Levels Of Bactericidal/permeability-increasing Protein Research Articles

Positive correlations between plasma BPI level and MPO-DNA and S100A8/A9 in myocardial infarction

Bactericidal/permeability-increasing protein (BPI) exhibits a number of important characteristics. RNA-seq analysis revealed that the BPI expression was increased in platelets of (non)ST-elevated myocardial infarction (NSTEMI/STEMI) patients. Activated platelets can induce NETosis which may be accompanied by the release of myeloperoxidase-DNA (MPO-DNA) and S100A8/A9. This study investigated the plasma BPI levels in myocardial infarction patients and its correlation with MPO-DNA and S100A8/A9. This prospective study recruited 80 control individuals, as well as 63 NSTEMI and 59 STEMI patients who were admitted to the First Affiliated Hospital of Bengbu Medical College for coronary angiography (CAG) and/or percutaneous coronary intervention (PCI) between May 1, 2020 and August 31, 2020. Demographic and clinical characteristics, clinical indicators, hs-CRP, IL-1β, MPO-DNA (a circulated marker of NETs), circulating levels of S100A8/A9 and BPI were measured from each individual. The severity of coronary lesions was evaluated by the Gensini score, based on the results of the CAG. Pearson’s or spearman’s correlation was used to examine the correlation between BPI and the above-mentioned parameters, as well as the severity of coronary artery disease. Linear regression analysis was applied to identify the independent predictive factors of BPI. Received operating characteristic (ROC) curve analysis was used to evaluate the diagnostic efficacy of plasma BPI for MI. The plasma BPI levels increased by 8.76 times in the STEMI group and 5.38 times in the NSTEMI group compared to the control group. The plasma level of hs-CRP and IL-1β in both STEMI and NSTEMI groups were also significantly higher than the control group. In addition, the plasma levels of MPO-DNA and S100A8/A9 in the STEMI and NSTEMI groups were significantly higher than the control group. Plasma levels of BPI were positively correlated with IL-1β, hs-CRP, MPO-DNA and S100A8/A9. The correlation between BPI and the severity of coronary artery disease was also significant. The optimal cutoff value of plasma BPI was 35.1705 ng/ml for MI patients from the ROC curve analysis. Plasma BPI levels are increased in myocardial infarction patients and positively correlated with MPO-DNA and S100A8/A9. Plasma BPI level may serve as a potential biomarker of myocardial infarction.

Lipopolysaccharide Binding Protein and Bactericidal/Permeability-Increasing Protein as Biomarkers for Invasive Pulmonary Aspergillosis.

Early diagnosis of invasive pulmonary aspergillosis (IPA) is crucial to prevent lethal disease in immunocompromized hosts. So far, lipopolysaccharide binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) levels have not been evaluated as biomarkers for IPA. IL-8, previously introduced as a biomarker for IPA, was also included in this study. Bronchoalveolar lavage fluid (BALF) of IPA patients and control patients with non-infectious lung disease was collected according to clinical indications. Measurements in BALF displayed significantly higher levels of LBP (p < 0.0001), BPI (p = 0.0002) and IL-8 (p < 0.0001) in IPA compared to control patients. Receiver operating characteristic curve analysis revealed higher AUC for LBP (0.98, 95% CI 0.95–1.00) than BPI (0.84, 95% CI 0.70–0.97; p = 0.0301). Although not significantly different, AUC of IL-8 (0.93, 95% CI 0.85–1.00) also tended to be higher than AUC for BPI (p = 0.0624). When the subgroup of non-hematological patients was analyzed, test performance of LBP (AUC 0.99, 95% CI 0.97–1.00), BPI (AUC 0.97, 95% CI 0.91–1.00) and IL-8 (AUC 0.96, 95% CI: 0.90–1.00) converged. In conclusion, LBP and—to a lesser extend—BPI displayed high AUCs that were comparable to those of IL-8 for diagnosis of IPA in BALF. Further investigations are worthwhile, especially in non-hematological patients in whom sensitive biomarkers for IPA are lacking.

