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D. O. Zamora, S. J. Binek, N. S. Carter, J. T. Rosenbaum, J. R. Smith; Toxoplasma gondii Tachyzoites Preferentially Bind Human Retinal Vascular Endothelial Cells in Comparison to Dermal Vascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2672.
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© ARVO (1962-2015); The Authors (2016-present)
Toxoplasma gondii infection of the retina is a leading cause of posterior uveitis. Previously we reported that human retinal vascular endothelial cells (HRECs) were relatively more supportive of infection with T. gondii tachyzoites than other EC subtypes. In this study we hypothesized that enhanced susceptibility to infection was the result of increased binding affinity of tachyzoites for HRECs.
Commerically procured HREC and human dermal EC (HDEC) were grown to confluence in 96-well plates. Yellow fluorescent protein (YFP)-expressing RH strain T. gondii tachyzoites (gift of Dr. Boris Striepen, University of Georgia), maintained by serial passage in cultured fibroblasts, were suspended in phenol-free MCDB-131 medium with 5% heat-inactivated FBS and added to quadruplicate wells of HREC or HDEC at MOI of 50:1. Control wells were treated with medium alone. After a 1 hr binding incubation at 37°C and 5% CO2, wells were washed, and relative fluorescence intensity was measured by microplate reader (Ex: 480/Em: 530). In addition, numbers of parasites bound to HREC and HDEC monolayers were counted blindly using digital images obtained under epifluorescence microscopy. Cultures were subsequently incubated for an additional 71 hrs, at which time relative fluorescence intensity provided an index of tachyzoite proliferation in each cell type.
After subtraction of background levels, relative fluorescence intensity of tachyzoite-HREC cultures was 45% higher than tachyzoite-HDEC cultures after 1 hr incubation (p = 0.02, paired t-test, 3 separate experiments), indicative of preferential binding of tachyzoites to the HREC in comparison to HDEC. Manual counting also suggested more tachyzoites were bound to a fixed area of HREC versus HDEC monolayer (730 and 659 parasites/mm2, respectively, p = 0.068, t-test, single experiment). Consistent with increased binding and our previous observations, lysis of HREC was accelerated in comparison to HDEC, and at 72 hours, relative fluorescence intensity remained significantly higher for tachyzoite-HREC cultures (p ≤ 0.002, t-test, 3 experiments), indicative of higher numbers of tachyzoites within HREC than HDEC.
Of tested microvascular EC subtypes, T. gondii tachyzoites bound more avidly to HRECs than HDECs. This observation may explain the relative susceptibility of HRECs to infection with the parasite and may be relevant to understanding why T. gondii preferentially infects retina and brain in human subjects.
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