Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-3

Otein expression as compared to control siRNA in both mock and virus infected cells. (B) IL-8 production in virus infected keratocytes was reduced to mock infected levels by p38 MAPK-specific siRNA but not by control siRNA (*p = 0.0009).<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-0

against phospho- and total p38 MAPK reveals phosphorylation in HAdV-19 infected cells, reduced in the presence of the Src inhibitor PP2 (10 μM). Densitometry values for the phosphorylated protein (normalized to the corresponding total protein) are shown above each lane. (B) An p38 MAPK assay performed at 15 and 30 min after infection shows increased phosphorylation of the ATF-2 substrate signifying p38 MAPK activity upon HAdV-19 infection of keratocytes. (C) Densitometric quantification from three experiments of the phosphorylated ATF-2 band in the p38 MAPK assay revealed increased activity in virus infected keratocytes at both 15 and 30 min post-infection (p = 0.0012 and p = 0.0025, respectively). (D & E) Western blot analysis using phospho- and total antibodies against HSP27 and ATF-2 respectively, reveals increases in phosphorylation in HAdV-19 infected cells that was absent in the presence of PP2.<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

UV-inactivated adenovirus induces leukocyte infiltration and cytokine expression.

<p>(A) Representative dot plots of single cell suspensions prepared from corneas at 4 dpi stained with Gr1 and F4/80 and gated on CD45^high labeled cells. Corneas were infected with virus free buffer (M), or intact (V), UV-inactivated (UV), and heat-inactivated (H) HAdV-37. (B) Quantification of average numbers of Gr1 and F4/80 stained corneal cells in intact (V), UV-inactivated (UV), or heat-inactivated (H) virus injected corneas at 4 dpi (n = 6 mice/group). Data is derived from three separate experiments, and error bars represent SD. (C) Myeloperoxidase (MPO) levels assessed 24 hours post injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) are shown (n = 9 mice/group). Data represents mean of three independent experiments ± SD. (D–F) Cytokine expression in corneas after injection with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) as measured at 16 hpi by ELISA for CXCL1 (D), CCL2 (E), and IL-6 (F) protein (n = 9 mice/group). Data represents mean of three independent experiments ± SD. * p<.05, ANOVA.</p

Empty adenoviral capsid is sufficient to induce keratitis.

<p>(A) Silver stained polyacrylamide gel of proteins from intact HAdV-37 (V) or empty capsid (EC). First lane (M) shows protein standards. Arrows on the right point to capsid proteins missing from the empty capsid; capsid proteins V and VII are marked by the second and fourth arrows from the top, respectively. (B) Mouse cornea injected with Cy3 dye-labeled empty capsid (EC). Intracellular virus position was visualized with confocal microscopy at 90 min pi (n = 3 corneas). Red: Cy3-labeled empty capsid. Green: intracellular actin (phalloidin stain). Blue: nuclei (TO-PRO3 stain). Scale bar 20 µM. (C) Clinical appearance and (D) histopathology of mouse corneas at 4 dpi. Corneas were injected with virus free buffer (M), intact virus (V), or empty capsid (EC) (n = 5 mice/group).</p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-4

against phospho- and total p38 MAPK reveals phosphorylation in HAdV-19 infected cells, reduced in the presence of the Src inhibitor PP2 (10 μM). Densitometry values for the phosphorylated protein (normalized to the corresponding total protein) are shown above each lane. (B) An p38 MAPK assay performed at 15 and 30 min after infection shows increased phosphorylation of the ATF-2 substrate signifying p38 MAPK activity upon HAdV-19 infection of keratocytes. (C) Densitometric quantification from three experiments of the phosphorylated ATF-2 band in the p38 MAPK assay revealed increased activity in virus infected keratocytes at both 15 and 30 min post-infection (p = 0.0012 and p = 0.0025, respectively). (D & E) Western blot analysis using phospho- and total antibodies against HSP27 and ATF-2 respectively, reveals increases in phosphorylation in HAdV-19 infected cells that was absent in the presence of PP2.<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Adenoviral genomic DNA is not sufficient to induce keratitis in mice.

<p>(A) Confocal microscopy of mouse corneal stroma at 1 day after mock treatment with transfection reagent alone (M), HAdV-5 vector expressing eGFP (V), or plasmid vector EGFP-C1 (90 or 500 ng DNA). Photographs are representative of three corneas in each group. Scale bar, 200 µM. (B) Flow cytometric analysis of corneas at 1 day after injection with transfection reagent alone (M), plasmid vector (90 or 500 ng DNA) and transfection reagent, or HAdV-5 (V) vector expressing eGFP. Numbers in histograms denote percentage of total cells expressing eGFP. (C and D) C57BL/6J mouse corneas were injected with virus free buffer (M), HAdV-37 (V) or 90 ng and 500 ng of HAdV-37 genomic DNA with transfection reagent and observed up to 4 dpi. Representative photographs (C) and histopathology sections (D) of corneas at 4 dpi are shown (n = 5 mice/group).</p

Empty viral capsid induces chemokine expression and infiltration of leukocytes into the cornea.