Antimicrobial peptides in pregnant women with TORCH infections

Introduction. Analysis for TORCH infections is ordered to simultaneously detect several common infections: Toxoplasmosis, Other infections (syphilis, hepatitis B, chickenpox, Epstein-Barr virus, parvovirus and some others), Rubella, Cytomegalovirus and Herpes simplex virus. Contracting a TORCH infection in pregnancy triggers the synthesis of immune factors including antimicrobial peptides (AMPs).Aim: to determine the levels of AMPs – lactoferrin, defensin, endotoxin, BPI (bactericidal/permeability-increasing protein) and hepcidin – in the blood serum of pregnant women with TORCH infections.Materials and methods. The main group included 40 pregnant women with TORCH infections; in 33 of those, pregnancy continued until full-term delivery, and 7 women had miscarriages at the end of the first trimester. The comparison group consisted of 29 pregnant women free of TORCH infections. Blood for AMP measurement was taken in all pregnant women in the first trimester and in women with an ongoing pregnancy also in the III trimester. The control group consisted of 19 healthy non-pregnant women. Serum AMP was determined by enzyme-linked immunosorbent assay (ELISA).Results. In pregnant women with TORCH infections, there was an increase in the serum levels of lactoferrin, defensin, hepcidin, BPI, and endotoxin; the increase was most pronounced in the first trimester of pregnancy. The decrease in AMP levels observed in the III trimester could be due to the antiviral treatment given to the patients in order to prevent the immunological rejection of the embryo and maintain the normal course of pregnancy.Conclusion. The increased level of AMPs reflects the enhanced activity of the immune system and represents one of the pathogenetic links of spontaneous abortion.

Antimicrobial peptide plasma concentrations in patients with community-acquired pneumonia

Community-acquired pneumonia (CAP) is a common and potentially life-threatening infection. Innate immunity is the first line of defence, and antimicrobial peptides (AMPs) produced by white blood cells and at epithelial barriers participate by killing microorganisms and neutralizing bacterial toxins. We wanted to investigate whether concentrations of AMPs (1) are increased in CAP, (2) predict the clinical outcome, and (3) differ depending on the causative microbe. Plasma concentrations of AMPs were measured using an enzyme-linked immunosorbent assay in 89 patients with CAP, 21 patients with non-respiratory tract infections (non-RTI), and 63 healthy control subjects. In subjects with CAP, mean plasma concentrations of secretory leukocyte protease inhibitor (SLPI) and bactericidal/ permeability-increasing protein (BPI) were significantly higher than in healthy control subjects (85 vs 45 ng/ml, p < 0.001 and 48 vs 10 ng/ml, p < 0.001, respectively), but less markedly increased in patients with non-RTI (68 ng/ml, p = 0.06 and 41 ng/ml, p = 0.43). LL-37 and human neutrophil peptides 1-3 (HNP 1-3) levels were not increased in subjects with CAP. Levels of BPI and SLPI did not correlate to severity of disease, and AMP levels did not differ depending on the causative agent. Interestingly, male subjects with CAP displayed increased concentrations of SLPI compared to females. This was not observed in subjects with non-RTI and healthy control subjects. Subjects with CAP showed increased plasma concentrations of SLPI and BPI compared to healthy control subjects. The finding of higher SLPI levels in male subjects with CAP implies that there are sex-dependent immunological differences in SLPI turnover.

Increased bactericidal/permeability increasing protein in patients with cirrhosis

High levels of endotoxin in patients with cirrhosis are thought to be responsible for the activation of tumour necrosis factor-alpha (TNF)-alpha-mediated pro-inflammatory pathways involved in haemodynamic alterations. Bactericidal/permeability increasing protein (BPI) is a protein found in neutrophils with endotoxin-binding and neutralization capacity. It is not known whether defective BPI production or release is present in cirrhosis. We investigated the levels of BPI in cirrhotic patients and its relation to other endotoxin-binding proteins and inflammatory markers. Plasmatic levels of BPI, lipopolysaccharide-binding protein, soluble CD14, TNF-alpha and BPI mRNA expression in neutrophils were determined in 130 patients and 30 healthy controls. The capacity of patients' plasma to inhibit lipopolysaccharide (LPS)-mediated TNF-alpha production by monocytes from healthy donors was assessed in vitro. Patients with cirrhosis exhibited an increase in BPI mRNA and plasma level of BPI when compared with healthy controls (P<0.05). Child C group displayed the highest frequency of patients with a high concentration of BPI. A positive correlation was found between TNF-alpha and plasma levels of BPI (P<0.01). High levels of BPI in plasma were able to significantly reduce in vitro TNF-alpha release by monocytes after a challenge with LPS (8.54 +/- 1.04 vs. 10.44 +/- 0.85 pg/ml, P=0.028). BPI is increased in cirrhotic patients, especially in those with more severe liver disease. The amount of BPI in the plasma correlated with the TNF-alpha level and was able to reduce LPS-mediated TNF production by monocytes. BPI possibly plays a regulatory role by antagonizing the pro-inflammatory mechanisms mediated by TNF-alpha.

