Gram-negative Bacterial Endotoxin Research Articles

PAC1 Receptor

Septic shock is a systemic response to severe bacterial infections, generally caused by Gram-negative bacterial endotoxins, with multiple manifestations such as hypotension, tissue injury, disseminated intravascular coagulation, and multi-organ failure. All these effects, are induced by the generation of pro-inflammatory and vasodilator mediators, cell adhesion molecules, coagulation factors, and acute-phase proteins. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two immunopeptides with anti-inflammatory properties exerted through type 1 and 2 VIP receptors (VPAC1 and VPAC2, respectively), and PACAP receptor (PAC1). The present results recapitulate the protective role of PAC1 in an experimental model of lethal endotoxemia using a knockout for the PAC1 receptor. Our results demonstrate that VIP and PACAP decrease lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production, neutrophil infiltration and intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and fibrinogen expression through PAC1 receptor, providing an advantage to design more specific drugs complementing standard intensive care therapy in septic shock.

Biological contamination parameters of cotton lint as biomarkers for fibre quality; a preliminary study

It has been reported for several decades that microbes, which naturally contaminate cotton fibres during crop growth and subsequent storage can have an adverse effect on the structural quality of cotton lint. Although several studies have analysed the relationship between numbers of Gram-negative bacteria or bacterial endotoxin and particular physical properties, these studies have been limited to cotton from the United States, and the possible effects of fungal contamination have not been examined in detail. This study quantified the Gram-negative bacteria and fungal cells, as well as measuring concentrations of bacterial endotoxin and fungal glucan, on cotton lint samples from international sources. Spearman’s rank correlation coefficients calculated between these results and quality data analysed by an automated testing instrument revealed several significant correlations. Findings included inverse correlations between the biological contamination parameters and fibre elongation, micronaire and reflectance. The possible causes and implications of these findings were also discussed.

Molecular Basis of Reduced Potency of Underacylated Endotoxins

Potent TLR4-dependent cell activation by gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.

Staphylococcus aureus lipotechoic acid induces differential expression of bovine serum amyloid A3 (SAA3) by mammary epithelial cells: Implications for early diagnosis of mastitis

Mastitis is one of the most costly diseases of agriculturally important animals and is a common problem for lactating cows. Current methods used to detect clinical and especially subclinical mastitis are either inadequate or problematic. Pathogens such as the gram-positive bacterium Staphylococcus aureus or the gram-negative bacterium Escherichia coli typically cause mastitis. E. coli induces clinical mastitis, whereas, S. aureus causes a subclinical, chronic infection of the mammary gland. In this study we report the differential expression and secretion of mammary-derived serum amyloid A3 (SAA3) by bovine mammary epithelial cells following stimulation with the S. aureus cell wall component, lipotechoic acid (LTA). Two-dimensional immunoblot analyses confirmed that bovine SAA3 is the predominant SAA isoform produced by LTA stimulated mammary epithelial cells. Our previous study showed that bovine SAA3 is also differentially expressed in response to the gram-negative bacterial endotoxin lipopolysaccharide. Collectively, these data indicate that the local production of SAA3 by mammary epithelial cells in response to either gram-positive or gram-negative bacterial components may provide a sensitive indicator for early detection and treatment of mastitis in vivo, minimizing chronic cases of infection, the spread of mastitis to other animals, and economic losses.

Monomeric endotoxin:protein complexes are essential for TLR4-dependent cell activation

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin—protein and protein—protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (Eagg) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14 is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOSagg) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.

Monomeric endotoxin:protein complexes are essential for TLR4-dependent cell activation

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin?protein and protein?protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (E(agg)) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOS(agg)) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.

