Plateletneutrophil complex formation

Examination ofthe neutrophils from whole blood stained with CD42b:PE and CDllb:FITC, allowed the detection of two populations of cells: free neutrophils and platelet-neutrophil complexes (PNCs). ADP stimulation increased the intensity ofCDl lb staining in both PNCs and free neutrophils. Addition of defatted breast milk led to a dose dependent increase in PNCs and an increase in neutrophil CD1lb expression. This occurred in the presence or absence of ADP. Experiments with BMO also increased PNCs although in the samples used low levels of endotoxin were detected (Figure2).

Tfil T

:" 111 i PBS 1% 5% 10% PBS 1% 5% 10% No ADP ADP

Figure 2: CD1 1b/CD42b expression on polymorphic neutrophiles and platelets after incubation with breast milk oligosaccharides

5. CONCLUSIONS

(1) This study investigated the capacity for different breast milk fractions to influence the processes of adhesion involved in endothelial -leukocyte interactions and in platelet-neutrophil complex formation. Both adhesive models require selectins, integrins and members of the immunoglobulin superfamily. We found that purified neutral oligosaccharides had the capacity to inhibit binding of neutrophils to TNF stimulated endothelium. The model used examined adhesion under low flow conditions and so it is difficult to be exactly certain how these sugars were operating. It is probable that selectin mediated adhesion was most effected but further studies are required to examine this further.

(2) We also found that defatted BM and purified oligosaccharides enhanced the formation of PNCs. Again both selectins and integrins are involved in PNC formation. How the oligosaccharides are mediating this effect is unclear. They may act to bridge CD62P to its ligands on neutrophils or more probably through the crosslinking of neutrophil selectins they activate a signalling cascade which leads to increased integrin expression and activation. More work is required to understand these processes in more detail.

(3) In summary, our results indicate that breast milk oligosaccharides can influence cellular adhesion but further work is required before we can be confident that the oligosaccharides are operating in isolation and are not acting in concert with non-oligosaccharide contaminants present in the samples as a result ofthe purification process.

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