3D) was greater than samples with equimolar RGES ( Fig 3E) The

3D) was greater than samples with equimolar RGES ( Fig. 3E). The decline in aggregation frequency and concomitantly more uniform distribution of the hemocytes treated with RGDS (5 mM; Fig. 3D) compared to the

samples with RGES (5 mM; Fig. 3E) and the buffer control (CTX only; Fig. 3C) likely represents inhibition of integrin-mediated in vitro microaggregate formation. The number of circulating hemocytes may vary, counts declining or increasing (a form of hemocyte mobilization) as the hemocytes adhere to or lose affinity for surrounding tissues [77] and [32]. Unlike the in vitro results ( Fig. 1), incubation duration for acute CTX (0, 6, 60 nM) effects on circulating hemocytes in vivo was greatest at 20 min ( Fig. 4). This discrepancy with the in vitro results ( Fig. 1) was expected since reaction times are shorter in vivo than in vitro due to possible differences caused by plasma factors and their concentrations Cilengitide [28]; 20 min was chosen for subsequent in vivo experiments. The influence of CTX and its moieties on circulating hemocytes in vivo was determined with increasing concentrations CTX and CTA (ranging from 1.2 to 120 nM), and CTB (ranging from 0 to 600 nM). Levels of circulating hemocytes peaked at 6 nM

CTX, followed by a drop at 12 nM and rose to a plateau level by 60 nM, the latter hemocyte levels being similar to those at 6 nM CTX ( Fig. 5A). CTB caused an initial decrease in circulating hemocytes plateauing Selleckchem FRAX597 from 6 nM to 150 nM, followed thereafter to a maximum increase that plateaued starting at 300 nM, the latter two effects being similar to the corresponding concentrations of CTX ( Fig. 5A). Molar equivalents of CTA had no statistically significant effect (p>0.05) on circulating hemocyte counts ( Fig. 5A). The effect of CTX in vivo on circulating hemocytes was mirror image the pattern seen in vitro ( Fig. 2A), albeit the reaction taking place

at higher levels of CTX in vivo suggesting a tissue dilution effect in vivo in which other tissues might interact with CTX lowering the amount of holotoxin available for reaction with the hemocytes. To determine if CTX or its moieties affected the adhesiveness of the hemocytes remaining in circulation larvae were injected with these molecules and the hemocyte counts determined. Concentrations of 0, 6, second 12, and 60 nM CTX (corresponding to 0, 30, 60, and 300 nM CTB) were chosen since 6 and 60 nM represent the most pronounced effects of CTX in vivo and 12 nM CTX represents a lower intermediate effect ( Fig. 5A). The total number of adhering hemocytes decreased in 6 nM CTX-injected insects compared with PBS-injected insects, whereas adhesion levels increased from insects injected with 12 and 60 nM CTX ( Fig. 5B). Levels of attached granular cells and plasmatocytes followed the same pattern in insects injected with 0, 6, and 12 nM CTX, levels of adhering granular cells between 6 and 60 nM CTX increased faster than plasmatocytes ( Fig. 5B).

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