11 research outputs found
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<sup>high</sup> 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
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
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
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
HAdV-37 enters human corneal fibroblasts through caveolin-1 containing, membrane associated vesicles.
<p>Human corneal fibroblasts were fixed in 2 % paraformaldehyde and the fixed, dehydrated cell pellet embedded and sectioned at 70-90 nm. Specimens were stained with uranyl acetate and Sato’s lead stain, and viewed by transmission electron microscopy. <b>A</b>. Photo-electron micrograph of cell membrane demonstrates multiple flask shaped vesicles resembling caveolae (1), and the formation of a cavesome-like structures (2). <b>B</b>-<b>D</b>. After 30 min of absorption at 4°C, and 30 min incubation at 37°C, virus can be seen within similar structures (3), and in some cases within what appear to be early or late fusions of caveolae to form caveosomes (4). Immunoelectron microscopy for caveolin-1 (<b>E</b>, <b>F</b>) was performed on cells infected as above, but treated with 2.3 M sucrose in PBS for 15 min, frozen, and sectioned at -120° C at 50-80 nm thickness. After immunostaining, protein-A nanogold was visualized by electron microscopy. (<b>E</b>) Uninfected cells stained for caveolin-1 demonstrate binding of 10 nm gold particles to flask-shaped vesicles. (<b>F</b>) After virus infection, virus (arrows) can be seen within vesicles also associated with gold particles. Scale bar = 500 nm. </p
HAdV-D37 uses primarily caveolin-1 to enter corneal cells.
<p><b>A</b>. Endosomal purification was performed on postnuclear extracts from corneal cells at 1 hr post infection in 62%, 35% and 25% sucrose solution. Fractions were immunoblotted for caveolin-1 (Cav 1), pSrc, RAB5, and LAMP1. <b>B</b>. Src kinase assay was performed on endosomal fraction #’s 5-11. In this assay, fluorescence is inversely proportional to kinase activity. As shown, fractions 10 and 11, which previously showed increased caveolin-1, also demonstrated greater Src kinase activity. <b>C</b>. Fractions that had maximum expression of LAMP1 (#6) and caveolin-1 (#10) were then subjected to real time PCR. For both fractions, the quantity of viral DNA was reduced by MβCD pretreatment (*p<.05). Cholesterol replenishment resulted in restoration of E1A expression (*p<.05). There was no difference in E1A expression between virus infected cells and those pretreated with MβCD but replenished with cholesterol and then virus infected. The data presented is representative of four experiments each run in duplicate. </p
Caveolin-1 and cholesterol dependent viral entry.
<p>siRNA and scrambled (sc)RNA were generated and transfected into human corneal cells. <b>A</b>. Real-time RT-PCR for caveolin-1 mRNA confirmed successful reduction of caveolin-1 message (~95%) as compared to scRNA transfected cells (*p=.0002). <b>B</b>. Western blot showed reduced expression of caveolin-1 in caveolin-1 specific siRNA treated cells (right lane) as compared to untransfected and uninfected cells (left lane) or scRNA transfected, virus infected cells (middle lane) <b>C</b>. IL-8 mRNA expression after HAdV-D37 infection was reduced to almost 50% by caveolin-1 siRNA transfection, compared to scRNA (*p=.0001). </p
HAdV-37 uses caveolin-1 to enter human corneal cells.
<p><b>A</b>. Human corneal and A549 cells grown on slide chambers were infected with Cy3-labeled HAdV-D37 (red). Cells were then stained with caveolin-1 or LAMP1 antibodies as indicated followed by alexa-fluor488 (green) secondary antibodies. Original magnification: 63X. Insets represent similarly magnified squares from each photomicrograph. <b>B</b>. Three random frames from different experiments were chosen in masked fashion from each experiment (n=3) to quantify co-localization using Amira 5.2.2. In A549 cells, HAdV-C2 co-localized predominantly with caveolin-1, while HAdV-D37 co-localized with LAMP1 (*p=.02 and *p=.0025, respectively). In corneal cells (CC), Cy3-labeled HAdV-D37 co-localized predominantly with caveolin-1 (*p=.0068), and HAdV-C2 was not seen. Students <i>t</i> test was used for all comparisons. <b>C</b>. Human corneal cells were grown in chamber slides, Cy3-labelled HAdV-D37 and 488-Cholera Toxin B (CTXB) were added to the cells on ice and then warmed to 37°C, and incubated for 30, 60, and 90 min, prior confocal microscopy. HAdV-D37 (red) co-localized with CTXB (green) at all time points. At 30 min after warming, HAdV-D37 and CTXB co-localization was observed at the cell membrane, at 60 min in the cytoplasm, and at 90 min at the perinuclear region. </p