Comparison of the Modulatory Effects of Four Different Fast-Track Anesthetic Techniques on the Inflammatory Response to Cardiac Surgery With Cardiopulmonary Bypass

To test the hypothesis that the choice of anesthesia technique for coronary artery surgery influences the degree and magnitude of the subsequent inflammatory response and its consequences. Prospective, randomized, comparative study. Major university teaching hospital. Sixty patients undergoing elective surgery. Patients were randomized into an alfentanil group, a high-dose remifentanil group, a low-dose remifentanil group, or a thoracic epidural group, in combination with a propofol target-controlled infusion. The study was blinded for the opioid, except in the epidural group. Tight control of perioperative hemodynamic parameters was maintained, and the postoperative management was strictly standardized. Bactericidal permeability-increasing protein as an indicator of the polymorphonuclear neutrophil response, interleukin-6 as an inducer of the acute-phase response, and lipopolysaccharide-binding protein and C-reactive protein as parameters of the acute phase response were determined at regular intervals. Ventilator dependency and analgesia were evaluated as clinical outcome measures. Interleukin-6 levels increased in all groups. Plasma levels in the epidural group were significantly higher at all time points than in the other groups. The increase in the plasma levels of bactericidal permeability-increasing protein, lipopolysaccharide-binding protein, and C-reactive protein showed the same pattern in all groups, and no significant differences among the 4 groups were observed. Supplementation of a fast-track anesthetic technique with epidural analgesia preserves hemodynamic stability and is associated with faster extubation times (p = 0.003) and less postoperative pain (p = 0.045). Thoracic epidural analgesia was associated with significantly higher levels of IL-6 throughout the study period as compared with the total intravenous anesthesia groups. The exact clinical relevance of this finding remains unclear.

The levels of bactericidal/permeability increasing protein (BPI) in body fluids

Rationale BPI is a microbicidal protein detected in neutrophil granules and recently discovered in epithelia. BPI, together with LBP (lipopolysaccharide binding protein) and CD14, binds with high affinity LPS (lipopolysaccharide). BPI is the target of autoantibodies, BPI-ANCA being found in a subset of patients with cystic fibrosis (CF), autoimmune hepatitis, and rheumatoid arthritis. To test the hypothesis that ANCA might develop related to chronic exposure to excess target antigen derived from the epithelia, we investigated the levels of BPI in body fluids. Methods BPI serum levels were measured in 41 children who were CF BPI-ANCA negative, 39 who were BPI-ANCA positive, and 40 controls. Ten eluates from CF stools and 10 controls were also investigated for BPI presence and saliva samples were screened for BPI content. Results Serum levels of BPI did not differ in the tested subgroups of BPI-ANCA negative children (58.5 ng/ml), BPI-ANCA positive children (61.8 ng/ml) or controls (56.1 ng/ml). Only 2 CF and 1 normal stool eluate had very low levels of BPI. Saliva levels of BPI were remarkably stable, 77.4-78.9 ng/ml in all tested subject, with no difference between CF and controls. Conclusions Circulating BPI is stable and does not correlate with BPI-ANCA or clinical disease status. BPI most probably of epithelial origin is consistently present in saliva.

Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus: prevalence, antigen specificity, and clinical associations.

Fifty-five patients with systemic lupus erythematosus (SLE) were examined for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF). Enzyme-linked immunosorbent assay (ELISA) for ANCA against myeloperoxidase (MPO), lactoferrin (LF), proteinase 3 (PR3), elastase (HLE), and bactericidal/permeability-increasing protein (BPI) was performed. The prevalence of ANCA by IIF was 29.1% (16/55 patients). MPO-ANCA were found in 10.9% (6/55), LF-ANCA in 18.2% (10/55), PR3-ANCA in 12.7% (7/55), BPI-ANCA in 23.6% (13/55), and HLE-ANCA in 1.8% (1/55). The levels of BPI-, LF-, and PR3-ANCA correlated with disease activity. A significant association between serositis and the presence of BPI-, LF-, and PR3-ANCA was observed, and PR3-ANCA were found to be associated with arthritis as well. Our results demonstrate that ANCA of various specificities occur in SLE, and BPI appears to be an important target antigen.

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.

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.

Changes in endotoxin-binding proteins during major elective surgery: important role for soluble CD14 in regulation of biological activity of systemic endotoxin.

