Impaired Innate Immunity in the Newborn: Newborn Neutrophils Are Deficient in Bactericidal/Permeability-Increasing Protein
Objective. The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. A central element of innate immunity to bacterial infection is the neutrophil—a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against Gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary (azurophilic) granules of the 55-kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills Gram-negative bacteria. In light of the importance of BPI to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns.
Design. The cellular content of BPI was determined by Western blotting of neutrophils derived from full-term newborn cord blood (n = 21; mean gestational age: 38.6 weeks) and from adult peripheral blood (n = 22; mean age: 29 years). Extracellular levels of BPI in adult and newborn plasma were assessed by enzyme-linked immunosorbent assay. Neutrophil content of other azurophil granule markers also was assessed: myeloperoxidase by Western blotting and defensin peptides by acid-urea polyacrylamide gel electrophoresis and Coomassie staining. Acid extracts of newborn and adult neutrophils were analyzed for antibacterial activity against serum-resistant encapsulated isolate Escherichia coliK1/r.
Results. The neutrophils of newborns contain at least threefold to fourfold less BPI per cell than adult neutrophils (67 ± 13 ng per 106 cells vs 234 ± 27 ng per 106 cells). The relative BPI-deficiency of newborn neutrophils apparently was not attributable to perinatal stress-related degranulation of intracellular BPI stores because: 1) newborn and adult neutrophils contained nearly identical amounts of 2 microbicidal constituents derived from the same primary (azurophil) granule compartment as BPI (the enzyme myeloperoxidase as well as defensin peptides), and 2) levels of extracellular BPI in newborn plasma, measured by enzyme-linked immunosorbent assay, represent only ∼2% of cellular BPI content. As predicted by their lower BPI content, newborn neutrophil acid extracts demonstrated significantly lower antibacterial activity against E coli K1/r than did adult neutrophil acid extracts.
Conclusion. These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against Gram-negative bacteria. BPI deficiency correlates with decreased antibacterial activity of newborn neutrophil extracts against serum-resistant E coli and could contribute to the increased incidence of Gram-negative sepsis among newborns relative to healthy adults. neonatal sepsis, Gram-negative bacteria, endotoxin, neutrophil, polymorphonuclear leukocyte, innate immunity, bactericidal/permeability-increasing protein, defensin, myeloperoxidase.
Effective host defense against bacterial infection requires the presence of a phagocytic system. The importance of neutrophils to antimicrobial defense is evident in the increased frequency and severity of infection when neutrophil activity is limited by decreased quantity or function.1 In addition to the oxygen-dependent phagocyte oxidase2 and myeloperoxidase (MPO)3systems, neutrophils have increasingly been shown to contain a variety of granule-associated oxygen-independent antimicrobial proteins and peptides.4 These include the broadly cytotoxic 4-kDa defensin peptides5 as well as the more highly selective (vs Gram-negative bacteria) 55-kDa bactericidal/permeability-increasing protein (BPI).6,,7
Although the neutrophil defense system is innate, there are indications that its function at birth is immature and suboptimal.8–10 Much of the work on newborn neutrophils has focused on functional assays demonstrating impaired adherence, chemotaxis, and phagocytosis. Impaired stimulus-induced adhesion and migration has been associated with decreased surface expression of L-selectin and the β2-integrin Mac-1.1 These findings may explain the difficulty in mobilizing neutrophils to sites of bacterial infection but do not explain the decreased neutrophil phagocytic activity (especially toward Gram-negative bacteria)11 as well as the decreased bactericidal activity of neutrophils derived from stressed newborns.8
Most studies of the microbicidal mechanisms of newborn neutrophils have focused on oxidative mechanisms (ie, the phagocyte oxidase/MPO/hydroxyl radical system), with conflicting data indicating either increased or decreased capacity of this oxygen-dependent mechanism in newborns.1,,12 Despite a growing literature on antibiotic proteins and peptides,4 little is known about the oxygen-independent microbicidal mechanisms of newborn neutrophils. A slightly decreased content of specific (secondary) granules in the neutrophils of newborns has been documented, with an associated modest (≤2-fold) decrease in lysozyme and lactoferrin content relative to adult neutrophils.12 However, to our knowledge, the major elements of the oxygen-independent antimicrobial arsenal of neutrophil primary granules, including BPI and the defensin peptides, have not been assessed in neonates. A study by Qing et al13revealed that newborn neutrophils, in contrast to adult neutrophils, have lower levels of a membrane-associated 55-kDa protein capable of binding lipopolysaccharide (LPS). Although this study did not identify the missing protein, its characteristics (size, binding properties, and localization) seemed to be similar to those of BPI and to the surface LPS receptor CD-14.
