Plasma levels of bactericidal/permeability-increasing protein correlate with systemic inflammation in acute coronary syndrome.

Background: Neutrophils play important roles in atherosclerosis and atherothrombosis. Bactericidal/permeability-increasing protein (BPI) is mainly expressed in the granules of human neutrophils in response to inflammatory stress. This observational, cross-sectional study investigated the plasma level of BPI in patients with acute coronary syndrome (ACS) and its correlation with blood neutrophil counts and circulating inflammatory biomarkers. Methods: A total of 367 patients who had acute chest pain and who were admitted to our hospital for coronary angiography (CAG) and/or percutaneous coronary intervention (PCI) from May 1, 2020 to August 31, 2020 were recruited. Among them, 256 had a cardiac troponin value above the 99th percentile upper reference limit and were diagnosed with ACS. The remaining patients (n = 111) were classified as non-ACS. The TIMI and GRACE scores were calculated at admission. The Gensini score based on CAG was used to determine atherosclerotic burden. Plasma levels of interleukin (IL)-1ß, myeloperoxidase-DNA (MPO-DNA), high sensitivity C-reactive protein (hs-CRP), S100A8/A9, and BPI were measured using enzyme-linked immunosorbent assays. Correlations of plasma BPI levels with examination scores and levels of circulating inflammatory biomarkers were explored. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic efficacy of BPI for ACS and myocardial infarction. Results: Patients in the ACS group showed significantly higher plasma BPI levels compared to the non-ACS group (46.42 ± 16.61 vs. 16.23 ± 6.19 ng/mL, p < 0.05). Plasma levels of IL-1ß, MPO-DNA, hs-CRP, and S100A8/A9 in the ACS group were also significantly higher than those in the non-ACS group (all p < 0.05). In addition, plasma BPI levels were positively correlated with the TIMI, GRACE, and Gensini scores (r = 0.176, p = 0.003; r = 0.320, p < 0.001; r = 0.263, p < 0.001, respectively) in patients with ACS. Plasma BPI levels were also positively correlated with blood neutrophil counts (r = 0.266, p < 0.001) and levels of circulating inflammatory biomarkers (IL-1ß, r = 0.512; MPO-DNA, r = 0.452; hs-CRP, r = 0.554; S100A8/A9, r = 0.434; all p < 0.001) in patients with ACS. ROC curve analysis revealed that the diagnostic efficacy of BPI for ACS was not inferior to that of IL-1ß, MPO-DNA, hs-CRP, S100A8/A9, or blood neutrophil counts. ROC analysis also showed that the diagnostic efficacy of BPI for myocardial infarction was not inferior to that of creatine kinase (CK)-MB or cardiac troponin I. Conclusion: BPI is associated with systemic inflammation in ACS and may be involved in the process of atherosclerosis and atherothrombosis. The potential of BPI as a prognostic and diagnostic biomarker for ACS should be investigated in clinical settings.

Salmonella Typhimurium PgtE is an essential arsenal to defend against the host resident antimicrobial peptides.

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and occasionally causes systemic infection. Salmonella's ability to survive and replicate within macrophages is an important characteristic during systemic infection. The outer membrane protease PgtE of S. enterica is a member of the Omptin family of outer membrane aspartate proteases which has well-characterized proteolytic activities in-vitro against a wide range of physiologically relevant substrates. However, no study has been done so far that draws a direct correlation between these in-vitro observations and the biology of the pathogen in-vivo. The main goals of this study were to characterize the pathogenesis-associated functions of pgtE and study its role in the intracellular survival and in-vivo virulence of Salmonella Typhimurium. Our study elucidated a possible role of Salmonella Typhimurium pgtE in combating host antimicrobial peptide- bactericidal/ permeability increasing protein (BPI) to survive in human macrophages. The pgtE-deficient strain of Salmonella showed attenuated proliferation and enhanced colocalization with BPI in U937 and Thp1 cells. In the presence of polymixin B, the attenuated in-vitro survival of STM ΔpgtE suggested a role of PgtE against the antimicrobial peptides. In addition, our study revealed that compared to the wild type Salmonella, the pgtE mutant is replication-deficient in C57BL/6 mice. Further, we showed that PgtE interacts directly with several antimicrobial peptides (AMPs) in the host gut. This gives the pathogen a survival advantage and helps to mount a successful infection in the host.

Killing three birds with one BPI: Bactericidal, opsonic, and anti-inflammatory functions.