The effect of lipopolysaccharide (LPS) on cord-blood hemopoietic progenitors

RATIONALE: The Hygiene Hypothesis has focused attention on the interaction of the innate immune system and the microbial environment. Since hemopoietic mechanisms are involved in the maintenance and development of atopy, we studied the effect of the gram-negative bacterial endotoxin, lipopolysaccharide, on cord-blood hemopoietic progenitors. METHODS: Non-adherent mononuclear (NAMNC) human cord blood cells were enriched for CD34+ hemopoietic progenitors using magnetic activated cell sorting (MACS), either via depletion of CD3+/CD16+ cells (n=9) or via positive CD34+ selection (n=14), then incubated for 24 hours in varying doses of LPS (0-, 0.01, 0.1, 1 and 10 mcg/ml), followed by washing and incubation in methylcellulose colony-forming assays (CFU) in the presence of IL-3, IL-5 or GM-CSF. Eosinophil/basophil (Eo/B) CFU were enumerated at 14 days, and analysis performed using Friedman's ANOVA. RESULTS: There was a significantly positive dose-response of Eo/B CFU to LPS using either enrichment method for CD34+ cord blood populations. For CD3/CD16 depleted NAMNC, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (2.5 compared to 11.5 CFU/2x105 cells plated, p= 0.018); overall LPS effect (p=0.047). For CD34-selected cells, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (3 compared to 8.25 CFU/104 cells plated, p= 0.033); overall LPS effect (p=0.048). CONCLUSIONS: CB NAMNC, specifically CD34+ cells, are capable of responding to LPS and hemopoietic cytokines functionally, by differentiating into mature eosinophils and basophils. The findings highlight a potential mechanism through which microbial stimuli could directly modulate atopy and/or allergic inflammation.

Hyperbaric oxygen protects against lipopolysaccharide-stimulated oxidative stress and mortality in rats

Free radicals and proinflammatory mediators have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. Hyperbaric oxygen is used as an adjuvant therapy for various inflammatory diseases and shows beneficial effects in lipopolysaccharide-induced shock syndrome. However, the underlying mechanisms for these effects are still to be defined. In this study, we investigated the effect of hyperbaric oxygen on inflammatory mediators, free radicals, and mortality in endotoxic rats. Wistar–Kyoto rats were injected with lipopolysaccharide (10 mg/kg) and then exposed to aminoguanidine, an inhibitor of inducible nitric oxide (NO) synthase (bolus injection 2 h after lipopolysaccharide), or hyperbaric oxygen (2 ATA for 60 min 1, 4, 9, and 24 h after lipopolysaccharide). Plasma tumor necrosis factor alpha (TNF-α), NO, and superoxide anion were detected and the vasorelaxation response and survival rate were assessed. The results demonstrated that increases in plasma TNF-α and NO, and the vasohyporeactivity induced by lipopolysaccharide treatment were significantly inhibited by hyperbaric oxygen and aminoguanidine. Mortality and vascular superoxide anion production of lipopolysaccharide treatment were also markedly reduced by hyperbaric oxygen treatment, but were not restored by aminoguanidine. None of the parameters was changed by hyperbaric oxygen treatment alone. Thus, repeated hyperbaric oxygen exposure significantly attenuated the inflammatory mediators, free radicals, and mortality in endotoxic rats.

Comprehending the role of LPS in Gram-negative bacterial vaginosis: ogling into the causes of unfulfilled child-wish.

Intrauterine infection is frequently associated with pregnancy loss in pregnant women. This article reviews the role of Gram-negative bacterial infection in various complications related to early pregnancy and subsequent pregnancy loss. Here we discuss the pathways of ascending intrauterine infection, microbiology and the pathophysiology of such infections. The clinical impact, therapy, consequences, prevention and implications of Gram-negative bacterial infections in women during their reproductive life span is also discussed. This article also makes an attempt to discuss our studies and findings, related to the effect of the LPS component of the Gram-negative bacterial endotoxin on preimplantation stage embryonic development and implantation. This early phase of pregnancy remains mostly unnoticed by the mother as well as the health care provider, and therefore holds more threat to the life of the fetus and the mother. The molecular mechanisms of LPS-induced pregnancy losses through abnormal embryonic development, implantation failure, and preterm labor and birth with specific references to the role of proinflammatory cytokines like IL-1 and TNF are discussed. Once these inflammatory mediators have increased in the feto-maternal tissues, it may be too late or harmful to try and prevent the adverse outcomes of pregnancy.

Pharmacodynamics of Isoflupredone Acetate in an Endotoxin-Induced Mastitis Model

The effect of intravenous administration of the steroidal drug isoflupredone acetate on lactating dairy cows with mastitis induced using gram-negative bacterial endotoxin was investigated. Cows were randomly assigned to one of four treatment groups: untreated controls, isoflupredone acetate only, mastitis only, and mastitis plus isoflupredone acetate. Isoflupredone acetate was given to treated groups at a dose of 20mg intravenously, once. Mastitic cows receiving treatment were given isoflupredone acetate after the development of clinical signs. When compared with untreated mastitic controls, cows with endotoxin-induced mastitis treated with isoflupredone acetate did not exhibit measurable differences in heart rate, rectal temperature, rumen motility, or changes in mammary gland surface area in the 14h following the administration of intramammary endotoxin. Healthy cows treated with isoflupredone acetate had a higher heart rate over the 14h after drug administration than did untreated healthy controls. When compared with untreated mastitic controls, cows treated with isoflupredone acetate did not exhibit statistically significant differences in milk production following endotoxin-induced mastitis.

Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress.

Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS.

Byssinosis among jute mill workers.

Although byssinosis in jute mill workers remains controversial, studies in a few jute mills in West-Bengal, India, revealed typical byssinotic syndrome associated with acute changes in FEV1 on the first working day after rest. The present study on 148 jute mill workers is reported to confirm the occurrence of byssinosis in jute mill workers. Work related respiratory symptoms; acute and chronic pulmonary function changes among exposed workers were studied on the basis of standard questionnaire and spirometric method along with dust level, particle mass size distributions and gram-negative bacterial endotoxins. The pulmonary function test (PFT) changes were defined as per the recommendation of World Health Organization and of Bouhys et al. Total dust in jute mill air were monitored by high volume sampling, technique (Staplex, USA), Andersen cascade impactor was used for particle size distribution and personal exposure level was determined by personal sampler (Casella, London). Endotoxin in airborne jute dust was analysed by Lymulus Amebocyte Lysate (LAL) "Gel Clot" technique. Batching is the dustiest process in the mill. Size distribution showed that about 70-80% dust in diameter of < 10 microm, 40-50%, < 5 microm and 10-20%, < 2 microm. Mean endotoxin levels found in hatching, spinning and weaving, and beaming were 2.319 microg/m3, 0.956 microg/ m3, 0.041 microg/m3 respectively and are comparable to the values obtained up to date in Indian cotton mills. Respiratory morbidity study reported typical byssinotic symptoms along with acute post shift FEV1 changes (31.8%) and chronic changes in FEV1 (43.2%) among exposed workers. The group with higher exposure showed significantly lower FVC, FEV1, PEFR and FEF25-75% values. The study confirmed the findings of the earlier studies and clearly indicated that the Indian jute mill workers are also suffering from byssinosis as observed in cotton, flask and hemp workers.

Effect of bacteria and antibiotic treatment on whole blood-mediated endothelial permeability in vitro

Abstract Background During Gram-negative sepsis bacterial endotoxin or lipopolysaccharide (LPS) may activate and damage endothelial cells resulting in systemic vascular leakage. Antibiotic treatment of Gram-negative bacteria results in significant release of LPS, inducing production of cytokines by leucocytes in vitro. The biological relevance of this finding is not well understood. A whole blood assay and cultured endothelial monolayers have been used to evaluate the effect of antibiotic treatment of bacteria on endothelial permeability. Methods Heat-inactivated (HI) Escherichia coli (ATCC 25922) was treated with either cefuroxime (CXM), imipenem–cilastin (IPM) or polymyxin B (poly B). The cultures were incubated with heparinized whole blood from healthy donors in a final concentration of 105 colony-forming units ml−1 and incubated at 37°C for 24 h. Plasma was then collected and added to human umbilical vein endothelial cell monolayers cultured on semipermeable Transwell COL membranes. The permeability of the monolayers was determined using fluorescein isothiocyanate-labelled (FITC) albumin and rhodamine-β-isothiocyanate-labelled (RITC) dextran. The production of interleukin (IL) 1β, IL-6, IL-1ra and tumour necrosis factor (TNF) α in whole blood was measured using radioimmunoassay. Results In whole blood, HI bacteria alone induced the production of IL-1β, IL-6, IL-1ra and TNF-α. CXM treatment of HI E. coli before incubation in blood induced significantly more IL-1β, TNF-α and IL-6 than the HI control, while poly B treatment induced less cytokine production. IPM did not significantly alter the production of cytokines. In the permeability assay, whole blood treated with HI bacteria alone induced a permeability increase of 2·3 times control monolayer permeability (P &amp;lt; 0·05). While CXM or IPM treatment resulted in a significant further enhancement of the permeability, there was no significant difference between these antibiotic treatments (permeability increase 2·9 and 2·8 times the control respectively). In contrast, treatment with poly B almost completely abrogated the permeability effect of HI bacteria (1·1 times control; P &amp;gt; 0·05). Conclusion Since neutralization of LPS by poly B reduces the E. coli-induced production of cytokines in whole blood and prevents increased permeability, free LPS may be the driving force of cytokine-mediated endothelial permeability. Treatment of bacteria with CXM or IPM induces a significantly different cytokine production in whole blood. However, in both cases the resulting plasma leads to a similar increase in permeability. Thus, the fact that individual antibiotics can release different amounts of LPS may be irrelevant to endothelial permeability.