The selective action of BPI against Gram-negative bacteria has been explained by its high affinity toward the lipid A portion common to the LPS or endotoxin of all Gram-negative bacteria.7 The cationic N-terminal half of BPI not only binds to and kills Gram-negative bacteria, it also binds and thereby neutralizes LPS. The hydrophobic C-terminal portion of BPI recently has been shown to enhance the opsonic activity of BPI, whereby binding of BPI (via its N-terminal portion) to Gram-negative bacteria results in enhanced phagocytosis.14 Judging by the dramatic effects of adding neutralizing anti-BPI serum to crude neutrophil extracts, BPI is believed to play a dominant role in the bactericidal activity of whole neutrophils against Gram-negative bacteria such as Escherichia coli.7 The high affinity and potency of BPI are manifest in biologic fluids such as plasma, serum, and whole blood, suggesting that recombinant forms of BPI may serve as antimicrobial/antiinflammatory agents for treating Gram-negative infections and their complications.15
In this study, we directly assessed the cellular content of BPI in newborn neutrophils in relation to adult neutrophils, comparing observed discrepancies with the relative content of 2 other neutrophil primary (azurophil) granule-associated antimicrobial proteins: MPO and defensins. As a functional correlate of BPI content, we measured the antibacterial activity of neutrophil extracts against E coliK1/r.
Collection of Neonatal Cord Blood
After institutional review board approval at the Brigham and Women's Hospital, cord blood samples were collected immediately after cesarean section (n = 13) or vaginal delivery (n = 8). Cord blood, from both female (n = 12) and male (n = 9) newborns, was collected into sterile tubes anticoagulated with sodium heparin (Becton Dickinson, Cockeysville, MD) and placed on ice. All samples were labeled numerically and the results kept anonymous. Adult peripheral blood was obtained by venous phlebotomy of nonpatient adult volunteers.
Isolation of Neutrophils From Whole Blood
Neutrophils were isolated from whole blood as previously described.16 Anticoagulated blood was promptly (within 30–60 minutes) processed by dextran sedimentation (3% pyrogen-free dextran (United States Biochemical, Cleveland, OH) diluted in Hanks' Balanced Salt Solution without divalent cations (to avoid neutrophil clumping; Gibco BRL, Gaithersburg, MD). Ficoll-hypaque (endotoxin-free Ficoll-Paque Plus; Pharmacia Biotech, Piscataway, NJ) gradient centrifugation was used to generate a neutrophil-rich fraction. Brief hypotonic lysis (∼45 seconds on ice) was used to remove red blood cells. An automated total white blood cell count and differential (Technion H3 RTX automated cell counter; Miles, Tarrytown, NY) was obtained on every sample before pelleting by centrifugation. White cell differential counts often were confirmed by Wright stain and manual assessment. Viability was assessed by trypan blue exclusion. Neutrophil pellets (typically >85% pure) were frozen in Eppendorf tubes at −70°C before batch analysis.
