A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood.
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.
Laboratory of Pediatric Infectious Diseases, Radboud Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands.
Erika
E
van der Maten
Laboratory of Pediatric Infectious Diseases, Radboud Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands.
Marien I
MI
de Jonge
Laboratory of Pediatric Infectious Diseases, Radboud Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands.
Ronald
R
de Groot
Laboratory of Pediatric Infectious Diseases, Radboud Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands.|Pediatric Infectious Diseases and Immunology, Department of Pediatrics, Radboud university medical center, Nijmegen, The Netherlands.
Michiel
M
van der Flier
Laboratory of Pediatric Infectious Diseases, Radboud Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands.
Jeroen D
JD
Langereis
D000328:Adult; D001681:Biological Assay; D003167:Complement Activation; D003176:Complement C3 / Q000737:chemistry; D004926:Escherichia coli / Q000254:growth & development / Q000276:immunology; D005260:Female; D005343:Fibrinolytic Agents / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006193:Haemophilus influenzae / Q000254:growth & development / Q000276:immunology; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D057131:Immune Evasion; D007074:Immunoglobulin G / Q000737:chemistry; D007075:Immunoglobulin M / Q000737:chemistry; D007711:Klebsiella pneumoniae / Q000254:growth & development / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000382:microbiology; D008297:Male; D009345:Neisseria meningitidis / Q000254:growth & development / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000276:immunology; D013211:Staphylococcus aureus / Q000254:growth & development / Q000276:immunology; D013296:Streptococcus pneumoniae / Q000254:growth & development / Q000276:immunology; D013388:Succinimides / Q000737:chemistry
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D003176:Complement C3; D005343:Fibrinolytic Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006629:Hirudins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D013388:Succinimides