Figure 1
Figure 1. IV.3 induces Fc-mediated anaphylaxis and thrombocytopenia in FCGR2A mice. (A) Dose-dependent reduction in CBT induced by IV injection of mouse IV.3 in FCGR2A mice. (B) Platelet counts of mice after IV injection of IV.3. Animals represented are those of panel A (n = 1 per dose). (C) Representative flow cytometric analysis of CD41+ platelets (oval gate) in FCGR2A mouse whole blood before and 30 minutes after injection of 70 µg of IV.3. Cells in the upper right area are red blood cells (RBCs) and white blood cells (WBCs). Fluorescent beads (1.9 μm, Life Technologies; small boxed population above platelet oval) were used as a size reference and also to ensure consistent volume sampling between specimens. (D) Cytometric platelet count (as in panel C) of representative FCGR2A mouse before and 30 minutes after agly-IV.3 injection (70 μg). (E) Comparison of cytometric platelet counts (mean + SD) in whole blood from PBS, agly-IV.3, and IV.3-injected FCGR2A mice. *Significant difference (P < .05). A Kruskal-Wallis 1-way ANOVA on ranks identified a statistically significant difference (P = .003) in median values among all treatment groups. A t test showed that 70 µg of IV.3 differed significantly from 70 µg of agly-IV.3 (P < .001). A Mann-Whitney rank sum test showed that IV.3 (all mice injected with 35, 50, 70, or 100 µg of IV.3) differed significantly (P < .001) from 70 µg of agly-IV.3. (F) Comparison of body temperature change of mice injected with agly-IV.3 (70 µg/mouse; n = 7) or IV.3 (35 µg each; n = 4). Data in this panel (agly-IV.3 and IV.3) include mice from panel E. The 2 groups (agly-IV.3 vs IV.3) are statistically different based on a repeated measures test (F = 59.1; degrees of freedom = 1,9; P < .001). (G) High-resolution projection image of STED deconvolved microscopy sections (z stack; see supplemental Video) of FCGR2A mouse washed platelets (red) collected 24 hours after injection of 70 µg of agly-IV.3-A488 (green). Microscopic analysis shows agly-IV.3-A488 signal from objects ∼80-200nm in size and located in close physical proximity to platelet actin (labeled with phalloidin-Alexa 555). FS, forward scatter; SS, side scatter.

IV.3 induces Fc-mediated anaphylaxis and thrombocytopenia in FCGR2A mice. (A) Dose-dependent reduction in CBT induced by IV injection of mouse IV.3 in FCGR2A mice. (B) Platelet counts of mice after IV injection of IV.3. Animals represented are those of panel A (n = 1 per dose). (C) Representative flow cytometric analysis of CD41+ platelets (oval gate) in FCGR2A mouse whole blood before and 30 minutes after injection of 70 µg of IV.3. Cells in the upper right area are red blood cells (RBCs) and white blood cells (WBCs). Fluorescent beads (1.9 μm, Life Technologies; small boxed population above platelet oval) were used as a size reference and also to ensure consistent volume sampling between specimens. (D) Cytometric platelet count (as in panel C) of representative FCGR2A mouse before and 30 minutes after agly-IV.3 injection (70 μg). (E) Comparison of cytometric platelet counts (mean + SD) in whole blood from PBS, agly-IV.3, and IV.3-injected FCGR2A mice. *Significant difference (P < .05). A Kruskal-Wallis 1-way ANOVA on ranks identified a statistically significant difference (P = .003) in median values among all treatment groups. A t test showed that 70 µg of IV.3 differed significantly from 70 µg of agly-IV.3 (P < .001). A Mann-Whitney rank sum test showed that IV.3 (all mice injected with 35, 50, 70, or 100 µg of IV.3) differed significantly (P < .001) from 70 µg of agly-IV.3. (F) Comparison of body temperature change of mice injected with agly-IV.3 (70 µg/mouse; n = 7) or IV.3 (35 µg each; n = 4). Data in this panel (agly-IV.3 and IV.3) include mice from panel E. The 2 groups (agly-IV.3 vs IV.3) are statistically different based on a repeated measures test (F = 59.1; degrees of freedom = 1,9; P < .001). (G) High-resolution projection image of STED deconvolved microscopy sections (z stack; see supplemental Video) of FCGR2A mouse washed platelets (red) collected 24 hours after injection of 70 µg of agly-IV.3-A488 (green). Microscopic analysis shows agly-IV.3-A488 signal from objects ∼80-200nm in size and located in close physical proximity to platelet actin (labeled with phalloidin-Alexa 555). FS, forward scatter; SS, side scatter.

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