Neutrophils were thawed and solubilized with 4X sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (.8% SDS, .34 (v/v) glycerol, .04% Bromphenol Blue, .02 M DTT, and .2 M Tris pH 6.8) before fractionation over a 10% SDS-PAGE gel (PAGE-ONE precast 10% gels; Owl Separation Systems, Portsmouth, NH). After Western transfer onto nitrocellulose (Protran BA85, pore size .45 μm, Schleicher and Schuell, Keene, NH), and blocking of nonspecific sites with 3% bovine serum albumin/Tris-buffered saline pH 7.4, BPI was detected using .1% (v/v) whole anti-BPI goat serum (provided by Drs Peter Elsbach and Jerrold Weiss; New York University) as previously described.17 Bound antibody was detected using: 1) .05% (v/v) peroxidase-conjugated protein G followed by metal-enhanced diaminobenzoic acid (DAB; Pierce, Rockford, IL), 2) 1:35 000 dilution of peroxidase-conjugated protein G as part of the SuperSignal chemiluminescent system (Pierce), or ) .1% (v/v) I-125 protein G. For detection methods 2 and 3, signal was detected by exposing the blots to Kodak XAR film (Kodak, Rochester, NY). This Western transfer protocol provided detection in the range of 10 to 200 ng with readily apparent differences in signal intensity between twofold dilutions of a BPI standard, thus allowing interpolation of BPI content in test samples. Native human BPI was provided by Drs Peter Elsbach and Jerrold Weiss, New York University. Recombinant human BPI (rBPI) was prepared as previously described.18
MPO was detected by Western blotting using .1% (v/v) rabbit anti-MPO serum (provided by Dr William Nauseef, University of Iowa)2 followed by .1% (v/v) I-125 protein G. As control for MPO blots, a twofold dose curve of adult azurophil granule fraction (prepared as previously described)20 was solubilized in 4X SDS-PAGE buffer and analyzed as well. For purposes of quantitation, MPO content in neutrophil samples was expressed in antigenic units defined in relation to an adult azurophil granule extract standard: 1 antigenic unit was set equal to the band intensity of an azurophil granule extract sample representing 106 adult neutrophil equivalents.
Detection of Defensins
Defensins were detected by subjecting neutrophil extracts to acid-urea (AU)-PAGE as previously described.21 Briefly, neutrophils were sonicated in 5% acetic acid before overnight extraction at 4°C. Insoluble components were removed by centrifugation, supernatants lyophilized, and resuspended in AU-PAGE buffer before electrophoresis and Coomassie Brilliant Blue R stain.
BPI Enzyme-linked Immunosorbent Assay (ELISA)
To determine the levels of extracellular BPI, newborn and adult plasma were collected within 30 to 60 minutes of drawing cord or peripheral venous blood, respectively. Samples were stored in cryogenic microtubes (Sarstedt) at −70°C before batch analysis. BPI content of plasma and of polymorphonuclear leukocyte acid extracts was determined employing a biotinylated anti-BPI antibody in a sandwich ELISA format as previously described.22 This ELISA system yielded a linear range from .1 to 6 ng BPI/mL and showed negligible cross-reactivity with the homologous lipopolysaccharide-binding protein.
Solubilization of BPI by Acid Extraction of Neutrophils
BPI was extracted from neutrophils using sulfuric acid as previously described.17 In brief, neutrophil pellets (∼5 × 107 cells), stored frozen at −80°C, were thawed on ice and homogenized by adding sterile water (167 μL of H2O added per 107 cells) and sonicating for 15 seconds bursts in an ice water bath sonicator at ∼40% maximal output (Sonic Dismembrator 550; Fisher, Pittsburgh, PA) until the sample was uniformly milky white and free flowing. Chilled (4oC) .4 N sulfuric acid then was added to a final concentration of .16 N and the samples extracted on ice for 30 minutes with intermittent vortexing. Extracted samples then were centrifuged at 14 000 rpm at 4°C and the supernatant recovered as the neutrophil acid extract.
To compare analysis of BPI content by 2 independent techniques, a number of neutrophil acid extracts were analyzed for BPI content by both Western blotting and ELISA. By both assay methods, similar levels were obtained, suggesting that the differences noted between adult and newborns were not attributable to the method of analysis.
Measurement of Antibacterial Activity
Antibacterial activity of neutrophils was determined by measuring the ability of neutrophil acid extracts to inhibit growth of the serum-resistant bacteremic isolate E coli K1/r, a K1-encapsulated rough LPS chemotype strain23 (provided by Dr Alan Cross, Department of Bacterial Diseases, Walter Reed Army Medical Center, Washington, DC). As previously described,17 bacteria were subcultured in trypticase soy broth (Becton Dickinson) and grown to late logarithmic phase (∼4 hours) at 37°C. Bacterial concentrations were determined by measuring OD550 in a spectrophotometer. Subcultures were harvested by centrifugation and resuspended in sterile physiologic saline to the desired concentration. For antibacterial assay, bacteria were incubated at 104/mL in incubation buffer (20 mM sodium phosphate pH 7.4 with .9% wt/vol saline) at 37°C, shaking for 1 hour. Bacterial viability was assessed by plating 10 μL of each sample onto a Petri dish and dispersing this sample with ∼9 mL of molten (50oC) Bactoagar (Difco, Detroit, MI) containing .8% (wt/vol) nutrient broth (Difco) and .5% (wt/vol) NaCl. The agar was allowed to solidify at room temperature, and bacterial viability was measured as the number of colonies formed after incubation of plates at 37°C for 18 to 24 hours.
