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. 2000 Sep;68(9):5120-5.
doi: 10.1128/iai.68.9.5120-5125.2000.

Enhancement of Neonatal Innate Defense: Effects of Adding an N-terminal Recombinant Fragment of Bactericidal/Permeability-Increasing Protein on Growth and Tumor Necrosis Factor-Inducing Activity of Gram-Negative Bacteria Tested in Neonatal Cord Blood Ex Vivo

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Free PMC article

Enhancement of Neonatal Innate Defense: Effects of Adding an N-terminal Recombinant Fragment of Bactericidal/Permeability-Increasing Protein on Growth and Tumor Necrosis Factor-Inducing Activity of Gram-Negative Bacteria Tested in Neonatal Cord Blood Ex Vivo

O Levy et al. Infect Immun. .
Free PMC article

Abstract

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC(50)], approximately 10 nM), C. koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM). The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Figures

FIG. 1
FIG. 1
Effect of added rBPI21 on the survival of E. coli K1/r in adult and newborn cord blood. E. coli K1/r cells (104 bacteria/ml) were incubated in citrated adult blood (A) or newborn cord blood (B) with increasing concentrations of rBPI21. Samples were periodically plated to assess bacterial survival (CFU) as a percentage of that of a control sample incubated in buffered saline alone. The results shown represent the mean of 2 to 12 independent determinations (error bars omitted to enhance figure clarity).
FIG. 2
FIG. 2
Effect of added rBPI21 on the survival of C. koseri in adult and newborn cord blood. C. koseri cells (104 bacteria/ml) were incubated in citrated adult (A) or newborn cord blood (B) with increasing concentrations of rBPI21. The results represent the mean of three to four independent determinations (error bars omitted to enhance figure clarity).
FIG. 3
FIG. 3
Effect of added rBPI21 on TNF-α induction by E. coli K1/r in adult and newborn cord blood. (A) E. coli K1/r (101 to 104 bacteria/ml) were incubated in citrated adult or newborn cord blood for 5 h, at which point samples were diluted with RPMI and the extracellular fluid was collected by centrifugation. TNF-α release was measured by ELISA. In order to determine the effects of rBPI21 on bacterially induced TNF release, E. coli K1/r cells (104 bacteria/ml) were incubated in citrated adult or newborn cord blood with increasing concentrations of rBPI21. (B) TNF release is expressed as a percentage of that of a control sample incubated without rBPI21. Results represent the mean ± standard error of two to four determinations.
FIG. 4
FIG. 4
Gram-negative bacterial strains are potent inducers of TNF-α in both adult and newborn cord blood, as shown by inhibition by rBPI21. (A) Data points represent averages of TNF-α release induced at each bacterial concentration by the six species tested. (B) Average percentage of TNF release induced by six bacterial species incubated in the presence of increasing concentrations of rBPI21. Results represent the mean ± standard error of 22 to 25 independent determinations.
FIG. 5
FIG. 5
Combined effects of ampicillin-gentamicin (A/G) and rBPI21 on bacterially induced TNF-α release. E. coli K1/r (A) and K. pneumoniae (B) were incubated at a concentration of 104/ml in newborn cord blood either alone (white bar), with a bactericidal concentration of ampicillin and gentamicin (100 μg of ampicillin and 5 μg of gentamicin per ml in panel A and 1,000 μg of ampicillin and 50 μg of gentamicin per ml in panel B [grey bar]), with an endotoxin-neutralizing concentration of rBPI21 alone (100 nM in panel A and 1,000 nM in panel B [striped bar]), or with a combination of ampicillin-gentamicin and rBPI21 (black bar). Results represent the mean ± standard error of three independent determinations.

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