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Joao M. Furtado, Arpita S. Bharadwaj, Liam M. Ashander, Hoda A. Ilias, Yuzhen Pan, Justine R. Smith; Toxoplasma gondii Migration Across Simulated Human Retinal Endothelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5865.
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© ARVO (1962-2015); The Authors (2016-present)
Toxoplasmic retinochoroiditis is a common type of posterior uveitis. The causative parasite, Toxoplasma gondii, reaches the retina in tachyzoite form via the circulation; the route by which tachyzoites cross the retinal endothelium is unknown. Using clonal and natural isolates, we investigated the ability of free tachyzoites and infected monocytes to transit a stimulated human retinal endothelium.
Retinal endothelial transmigration assays were performed in transwells. Transwell membranes (0.3 cm2, 3 µm pores) were coated with collagen I, seeded with human retinal endothelial cells, and incubated for 4 days at 37oC to create a relatively impermeable endothelial monolayer. T. gondii tachyzoites were maintained by serial passage in confluent foreskin fibroblasts in DMEM + 1% heat-inactivated FBS. Live or heat-killed tachyzoites, suspended in EBM + endothelial growth factors + 2.5% FBS, were applied to upper chambers of transwells. Impact of number, time and strain on migration to lower chambers was compared, using RH as index strain. THP1 monocytes were infected with tachyzoites (MOI=10:1) for 4 hours, and separated from free parasites by density gradient centrifugation. 106 infected or uninfected THP1 cells were migrated in transwells for 18 hours. Rate of diffusion of 70 KDa dextran-Texas Red between chambers was measured to assess intactness of endothelial monolayers.
4 hours after application of 106 cloned strain RH tachyzoites to upper chambers, significantly more live (2.9±0.5%) than killed (0.2±0.06%) parasites were recovered from lower chambers (n=4) (p=0.002, representative of 3 experiments). The same result was observed for 3 natural isolates, including common haplogroup 1 strain (GT-1) and exotic haplogroup 6 strains (TgCatBR2, GPHT). Number of migrating RH parasites was directly proportional to input number, and increased over time, achieving 31.9% by 24 hours. Although killed parasites were recovered from lower chambers, no significant increase in diffusion of Texas Red-dextran suggested an intact endothelial monolayer. Infected THP1 cells migrated across endothelial monolayers, but in significantly smaller numbers than uninfected cells (n=3, p<0.001). Per plaque assay, parasite viability was ≥23% at time of assay, consistent with published reports.
Our findings suggest that T. gondii tachyzoites access retina both as free parasites or in leukocyte taxis, to initiate retinochoroiditis. This study is the first demonstration that T. gondii tachyzoites are capable of direct interaction with endothelium.
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