Abstract
Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.
Highlights
Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis
Gram-positive bacteria are protected from complement-mediated lysis by the presence of a thick outer cell wall consisting of peptidoglycan, which prevents the bacterial membrane from lysis by the pore-forming membrane attack complex[1]
Several bacterial species express a polysaccharide capsule, that protects them from recognition by opsonizing antibodies and in Gram-negative bacteria such as Haemophilus influenzae from insertion of the membrane attack complex[3]
Summary
Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis When these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. We used a selective thrombin inhibitor hirudin, which preserved complement activity of whole blood, in contrast to lithium heparin, sodium heparin, EDTA or sodium citrate
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