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"There are several reasons ophthalmologists need to be acquainted with the pharmacologic profiles of PDE5 inhibitors and their potential side effects," the authors wrote "The frequency of erectile dysfunction, which is a form of peripheral vascular disease that impairs men's abilities to achieve and maintain an erection, increases dramatically with age and in the presence of cardiovascular risk factors. Therefore, many men who take PDE5 inhibitors to treat their erectile dysfunction will also be followed up by ophthalmologists for ocular disorders such as diabetic retinopathy, macular degeneration and ocular vascular disease."
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Cell purification. CD11b+ purification was performed with mouse CD11b MicroBeads (Miltenyi Biotec). The positive and negative fractions were sorted with the LS columns according to the manufacturer's instructions. CD11c+ cells were positively selected from splenocytes of tumor-bearing mice using mouse CD11c MicroBeads (Miltenyi Biotec). CD4+ or CD8+ T cells were negatively selected, using the CD4+ or CD8+ T cell isolation kit (Miltenyi Biotec), from CD11b-depleted splenocytes. CD14 depletion of human PBMCs was obtained by adding PE-labeled, anti–human CD14 antibody (BD Biosciences). The CD14-negative population was collected using a cell sorter (FACSVantage SE; BD Biosciences).
To determine whether the immunomodulatory effect of PDE5 inhibition affected T cell activation within the tumor microenvironment, we examined IL-2 production by TILs using a transgenic mouse in which expression of GFP is under an IL-2 promoter (BALB/c–IL-2p/GFP) (25). In this model, T cell stimulation activates the IL-2 promoter and results in expression of the reporter transgene, GFP, which is easily detectable by flow cytometry. C26GM-primed BALB/c–IL-2p/GFP splenocytes were adoptively transferred into tumor-bearing recipients that were either left untreated or treated with sildenafil for 9 d. Single cell suspensions of the tumor-infiltrating CD8+ T cells were analyzed by flow cytometry for GFP expression. Adoptively transferred, vaccine-primed T cells were activated in the tumor microenvironment only in the presence of PDE5 inhibition, whereas in its absence they produced no IL-2 and, hence, were bona fide anergic T cells (Fig. 4 D). To further prove that these effects were dependent on CD8+ T cells, mice were challenged with C26GM and were (a) left untreated, (b) given sildenafil, (c) given an anti-CD8+ depleting antibody, or (d) given both sildenafil and the CD8+ depleting antibody. Sildenafil treatment again demonstrated a statistically significant reduction in tumor outgrowth, an effect completely abrogated by CD8+ depletion (Fig. 4 E). These experiments demonstrate that PDE5 inhibition enhances the tumor-specific T cell response, increases intratumoral T cell infiltration and activation, and underscores the role of CD8+ T cells in sildenafil-mediated antitumor responses.
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