Assessment of circulating endotoxin during the perioperative period, which is only demonstrated by the Limulus amebocyte lysate (LAL) test, may be modulated by several endotoxin-binding proteins. Endotoxin-neutralizing capacity (ENC) and the plasma levels of soluble CD14 (sCD14), lipopolysaccharide-binding protein, and bactericidal/permeability-increasing protein (BPI) were determined in 40 patients 6 h prior to skin incision for major abdominal surgery. The bioactivity of plasma endotoxin was tested by the polymyxin B-inhibited stimulatory activity of the plasma samples on healthy monocytes as measured by the release of tumor necrosis factor alpha. Plasma endotoxin levels in almost all patients increased from 0.05 +/- 0.01 to 0.23 +/- 0.03 experimental units (EU) per ml (P < 0.001); more specifically, 17 of 40 samples showed endotoxin levels of greater than 0.2 EU per ml and corresponding reductions in ENC. Soluble CD14 plasma levels were decreased from 5. 6 +/- 0.3 to 4.6 +/- 0.3 microg per ml (P < 0.05). ENC was strongly correlated with the sCD14 plasma concentration throughout the period of observation. The addition of sCD14-neutralizing monoclonal anti-sCD14 antibodies reduced ENC both pre- and postoperatively. No correlation could be established between ENC and the plasma levels of BPI, high-density lipoproteins, or low-density lipoproteins determined by measuring the concentrations of apoprotein A and apoprotein B. Biologically active endotoxin was found in only 6 of 17 samples with endotoxin levels greater than 0.2 EU per ml in the LAL test. These samples could be characterized by their perioperative loss of at least 35% of their sCD14. No change in sCD14 was detected in the remaining 11 samples. The perioperative loss of ENC is partly caused by the loss of sCD14 resulting from its consumption by endotoxin reaching the bloodstream. This study demonstrated the role of sCD14 on the bioactivity of circulating endotoxin in a human model of endotoxemia after major abdominal surgery.

Priming of cytokine release and increased levels of bactericidal permeability-increasing protein in the blood of animal facility workers.

To investigate cellular immune responses in laboratory animal workers who are exposed to high levels of animal allergens but also to other biologically active substances containing lipopolysaccharides (LPS), i.e., endotoxins. A survey among 20 animal facility workers and 20 matched (gender, smoking) controls was conducted using exposure measurements (endotoxin, colony-forming units of bacteria and fungi) and a questionnaire on respiratory symptoms. Blood samples were taken to determine the ex vivo whole-blood release of tumor necrosis factor-alpha (TNF) and interleukin-8 (IL-8) as well as plasma levels of LPS-binding protein (LBP), bactericidal permeability-increasing protein (BPI), the 75-kDa soluble TNF receptor (sTNFR75), and total/specific IgE. Although exposure to endotoxin was low (range 0.05-2.8 ng/m(3)), a significant (P < 0.05) increase in plasma BPI (4-fold) and srTNF75 (1.2-fold) was found, suggesting a response to inhalation of LPS. Also, the capacity of blood leukocytes to release TNF and IL-8 in response to ex vivo exposure to workplace dust was increased. Data were not confounded by specific allergies, levels of IgE, smoking, or respiratory symptoms. A profound effect on the cellular immune response was seen in animal workers with low endotoxin exposure and a high antigenic load. It remains to be determined which other biologically active substance(s) are involved in this effect.

Inflammatory bowel disease is associated with increased mucosal levels of bactericidal/permeability-increasing protein

BACKGROUND & AIMS: Clinical sepsis seldom accompanies inflammatory bowel disease. The aim of this study was to measure colonic mucosal levels of the neutrophil product bactericidal/permeability-increasing protein (BPI), which kills gram-negative bacteria in addition to inactivating endotoxin. METHODS: Enzyme-linked immunosorbent assay and immunohistochemistry for BPI were performed on homogenates and tissue secretions of biopsy specimens from patients with ulcerative colitis (n=11) and Crohn's disease (n=5) and from normal controls (n=5). RESULTS: Mucosal neutrophil content (144 +/- 23 vs. 35 +/- 9 neutrophils/mg protein; P<0.007) and BPI content (2.07 +/- 0.75 vs. 0.12 +/- 0.02 ng/mg protein; P<0.002) were greater in the colitis groups and correlated closely (r=0.68; P<0.001). This relationship held for both ulcerative colitis (P<0.002) and Crohn's disease (P<0.01) with a trend towards greater levels in Crohn's disease. There was a trend towards higher BPI levels with an increasing endoscopic inflammation score (grade I, 1.32 +/- 0.6 ng/mg protein; grade II, 2.82 +/- 1.4 ng/mg protein). Immunohistochemistry and the biopsy culture showed BPI to be both intracellular and extracellular, to be present in the crypt lumen, and to be released into incubating medium. CONCLUSIONS: Mucosal levels of BPI are increased in colitis. Such localization may ameliorate mucosal responses to gram-negative bacteria and their products. (Gastroenterology 1996 Mar;110(3):733-9)

