9) were stained anti-IE antibody-FITC (green), anticleaved caspase-9 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. E, Time-dependent increases in expression and cleavage of caspase-8 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibody against caspase-8. F, The involvement of caspases-3, -9, and -8 in HCMV-induced endothelial apoptosis. HAECs were infected with VHL/E for 8 days and treated with 10 μmol/L of caspase inhibitor for 3 days (fresh inhibitors were added daily). TUNEL-positive cells and total cells were counted in 4 fields per coverslip. Results were expressed as the mean±SEM percentage of TUNEL-positive cells over total cells. Using ANOVA, all 3 caspase inhibitors resulted in significantly less apoptosis (P<0.001); the inhibiting effect by caspase-3 or 9 inhibitors was greater than that by caspase-8 (P=0.024).

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Activation Assay, Infection, Expressing, Staining, TUNEL Assay

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Increased Bax and Bak levels in HCMV-infected endothelial cells. A, Increase of Bax and Bak expression in HCMV-infected endothelial cells. Total protein from uninfected (cultured for the same duration) and infected (by AD169, VHL/E, and Towne strains) HAECs (day 9) was extracted and blotted with anti-Bax and anti-Bak antibodies. B, Time-dependent increase of Bax and Bak expression in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected HAECs was extracted and blotted with anti-Bax and anti-Bak antibodies. C, Increased Bax level in HCMV-infected HAECs. VHL/E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-Bax antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, Increased Bak level in HCMV-infected HAEC. VHL/E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-Bak antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Expressing, Cell Culture, Staining

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: HCMV infection induced p53 phosphorylation in endothelial cells. A, Increase of p53 phosphorylation in HCMV-infected endothelial cells. Total protein from mock-infected and VHL/E-infected HAECs (day 9) was extracted and blotted with anti-phospho p53 (ser15), anti-phospho p53 (ser20), and anti-total p53 antibodies. B, Time-dependent increase in p53 phosphorylation in HCMV-infected cells. HAECs were infected with VHL/E as indicated, and total cell lysates were blotted with antibodies specific for p53 phosphorylated on ser15, ser20, total p53, or β-actin. C, Increased p53 phosphorylation in HCMV-infected HAECs. VHL/ E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-phosphor p53 (ser15) antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, VHL/E-infected HAECs (day 9) were stained with anti-IE (green), anti-phospho p53 (ser 20) (red) antibodies, and DAPI (blue). Merged image shows colocalization.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Staining

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: The involvement of p53 pathway in HCMV-induced endothelial apoptosis. HAECs were infected with VHL/E for 5 days before transfected with different amount of p53 siRNA. Twenty-four hours after transfection, total protein lysates from mock-infected and infected cells were prepared and blotted with antibodies against p53, Bax, Bak, caspase-3, and β-actin.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Transfection

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Involvement of the ATM/Chk2 pathway in HCMV-induced apoptosis. A, Time-dependent activation of the ATM pathway in HCMV-infected cells. HAECs were infected with VHL/E as indicated, and total cell lysates were blotted with antibodies specific for total ATM, phosphorylated Chk2, and β-actin. B, The involvement of the ATM pathway in HCMV-induced endothelial apoptosis. HAECs were treated with indicated concentrations of caffeine followed by infection with VHL/E. Five days after infection, cell lysates were subjected to immunoblotting with antibodies specific against phosphorylated Chk2, phospho p53 at ser15, and β-actin.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Activation Assay, Infection, Western Blot