Utility of an ex vivo human whole blood assay for bacterial killing ability qualification
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Infectious diseases caused by antibiotic resistant bacteria are difficult to treat using traditional antibiotics and have emerged as a global public health problem (Bartlett, Gilbert, & Spellberg, 2013). An alternative strategy to bypass antibiotic resistance in bacteria is to enhance the host immune system (Hancock, & Sahl, 2006), especially innate immunity (Ajesh, & Sreejith, 2009), in order to combat bacterial infections. To verify the augmentation of innate immunity under certain stimuli, a quantifiable measurement of bacterial killing ability is needed. A new method of using heparinized human peripheral whole blood and mild heated blood samples from the same individuals as comparisons has been developed. This assay was used to quantify the bacterial killing ability of phagocytic cells by measuring the bacterial load after co-culturing with the Gram negative bacteria Pseudomonas aeruginosa. The assay shows that peripheral whole blood of healthy humans is capable of being activated ex vivo upon bacterial challenge and suppressing the bacterial load, while blood gently heated at 48oC for one hour failed to do so. The morphology of the phagocytic cells was examined microscopically. Phagocytosis of bacteria in neutrophils is observed in intact blood, and no such changes were found in heated blood. The level of inflammatory cytokine interleukin 8 (IL-8) was elevated in intact blood after bacteria inoculation, while in the heated blood the cytokine level stayed at baseline, indicating a persistence of leukocyte viability in whole blood and damaged leukocyte activity in gently heated blood. The data show that the human peripheral whole blood assay is suitable as a method for ex vivo bacterial killing quantification with the mildly heated blood sample from the same individual as a comparison. When kept at 37oC ex vivo, human whole blood can be activated upon bacterial challenge, produce inflammatory cytokines, reduce bacterial load, and phagocytes in the whole blood can successfully maintain their cell viability and capacity to phagocytose bacteria.