Lipopolysaccharide Toxicity-Regulating Proteins in Bacteremia

The toxicity of lipopolysaccharide (LPS) is modified by several proteins, such as bactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP). BPI and LBP plasma levels were measured in patients with gram-negative (n = 36) or gram-positive (n = 28) bacteremia. Levels of BPI and LBP, which are proteins that neutralize and enhance LPS effects, respectively, were increased before bacteremia was first detected. The BPI/neutrophil ratio, reflecting neutrophil activation, was significantly associated with the presence of sepsis syndrome and death in bacteremic patients: 1.06 (0.11-6.49) versus 0.57 (0.06-3.82) in patients with and without sepsis syndrome (P < .01), respectively, and 0.64 (0.06-3.82) versus 1.02 (0.12-6.49) in survivors and nonsurvivors (P < .05), respectively (ratio in nanograms of BPI per 10(6) neutrophils). High LBP peak levels were significantly associated with the presence of sepsis syndrome (P < .01). No differences in BPI and LBP levels were observed in patients with gram-negative versus gram-positive bacteremia. BPI/neutrophil ratio, as a parameter of neutrophil activation, may be useful in monitoring infectious disease.

Characterization of two monoclonal antibodies directed against bactericidal/permeability-increasing protein.

In this study the production and characterization of two monoclonal antibodies (MAbs), 4E3 and 5D7, against the bactericidal/permeability-increasing protein (BPI) were described. Using ELISAs, both 4E3 and 5D7 were shown to detect recombinant (r) BPI. Furthermore, natural BPI present in polymorphonuclear leukocytes (PMNL) was detected by both 4E3 and 5D7. The use of both MAbs in flow cytometry revealed that PMNL expressed low levels of cell-surface BPI. Lipopolysaccharide (LPS) was shown to block the interaction between anti-BPI MAb and rBPI. In addition, the MAbs blocked biologic activity of rBPI. The inhibition by BPI of LPS activation of the limulus amebocyte lysate assay and of LPS-induced tumor necrosis factor-alpha release by monocytes was prevented by 4E3 and 5D7. Both MAbs are specifically directed against BPI and can inhibit BPI bioactivity.

Changes in polymorphonuclear leukocyte surface and plasma bactericidal/permeability-increasing protein and plasma lipopolysaccharide binding protein during endotoxemia or sepsis.

To evaluate changes in levels of polymorphonuclear leukocyte surface bactericidal/permeability-increasing protein (BPI), plasma BPI, and plasma lipopolysaccharide (LPS) binding protein (LBP) in normal human volunteers administered Escherichia coli LPS and in patients with sepsis and gram-negative infections. Survey; case series. Clinical research center and surgical intensive care unit of a medical school and an associated tertiary care hospital. Volunteers (n = 10) screened prior to study by history and physical examination to exclude those with underlying diseases or hematologic abnormalities. Consecutive sample of surgical intensive care unit patients (n = 10) meeting criteria for sepsis syndrome with gram-negative infection. An additional patient with systemic inflammatory response syndrome but no gram-negative infection. All patients were studied on meeting the criteria. Three of the patients with sepsis syndrome and the patient with systemic inflammatory response syndrome were evaluated on recovery (approximately 25 days after initial study). Because these studies in volunteers and patients overlapped temporally, the control values were those of volunteers evaluated prior to LPS administration. No matching was employed. Compared with controls, LPS-challenged volunteers and patients with sepsis both exhibited significant granulocytosis (P < .01) and increased concentrations of polymorphonuclear leukocyte surface BPI (P < .01) and of plasma LBP (P < .01). Plasma BPI concentrations were increased (P < .01) in volunteers following LPS administration. There was a trend toward increased concentrations of plasma BPI in patients, but this was not significant relative to controls. Maximum concentrations of plasma LBP were approximately 250- and 3000-fold higher than plasma BPI concentrations in endotoxemic volunteers and in patients, respectively. Circulating polymorphonuclear leukocytes increase expression of BPI in response to LPS or gram-negative sepsis. Subsequently, concentrations of plasma BPI and LBP increase. Because both LBP and BPI bind to LPS, it is suggested that endogenously derived plasma levels of BPI are likely to be inadequate to compete for LPS binding to the much more abundant LBP in the circulation.

Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI 23) in human body fluids. The linear range for the rBPI and rBPI 23 standard curves were 100–6000 pg/ml and 25–800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI 23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 μg/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI 23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9–72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet ( M r ≈ 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.