<p>(A) Infiltrating leukocytes were quantified using flow cytometry in corneas 4 days after injection with virus free buffer (M), intact HAdV-37 (V), or empty viral capsid (EC) (n = 6 corneas/group). Data represents the mean of three separate experiments, and error bars denote SD. (B) Myeloperoxidase (MPO) levels were quantified in mouse corneas 2 days after injection with virus free buffer (M), intact virus (V), or empty capsid (EC) (n = 6 corneas/group). Data represents the mean of two separate experiments, and error bars denote SD. (C–E) Cytokine protein levels as measured by ELISA in corneas 16 hours after injection with virus free buffer (M), intact virus (V), or empty capsid (EC). CXCL1 (C), CCL2 (D), and IL-6 (E) protein levels are shown (n = 9 corneas/group). Data shown represents the mean of three independent experiments, and error bars represent SD. * p<.05, ANOVA.</p

Viral gene expression is not essential for adenovirus keratitis.

<p>(A) Real-time PCR for the relative expression of viral transcript E1A10S at 4 hpi in mock (M), intact (V), UV-inactivated (UV), or heat-inactivated (H) HAdV-37 infected A549 cells. Data represents mean of three separate experiments ± SD. (B) Mouse corneas injected with Cy3-labeled intact (V), UV- inactivated (UV), or heat-inactivated (H) virus were analyzed by confocal microscopy at 90 min pi (n = 5 corneas/group). Red: Cy3-labeled virus. Green: intracellular actin (phalloidin stain). Blue: nuclei (TO-PRO3 stain). Scale bar 20 µM. (C) Representative photographs and (D) hematoxylin and eosin stained histopathological sections of mice corneas at 4 dpi, infected with virus free buffer (M), intact virus (V), UV-inactivated virus (UV), or heat-inactivated virus (H) (n = 5 mice/group).</p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-1

On, and lysates prepared 30 min after infection. (A) NFκB-p65 and IκB phosphorylation increased with virus compared to mock infection, and were reduced in cells pre-treated with SB203580 in dose-dependent fashion. Densitometry values for the phosphorylated protein (normalized to the corresponding actin levels) are shown above each lane. (B) Immunoprecipitation assay reveals association of p38 MAPK with NFκB-p65 in virus infected (V) but not in mock (M) infected cells. This association was blocked by the p38 MAPK inhibitor SB203580 (SB) but not by the JNK inhibitor SP600125 (SP). Isotype control did not immunoprecipitate any NFκB-p65. (C) NFκB-p65 activation in HAdV-19 infection analyzed by confocal microscopy. The left column shows DAPI staining for nuclei (blue), the middle column for p65 (yellow), and the right column a merging of the left 2 rows. (c) Mock infected keratocytes show mostly cytoplasmic localization of NFκB-p65. (f) HAdV-19 infected keratocytes at 20 min post-infection show nuclear localization of NFκB-p65. (i) Nuclear translocation of NFκB-p65 was reduced in the presence of 10 μM, and (l) completely blocked with 20 μM SB203580. Bottom row (m, n, o), represent isotype control. (D) Electromobility shift assay showing NFκB-p65 binding to IL-8 promoter in HAdV-19 infected keratocytes. Extracts from HAdV-19 infected cell nuclei show more binding to NFκB-specific IL-8 probe (lane 2) as compared to nuclear protein from mock infected cells (lane 6) or when pretreated with SB203580 (lane 10). Binding specificity of the probe is shown with 100 molar excess of unlabelled probe (lanes 3 and 7). SB203580 (SB) blocked NFκB binding in virus infected nuclear extracts (lane 10). Dose dependent supershifts using increasing amounts of NFκB-p65 antibody are shown in virus infected nuclear extracts (lanes 4 and 5). No shifts were observed in nuclear extracts from mock infected cells (lanes 8 and 9) or SB203580 treated cells (lane 11).<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Adenoviral genomic DNA induces differential expression of cytokines but does not cause infiltration of leukocytes into the cornea.

<p>(A) Flow cytometric analysis of Gr1 and F4/80 positive cells in mouse corneas at 4 days after injection with virus free buffer (M), virus free buffer and transfection reagent (M+T), intact HAdV-37 (V), intact HAdV-37 and transfection reagent (V+T), and 90 ng or 500 ng of HAdV-37 genomic DNA with transfection reagent (n = 6 corneas/group). Data shown represents the mean of three independent experiments, and error bars represent SD. (B–E) Protein levels of cytokines IL-6 (B), CXCL1 (C), CXCL2 (D) and CCL2 (E) in mouse corneas at 16 hpi. Corneas were injected with virus free buffer (M), virus free buffer and transfection reagent (M+T), intact HAdV-37 (V), intact HAdV-37 and transfection reagent (V+T), and 90 ng or 500 ng of HAdV-37 genomic DNA with transfection reagent (n = 9 corneas/group). Data shown represents the mean of three separate experiments, and error bars denote SD. * p<.05, ANOVA.</p