Measurement of Cell-Associated BPI in Adult and Neonatal Neutrophils
To compare the BPI content of neonatal and adult neutrophils, cell-associated BPI was measured by Western blot analysis of neutrophil detergent extracts. The neutrophil content of BPI then was estimated by visual comparison to twofold dilutions of purified BPI, allowing quantitation of sample values. A typical Western blot is shown in Fig 1, and indicates that the quantitation range for purified BPI was ∼10 to 200 ng. For comparison, several extracts from adult and newborn neutrophils also are shown inFig 1.
Composite data from multiple Western blotting experiments are shown inFig 2. All samples of adult neutrophils (n = 22; mean age: 29 years) contained quantifiable levels of BPI, the average of which was 234 ± 27 ng per 106 neutrophils. In contrast, newborn neutrophils (n = 21; mean gestational age: 38.6 weeks) contained significantly lower amounts of BPI: 67 ± 13 ng per 106 neutrophils (P < .001; 2-sided t test). Median values for BPI content per 106 neutrophils were 200 ng and 50 ng for adults and newborns, respectively. Thus, newborn neutrophils contained at least threefold less BPI than adult neutrophils.
Interestingly, it is also evident from Fig 2 that ∼40% (8 of 21) of the newborn neutrophils were markedly deficient in BPI. Among these 8 samples, 7 had no detectable BPI even after prolonged exposure. This number represents 33% of the newborn patients studied. For the purposes of quantitation, such samples were considered to contain one half of the lowest amount of BPI that was detectable in the standard curve (ie, ∼10 ng per 106 neutrophils). As a consequence, our analysis of BPI content is conservatively biased toward overestimating the amount of BPI in newborn neutrophils, with the actual difference in neutrophil BPI content between some newborns and adults perhaps being >10-fold.
To ensure that the discrepancy in BPI content between adults and newborns was not attributable to the presence in newborn neutrophils of an inhibitor of BPI detection (eg, a protease), a mixing experiment was performed. Addition of pure rBPI (80 ng) to a newborn neutrophil acid extract (prepared as previously described, 17) and incubation at 37°C for 15 minutes did not alter rBPI signal intensity or migration by Western blotting (not shown), indicating that the newborn neutrophil extract did not contain an inhibitor of BPI detection.
Determination of Extracellular BPI in Cord Blood
To determine whether the relatively low BPI content of newborn neutrophils was related to degranulation (eg, during collection or possibly secondary to perinatal stress), we measured the levels of extracellular BPI in newborn plasma samples (n = 13). Average cord plasma BPI content was 16 ± 3 ng/mL, which is elevated relative to the plasma BPI content of healthy adults22 and of critically ill children.24Nevertheless, calculated per milliliter of whole cord blood, the newborn plasma content of BPI represents <2% of cellular BPI content. Thus, there was no evidence for substantial extracellular degranulation of BPI at the time immediately preceding collection and processing of newborn cord blood.
Measurement of Other Bactericidal Constituents of Neutrophils
The relative BPI deficiency in newborn neutrophils raised the question as to the specificity of this finding to BPI. To assess whether other primary (azurophil) granule constituents also were decreased in newborn neutrophils, we measured the presence of MPO and of defensin peptides as follows.
First, MPO was evaluated by Western blotting of neutrophils from newborns (n = 7) and adults (n = 7). A representative Western blot is shown in Fig 3. As control, twofold dilutions of primary (azurophil) granule extracts also were analyzed. The MPO content of newborn neutrophils (6.0 ± 2.5 antigenic units per 106 cells) and of adult neutrophils (4.3 ± 1.6 antigenic units per 106 cells) was not statistically different. Thus, in accordance with previous observations by others,25 newborn and adult neutrophils apparently contain nearly identical amounts of MPO. Of note, MPO was readily detected in neutrophil samples from 3 newborns with undetectable BPI.
