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Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome by D. Maroeska W. M. te Loo, Leo A. H. Monnens, Thea J. A. M. van der Velden, Mario A. Vermeer, Frank Preyers, Pierre N. M. Demacker, Lambertus P. W. J. van den Heuvel, and Victor W. M. van Hinsbergh Blood Volume 95(11):3396-3402 June 1, 2000 ©2000 by American Society of Hematology Binding of VT-FITC to PMNs in whole blood was demonstrated by flow cytometric analysis.(A, B) Flow cytometric analysis of control blood (incubated with IgG-FITC alone) before addition of VTFITC. D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology Direct immunofluorescence of binding of VT-FITC to PMNs.Whole blood was incubated with CD13-PE (A), a specific marker for PMNs, and with VT-FITC (B) on ice for 20 minutes. D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology PMNs of 2 different donors were used to determine binding of 125I-VT-1 to PMNs.(A) PMNs were incubated with increasing concentrations of125I-VT-1 (0.3 to 70 nmol/L) at 4°C for 3 hours (▴). D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology 125I-VT-1 binding to neutral glycolipid extracts from human PMNs, GMVECs, and monocytes.Glycolipids were extracted and separated as described in “Materials and methods.” Binding of 125I-VT-1 was visualized, using a phosphor-imager. D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology The transfer of VT-1-FITC bound to PMNs to human GMVECs was studied by flow cytometry.Panels A, C, and F: FACS analysis of nonstimulated GMVECs incubated for 4 hours with PMNs loaded with VT-FITC on ice. D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology Phase-contrast microscopy of human GMVECs.Magnification × 50, except for (B) (magnification × 100). D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology Measurement of protein synthesis by incorporation of3H-leucine in newly synthesized proteins.(A) Nonstimulated GMVECs incubated with VT-1 loaded PMNs showed during 24-hour incubation a reduction of protein synthesis. D. Maroeska W. M. te Loo et al. Blood 2000;95:3396-3402 ©2000 by American Society of Hematology
