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Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment.

Millar JC, Pang IH, Wang WH, Wang Y, Clark AF - Mol. Vis. (2008)

Bottom Line: After day 7, GFP expression declined significantly.Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010).These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

View Article: PubMed Central - PubMed

Affiliation: Alcon Research, Ltd., Fort Worth, TX 76134, USA.

ABSTRACT

Purpose: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression.

Methods: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy.

Results: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4-7 with a fluorescence grade of 3.0 +/-0.0 (mean +/-SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/-0.7 (with anti-CD40L) and 1.2 +/-0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium.

Conclusions: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

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Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x107 pfu). The animals were euthanized at either 5 or 43 days after vector injection with or without anti-CD40L antibody treatment. Green fluorescence=GFP, blue fluorescence=DAPI staining of cell nuclei. The occasional yellowish-green fluorescence represents auto-fluorescence of certain tissues, such as those in the outer nuclear layer and retinal pigmented epithelium, which was also observable in non-injected eyes (Data not shown). Abbreviations: C=cornea; CB=ciliary body; I=iris; L=lens; R=retina; TM=trabecular meshwork.
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f5: Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x107 pfu). The animals were euthanized at either 5 or 43 days after vector injection with or without anti-CD40L antibody treatment. Green fluorescence=GFP, blue fluorescence=DAPI staining of cell nuclei. The occasional yellowish-green fluorescence represents auto-fluorescence of certain tissues, such as those in the outer nuclear layer and retinal pigmented epithelium, which was also observable in non-injected eyes (Data not shown). Abbreviations: C=cornea; CB=ciliary body; I=iris; L=lens; R=retina; TM=trabecular meshwork.

Mentions: Cross-sections of mouse eyes obtained from animals euthanized at days 5 and 43 following intraocular Ad5.CMV-GFP injection were examined by fluorescence microscopy, and GFP expression was typically localized to the cornea, TM, iris, ciliary body, and lens. No GFP fluorescence was detected in the retina regardless of injection route. Furthermore, the observed ocular distribution and intensity of GFP expression correlated very well with in vivo fluorescence grades of the different groups. For example, not much fluorescence in the anterior segment was observed at 5 and 43 days after IC injection of 1x107 pfu Ad5.CMV-GFP without anti-CD40L antibody treatment (Figure 5), while treatment with the anti-CD40L antibody enhanced the expression of GFP of the same injection regimen. Under this condition, a fraction of cells in the TM and corneal endothelium expressed GFP (Figure 5). IC injection at a higher titer of the vector (1x108 pfu), even without anti-CD40L antibody treatment, produced more noticeable GFP expression. At day 5, fluorescence was apparent in the corneal endothelium, TM and the ciliary body. However, the GFP fluorescence diminished and was barely visible in eyes 43 days after injection (Figure 6). Again, treatment with anti-CD40L antibody augmented the expression of GFP. In eyes 5 and 43 days after IC injection of 1x 108 pfu Ad5.CMV-GFP, the TM and corneal endothelium, and sporadically the lens epithelium and iris were positive with GFP fluorescence (Figure 6).


Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment.

Millar JC, Pang IH, Wang WH, Wang Y, Clark AF - Mol. Vis. (2008)

Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x107 pfu). The animals were euthanized at either 5 or 43 days after vector injection with or without anti-CD40L antibody treatment. Green fluorescence=GFP, blue fluorescence=DAPI staining of cell nuclei. The occasional yellowish-green fluorescence represents auto-fluorescence of certain tissues, such as those in the outer nuclear layer and retinal pigmented epithelium, which was also observable in non-injected eyes (Data not shown). Abbreviations: C=cornea; CB=ciliary body; I=iris; L=lens; R=retina; TM=trabecular meshwork.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267727&req=5

f5: Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x107 pfu). The animals were euthanized at either 5 or 43 days after vector injection with or without anti-CD40L antibody treatment. Green fluorescence=GFP, blue fluorescence=DAPI staining of cell nuclei. The occasional yellowish-green fluorescence represents auto-fluorescence of certain tissues, such as those in the outer nuclear layer and retinal pigmented epithelium, which was also observable in non-injected eyes (Data not shown). Abbreviations: C=cornea; CB=ciliary body; I=iris; L=lens; R=retina; TM=trabecular meshwork.
Mentions: Cross-sections of mouse eyes obtained from animals euthanized at days 5 and 43 following intraocular Ad5.CMV-GFP injection were examined by fluorescence microscopy, and GFP expression was typically localized to the cornea, TM, iris, ciliary body, and lens. No GFP fluorescence was detected in the retina regardless of injection route. Furthermore, the observed ocular distribution and intensity of GFP expression correlated very well with in vivo fluorescence grades of the different groups. For example, not much fluorescence in the anterior segment was observed at 5 and 43 days after IC injection of 1x107 pfu Ad5.CMV-GFP without anti-CD40L antibody treatment (Figure 5), while treatment with the anti-CD40L antibody enhanced the expression of GFP of the same injection regimen. Under this condition, a fraction of cells in the TM and corneal endothelium expressed GFP (Figure 5). IC injection at a higher titer of the vector (1x108 pfu), even without anti-CD40L antibody treatment, produced more noticeable GFP expression. At day 5, fluorescence was apparent in the corneal endothelium, TM and the ciliary body. However, the GFP fluorescence diminished and was barely visible in eyes 43 days after injection (Figure 6). Again, treatment with anti-CD40L antibody augmented the expression of GFP. In eyes 5 and 43 days after IC injection of 1x 108 pfu Ad5.CMV-GFP, the TM and corneal endothelium, and sporadically the lens epithelium and iris were positive with GFP fluorescence (Figure 6).

Bottom Line: After day 7, GFP expression declined significantly.Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010).These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

View Article: PubMed Central - PubMed

Affiliation: Alcon Research, Ltd., Fort Worth, TX 76134, USA.

ABSTRACT

Purpose: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression.

Methods: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy.

Results: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4-7 with a fluorescence grade of 3.0 +/-0.0 (mean +/-SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/-0.7 (with anti-CD40L) and 1.2 +/-0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium.

Conclusions: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

Show MeSH
Related in: MedlinePlus