Second, defensin content of newborn (n = 8) and adult (n = 8) neutrophils was evaluated by AU-PAGE and Coomassie staining (Fig 4). Similar to MPO, and despite the use of a sensitive detection technique that easily revealed twofold differences in defensin content, there was no discernible difference in the content of defensins in adult and newborn neutrophils. Of note, although the levels of lysozyme were somewhat decreased in 3 of the 4 newborn neutrophil samples depicted in Fig 4(in accordance with previous studies), the overall pattern of neutrophil proteins did not differ significantly in migration or band intensity between newborn and adults.
Measurement of Neutrophil Extract Antibacterial Activity
The deficiency of neonatal neutrophils in BPI suggested that these cells may have relatively low antibacterial activity toward Gram-negative bacteria. To investigate this possibility, we assayed the activity of neutrophil acid extracts (prepared as described in “Methods”) against the encapsulated clinical isolate E coli K1/r.23
As previously shown,23 pure BPI manifests potent antibacterial activity against this encapsulated bacterium (Fig 5A). Whereas adult neutrophil acid extracts were able to profoundly inhibit E coli K1/r viability, extracts derived from the neutrophils of neonates were significantly less potent, incapable of fully inhibiting bacterial viability at the doses tested (Fig 5B). When expressed as percentage killing (100 to percentage of survival) the composite data for individual patients (Fig 5C) reveal that adult neutrophil acid extracts tested at a 5% vol/vol dose (n = 7) uniformly killed the vast majority of bacteria (mean killing 93% ± 2.8%). In contrast, neonate neutrophil extracts (n = 8) varied in their activity averaging only 55% ± 7.3% killing.
This study demonstrates that newborn neutrophils are deficient in BPI. This is the first study, to our knowledge, to assess the presence of BPI in newborns and the first to demonstrate deficiency of a prominent antimicrobial component of the primary (azurophil) granules of newborn neutrophils. Qing and co-workers13 demonstrated that the membranes of newborn neutrophils lack a 55-kDa LPS-binding protein that was present in adult neutrophils, although the study of Qing et al did not identify the missing protein. Our current study demonstrates a selective deficiency of BPI in newborn neutrophils.
Although the discrepancy in BPI content could be explained by lesser recovery of BPI (eg, attributable to degranulation of newborn neutrophils in response to perinatal stress), we favor the interpretation that newborn neutrophils have intrinsically lesser quantities of BPI because: 1) a priori considerations would predict that BPI should remain intracellularly because it resides in the primary (azurophilic) granules that are known to be the least easily mobilized compartment of both adult and newborn neutrophils,25 2) newborn and adult neutrophils contain nearly identical amounts of both MPO and defensin, because these proteins share the same primary (azurophil) granule compartment as BPI, it is highly unlikely that selective degranulation of BPI occurred, and 3) levels of BPI in cord plasma represent only a small fraction (<2%) of total cellular BPI, suggesting that there was no substantial release of BPI from cellular stores to the extracellular space at the time immediately preceding cord blood collection.
The average BPI content of newborn neutrophils was significantly lower than those of adults but it was not uniform. As demonstrated in the scattergram (Fig 2), some newborns apparently contain larger BPI stores than others. Approximately 40% of newborns were markedly deficient (∼9- to 10-fold less BPI than adults), with several revealing no detectable BPI even after prolonged radiographic exposure. This variability suggests that BPI expression may be controlled by factors that are not uniformly distributed in newborns and may explain why some newborns are at greater risk of Gram-negative bacterial infection than are others. Although the exon/intron structure of the BPI gene has been defined,26 its transcriptional and translational regulation has not been characterized. Thus the relevant factors governing BPI expression in adults and newborns remain to be defined. Moreover, the age at which infants or children achieve normal levels of BPI also is unknown and is beyond the scope of this study.
Given the well documented role of BPI in oxygen-independent killing of Gram-negative bacteria,7 deficiency in BPI would be predicted to be associated with decreased activity of neonatal neutrophils against such pathogens. Indeed, the relatively low activity of newborn neutrophil acid extracts against the encapsulated serum-resistant clinical pathogen E coli K1/r provides support for such a functional defect. Of note, acid extraction and functional antibacterial assay are distinct from the SDS-PAGE buffer solubilization and Western blotting used to detect BPI antigen, thereby providing an independent comparison of the anti-Gram-negative bacterial properties of newborn and adult neutrophils.