Bactericidal/permeability-increasing protein (BPI) is an anti-microbial protein predominantly expressed in azurophilic granules of neutrophils. BPI has been shown to mediate cytocidal and opsonic activity against Gram-negative bacteria, while also blunting inflammatory activity of lipopolysaccharide (LPS). Despite awareness of these functions in vitro, the magnitude of the contribution of BPI to innate immunity remains unclear, and the nature of the functional role of BPI in vivo has been submitted to limited investigation. Understanding this role takes on particular interest with the recognition that autoimmunity to BPI is tightly linked to a specific infectious trigger like Pseudomonas aeruginosa in chronic lung infection. This has led to the notion that anti-BPI autoantibodies compromise the activity of BPI in innate immunity against P. aeruginosa, which is primarily mediated by neutrophils. In this review, we explore the three main mechanisms in bactericidal, opsonic, and anti-inflammatory of BPI. We address the etiology and the effects of BPI autoreactivity on BPI function. We explore BPI polymorphism and its link to multiple diseases. We summarize BPI therapeutic potential in both animal models and human studies, as well as offer therapeutic approaches to designing a sustainable and promising BPI molecule.

Molecular characterization and functional analysis of the bactericidal permeability-increasing protein/LPS-binding protein (BPI/LBP) from roughskin sculpin (Trachidermus fasciatus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide (LPS) binding proteins (LBP) both play important roles in innate immunity against bacterial infection. Herein, we identified a novel full-length cDNA sequence of BPI/LBP from Trachidermus fasciatus (designated as TfBPI/LBP). The full-length cDNA sequence of TfBPI/LBP was 1594bp, which contains an open reading frame (ORF) of 1422bp encoding a secreted protein with 473 amino acid residues. Similar to BPI/LBPs from other teleost and mammals, the peptide of TfBPI/LBP contains an N-terminal BPI/LBP/CETP domain with an LPS-binding motif and a C-terminal BPI/LBP/CETP domain BPI2. Multiple alignments and phylogenetic analysis supported that TfBPI/LBP was a new member of the vertebrate BPI/LBP family. TfBPI/LBP gene was ubiquitously expressed in all detected tissues, with the most abundant in the liver, and could be significantly induced in the skin, blood, liver, spleen post LPS challenge. The recombinant N-terminal domain of TfBPI/LBP (designated as rTfBPI/LBPN) was successfully expressed in Escherichia coli. Sugar binding assay showed that rTfBPI/LBPN could bind to LPS, peptidoglycan (PGN), and lipoteichoic acid (LTA), with the highest affinity to LPS. The results of bacteria binding and agglutinating assay revealed that rTfBPI/LBPN could bind and agglutinate to all of the 9 kinds of bacteria we used. Moreover, membrane integrity analysis indicated that rTfBPI/LBPN could increase the membrane permeability of bacteria. These results suggested that BPI/LBP may play crucial roles in host defense against microorganisms, possibly through non-selective bacterial recognition and induction of membrane penetration.

Bactericidal/Permeability-Increasing Protein Preeminently Mediates Clearance of Pseudomonas aeruginosa In Vivo via CD18-Dependent Phagocytosis.

Chronic Pseudomonas aeruginosa infection mysteriously occurs in the airways of patients with cystic fibrosis (CF), bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD) in the absence of neutrophil dysfunction or neutropenia and is strongly associated with autoimmunity to bactericidal permeability-increasing protein (BPI). Here, we define a critical role for BPI in in vivo immunity against P. aeruginosa. Wild type and BPI-deficient (Bpi-/-) mice were infected with P. aeruginosa, and bacterial clearance, cell infiltrates, cytokine production, and in vivo phagocytosis were quantified. Bpi-/- mice exhibited a decreased ability to clear P. aeruginosa in vivo in concert with increased neutrophil counts and cytokine release. Bpi-/- neutrophils displayed decreased phagocytosis that was corrected by exogenous BPI in vitro. Exogenous BPI also enhanced clearance of P. aeruginosa in Bpi-/- mice in vivo by increasing P. aeruginosa uptake by neutrophils in a CD18-dependent manner. These data indicate that BPI plays an essential role in innate immunity against P. aeruginosa through its opsonic activity and suggest that perturbations in BPI levels or function may contribute to chronic lung infection with P. aeruginosa.

Increased levels of BPI-ANCA in patients with primary Sjögren's syndrome are associated with lung involvement.

BACKGROUND: The goal of this study was to investigate the association between bactericidal permeability increasing (BPI)-antineutrophil cytoplasmic antibody (ANCA) protein levels and primary Sjogren's syndrome (pSS) with lung involvement, as well as the potential diagnostic performance of BPI-ANCA. METHODS: The levels of BPI-ANCA in pSS patients with (n = 36) and without (n = 85) lung involvement were measured using a commercial ELISA kit. Serological biomarkers and cytokines were measured in these patients as well. Lung involvement was determined by high-resolution computed tomography (HRCT) and/or clinical symptoms. The diagnostic performance of lung involvement was determined by receiver operating characteristic (ROC) curves. RESULTS: The percentage of neutrophils (NEUT%), neutrophil-lymphocyte ratio (NLR), erythrocyte sedimentation rate (ESR), and the levels of BPI-ANCA, C-reactive protein (CRP), interleukin-2 (IL-2) and IL-6 exhibited an upward trend, while the percentage of lymphocytes (LYMP%) and albumin (ALB) level exhibited a downward trend in the lung involvement group. The combination of BPI-ANCA, NEUT% and ALB significantly increased the area under the ROC curve (AUC) to 0.837 (95% confidence interval: 0.742-0.907, sensitivity: 82.14%, specificity: 81.36%, P < 0.001). CONCLUSIONS: Increased BPI-ANCA was found in pSS patients with lung involvement and was associated with inflammation. A combination of BPI-ANCA, NEUT% and ALB had the best AUC, and may serve as anadjunct to distinguish between pSS patients with and without lung involvement.

Dissociation of systemic and mucosal autoimmunity in cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa accounts for ~80% of cystic fibrosis (CF) airway infection. It shows a remarkable correlation with presence of autoantibody to bactericidal/permeability-increasing protein (BPI), which is not understood. In this study, we sought to better understand the characteristics of systemic and mucosal autoimmunity and their relation to humoral immunity to P. aeruginosa. METHODS: Antibody titers and isotypes to BPI and P. aeruginosa were characterized in sera and bronchoalveolar lavage (BAL) of adult and pediatric CF patients (n = 131), by ELISA and/or immunoblot. RESULTS: Serum BPI autoantibodies were common (~43%) in adult while rare (≪5%) in pediatric (≤18 yrs) CF patients. Serum BPI IgG autoantibodies were of high avidity and strongly correlated with anti-P. aeruginosa IgG responses. A parallel relationship was observed with IgA, but not IgG, responses in adult and pediatric CF patient in the BAL. Thus, BAL IgA anti-BPI antibodies were independent of age and correlated with the presence of BPI cleavage in BAL. CONCLUSIONS: IgG and IgA autoreactivity to BPI in CF patients was demonstrated in serum and BAL, respectively, and correlated with the isotype of the antibody response to P. aeruginosa. The co-occurrence of anti-BPI and anti-P. aeruginosa IgA in the BAL, but not serum, of pediatric CF patients suggests that BPI tolerance is broken in the P. aeruginosa-infected airway and that serologic IgG autoantibodies are later induced, potentially through a separate pathway. The relationship between P. aeruginosa, BPI cleavage, and IgA autoantibodies in the BAL suggests a role for cryptic epitope generation in the breaking of tolerance.

Calcium-activated chloride channel regulator 1 (CLCA1): More than a regulator of chloride transport and mucus production.

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl- transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases.

The Pathogenicity of BPI-ANCA in a Patient With Systemic Vasculitis.

Objective: ANCA associated vasculitis (AAV) is characterized by systemic necrotizing vasculitis with the presence of ANCA. Although BPI-ANCA is one of the atypical ANCAs and is occasionally seen in patients with vasculitis, the pathogenicity of BPI-ANCA remains unclear. This study was performed to examine the pathogenic role of BPI-ANCA against neutrophils. Methods: A 76-year-old Japanese man showed BPI-ANCA positive systemic vasculitis with a medical history of Pseudomonas aeruginosa infection. BPI-ANCA IgGs were eluted from the patient serum using an immunoadsorbent column. In vitro experiment, healthy donor neutrophils were treated with BPI-AAV IgGs, MPO-AAV IgGs, healthy control IgGs under TNFα stimulation. After 3 h incubation, neutrophil extracellular trap (NET) was assessed by immunofluorescent imaging. To determine the pathogenicity of BPI-ANCA, TNFα-primed neutrophils were incubated with monoclonal BPI-ANCA in the presence or absence of recombinant BPI. Results: BPI-AAV IgGs-treated neutrophils showed NET formation with histone citrullination. Interestingly, the monoclonal BPI-ANCA did not induce NET, but the immune complexes (ICs) of recombinant BPI and BPI-ANCA induced TNFα-dependent NET formation with hypercitrullination. Furthermore, TNFα increased the expression of BPIs in neutrophils and the BPIs were translocated to cell surface. Conclusion: BPI-ANCA could affect neutrophils leading to NET formation and may play a role in the development of systemic vasculitis as pathogenic autoantibody.