Although the majority of bacterial infections in newborns are caused by Gram-positive organisms, a variable (depending on time of sepsis onset, birth weight, and study population) but significant percentage (∼20%–40%)27,,28 are attributable to Gram-negative bacteria, particularly E coli, Haemophilus influenzae, Klebsiella spp, and Enterobacter spp. In fact, it is the Gram-negative infections that are, in some studies, associated with the highest mortality rate (∼40%).27,,28 Determining the contribution of variable BPI expression to the risk of a given newborn suffering bacterial sepsis will require measurement of neutrophil BPI content at birth and prospective assessment of clinical outcomes.
Seen in the context of previous studies of newborn neutrophils, our current study suggests that lower amounts of BPI may be available at inflammatory sites in newborns because: 1) relatively low bone marrow reserves29 render newborns with sepsis at risk for neutropenia, 2) impaired neutrophil migration and chemotaxis1 will limit the speed and magnitude of neutrophil response, 3) BPI can serve as an opsonin,14decreased BPI content per neutrophil might impair phagocytosis of Gram-negative bacteria, and 4) once a bacterium is ingested, a relatively low amount of BPI will be available for delivery to the phagolysosome. Thus, a deficiency in BPI raises the possibility that neonatal neutrophils have lesser bactericidal (as suggested in this study), as well as reduced antiendotoxic and opsonic activities toward Gram-negative bacteria. We are currently pursuing these studies.
Extending survival of extremely ill full-term as well as premature neonates defines a growing population at high risk for bacterial infection, including overwhelming sepsis with leukopenia. Although replacement of neutrophils by granulocyte transfusion in newborns with sepsis has apparently been beneficial in some studies,30this potential therapy has been complicated by difficulty in obtaining histocompatible neutrophils and by transfusion reactions, including induction of pulmonary inflammation. Of note in this regard, however, is that pure preparations of several neutrophil-derived antimicrobial proteins and peptides are currently being evaluated as possible therapeutic agents.15,,31,32 One of these agents (rBPI21) is derived from human BPI and has shown promise as a potential novel treatment for fulminant meningococcemia in a pediatric population.33 The demonstration of BPI deficiency among newborns raises the possibility that supplementation of BPI may be of clinical benefit for newborns, including those with Gram-negative bacterial infections, endotoxemia, or endotoxin-associated complications.
This project was supported by National Institutes of Health/National Center for Research Resources/General Clinical Research Center Grant M01RR02172.
We thank the following groups and individuals: Children's Hospital: Drs Philip Pizzo, Frederick Lovejoy, Mary Ellen Wohl, Joseph Majzoub, Greg Priebe, and David Weinstein for advice and encouragement, Dixon Yun for assistance with computer graphics, Dr David Zurakowski for assistance with statistical analysis, Martina Flynn and Cheryl Sweeney for administrative support, as well as Jennifer Langley, Irena Clark, and the technical staff of the Core Lab; The Brigham and Women's Hospital: Dr Steve Ringer for technical advice as well as the nursing, midwife, and obstetrical staff for assistance in cord blood collection; New York University: Drs Peter Elsbach, Jerrold Weiss, Yvette Weinrauch, Chen Shu, and Seth Katz, as well as Kol Zarember for technical advice and assistance; XOMA (US) LLC: Dr Patrick Scannon and Mark White for advice and encouragement.
- Received March 10, 1999.
- Accepted September 10, 1999.
Reprint requests to (O.L) Department of Medicine, The Children's Hospital, 300 Longwood Ave, Boston, MA 02115. E-mail:
- MPO =
- myeloperoxidase •
- BPI =
- bactericidal/permeability-increasing protein •
- LPS =
- lipopolysaccharide •
- SDS =
- sodium dodecyl sulfate •
- PAGE =
- polyacrylamide gel electrophoresis •
- rBPI =
- recombinant human BPI •
- AU-PAGE =
- acid-urea-PAGE •
- ELISA =
- enzyme-linked immunosorbent assay
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- Copyright © 1999 American Academy of Pediatrics