Molecular identification and function analysis of bactericidal permeability-increasing protein/LPS-binding protein 1 (BPI/LBP1) from turbot (Scophthalmus maximus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.

rBPI21 (Opebacan) Promotes Rapid Trilineage Hematopoietic Recovery in a Murine Model of High-Dose Total Body Irradiation.

The complexity of providing adequate care after radiation exposure has drawn increasing attention. While most therapeutic development has focused on improving survival at lethal radiation doses, acute hematopoietic syndrome (AHS) occurs at substantially lower exposures. Thus, it is likely that a large proportion of such a radiation-exposed population will manifest AHS of variable degree and that the medical and socioeconomic costs of AHS will accrue. Here, we examined the potential of rBPI21 (opebacan), used without supportive care, to accelerate hematopoietic recovery after radiation where expected survival was substantial (42-75%) at 30 days). rBPI21 administration was associated with accelerated recovery of hematopoietic precursors and normal marrow cellularity, with increases in megakaryocyte numbers particularly marked. This translated into attaining normal trilineage peripheral blood counts 2-3 weeks earlier than controls. Elevations of hematopoietic growth factors observed in plasma and the marrow microenvironment suggest the mechanism is likely multifactorial and not confined to known endotoxin-neutralizing and cytokine down-modulating activities of rBPI21 . These observations deserve further exploration in radiation models and other settings where inadequate hematopoiesis is a prominent feature. These experiments also model the potential of therapeutics to limit the allocation of scarce resources after catastrophic exposures as an endpoint independent of lethality mitigation. This article is protected by copyright. All rights reserved.

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin.

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.

Identification and expression analysis on bactericidal permeability-increasing protein (BPI)/lipopolysaccharide-binding protein (LBP) of ark shell, Scapharca broughtonii.

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are the numbers of the lipid transfer protein/lipopolysaccharide-binding protein family and play crucial roles in the innate immune response to Gram-negative bacteria. A novel Sb-BPI/LBP1 from ark shell Scapharca broughtonii was isolated by expressed sequence tag (EST) and RACE techniques. The Sb-BPI/LBP1 cDNA encoded a polypeptide of 484 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 463 amino acids. The deduced amino acid sequence of Sb-BPI/LBP1 contained an N-terminal BPI/LBP/CETP domain BPI1 with three functional regions that display LPS-binding activity, and a C-terminal BPI/LBP/CETP domain BPI2. In structure and sequence, Sb-BPI/LBP1 showed highly similar to those of the BPI/LBPs from invertebrate and non-mammalian vertebrate, the LBPs and BPIs from mammal. By quantitative real-time RT-PCR, Sb-BPI/LBP1 transcripts could be detected in all normal tested tissues, including hepatopancreas, adductor muscle, mantle margin, heart, gonad, gill and hemocytes, and was universally up-regulatable at 24 h post LPS challenge. The mRNA expression of Sb-BPI/LBP1 in hemocytes was the most sensitive to LPS challenge, significantly up-regulated at 12 h post LPS challenge and peaked at 24 h (16.76-fold, P < 0.05). These results suggested that Sb-BPI/LBP1 was a constitutive and inducible acute-phase protein contributing to the host immune defense against Gram-negative bacterial infection in ark shell S. broughtonii.

Bactericidal/permeability increasing protein: a multifaceted protein with functions beyond LPS neutralization.

Bactericidal permeability increasing protein (BPI), a 55-60 kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

Secretory expression of BPI_(m23) recombinant bactericidal protein in Pichia pastoris / 中国免疫学杂志

Objective:To express and secrete rBPI m23 bactericidal protein in Pichia pastoris expression system. Methods:pPICZ? synBPI m600 recombinant expression vector was constructed with prepared vectors (pUC18 synBPI 414 and PBV220 BPI m420 ) according to Molecular Cloning Laboratory Manual. The Pichia pastoris GS115 were electroporated with linearized pPICZ? synBPI m600 vectors , positive clones were screened on low salt LB medium with Zeocin , then the positive clones were identified by PCR and Mut phenotype analysis. rBPI m23 was induced to express in BMMY ,purified by SP Sepharose beads , identified by SDS PAGE and Western blot and measured with BCA method. Results:①pPICZ? synBPI m600 expression vector was successfully constructed.②Pichia pastoris GS115 strains integrated with synBPI m600 stably were obtained.③SDS PAGE showed the molecular weight of the recombinant protein was approximate 23 kD,and Western blot confirmed that the protein could bind to the anti BPI antibody specially.④The supernate protein concentration was about 2 9 mg/L.Conclusion:rBPI m23 was expressed and secreted effectively in Pichia pastoris GS115.