Defensins' Strategies

The innate immune system responses rapidly to pathogens, either by obstruction and direct killing of microbes or by activation of the adaptive arm of the immune system. Defensins have feet in both camps, and two studies add insight into how they work (see the Perspective by Ganz). Although chemokines play a role in the inhibition of human immunodeficiency virus-type 1 (HIV-1) replication by CD8 T cells, the full identity of the factors involved has been elusive. Zhang et al. used mass spectrometry and protein chip technology to examine culture supernatants from CD8 T cells isolated from patients who are long-term nonprogressors to AIDS. On the basis of amino acid sequencing and antibody recognition, they identified a set of defensins that only appeared upon T cell activation. Antibodies to these molecules blocked viral inhibition by CD8, and commercial preparations of α-defensins 1 and 2 inhibited different HIV-1 isolates. Biragyn et al. find that one defensin, β-defensin 2, could activate dendritic cells (DCs) by binding Toll-like receptor-4 (TLR-4), a cell-surface pattern-recognition protein hitherto considered to be limited to recognizing pathogen-derived molecules, such as the Gram-negative bacterial endotoxin, lipopolysaccharide (LPS). In DCs, the interaction of β-defensin 2 with TLR-4 induced much the same cellular activation program as LPS, with the stimulation of costimulatory molecule and proinflammatory cytokine expression. T. Ganz, Versatile defensins. Science 298 , 977-979 (2002). [Summary] [Full Text] L. Zhang, W. Yu, T. He, J. Yu, R. E. Caffrey, E. A. Dalmasso, S. Fu, T. Pham, J. Mei, J. J. Ho, W. Zhang, P. Lopez, D. D. Ho, Contribution of human α-defensin-1, -2, and -3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 298 , 995-1000 (2002). [Abstract] [Full Text] A. Biragyn, P. A. Ruffini, C. A. Leifer, E. Klyushnenkova, A. Shakhov, O. Chertov, A. K. Shirakawa, J. M. Farber, D. M. Segal, J. J. Oppenheim, L. W. Kwak, Toll-like receptor 4-dependent activation of dendritic cells by β-defensin 2. Science 298 , 1025-1029 (2002). [Abstract] [Full Text]

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.

Follow-up study of respiratory health of newly-hired female cotton textile workers.

Numerous studies have investigated adverse effects of exposure to cotton dust on respiratory health, but very limited longitudinal data are available with regard to the early pulmonary response to cotton dust. Moreover, the adverse effects of occupational exposure to cotton dust have been difficult to separate from the confounding effects of smoking. This setting provided a unique opportunity to evaluate early respiratory effects in newly hired and non-smoking female textile workers. To identify early pulmonary responses to cotton dust exposure and associated gram-negative bacterial endotoxin, respiratory symptoms and pulmonary function in 225 newly-hired textile workers were assessed at work initiation, and at three and twelve months later. All the workers were females and nonsmokers, with an average age of 18 years. Symptom incidence at three months was 3.6% for usual cough with phlegm, and 6.7% for usual dry cough. Lung function changes were detectable at one year: FEV1 declined by 70 ml and FVC by 124 ml over the year, and workers reporting respiratory symptoms at three months showed a significantly greater cross-shift drop in FEV1 (- 2.3%) than those without the symptoms (- 0.7%). These results suggest that the occurrence of respiratory symptoms represents the earliest response to cotton dust exposure, followed by lung function changes. Early respiratory symptoms may be a risk factor for subsequent loss of pulmonary function in cotton textile workers.

Cloning of anti-lPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro.

In this article we report the cloning and expression of a cDNA encoding Tachypleus anti-lipopolysaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxins. First, two degenerate primers were designed based on the sequence homology of anti-LPS factors purified from different species of horseshoe crab. The total RNA was extracted from amebocytes of Tachypleus tridentatus. The cDNA was then obtained by using the RT-PCR methods. Second, the cDNA of Tachypleus anti-LPS factor (TALF) was expressed in Bombyx mori larvae using baculovirus expression system, which showed a yield of up to 600 mg/L. Last, we determined the biological activity of the recombinant proteins by LPS neutralization assay and bacteriostatic assay in vitro.

The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies

The bactericidal/permeability-increasing protein (BPI) is an endotoxin-binding neutrophil leukocyte-granule protein with antibacterial and anti-endotoxin properties. A recombinant form of BPI (rBPI21) has been developed and is being tested as a therapeutic agent to treat gram-negative bacterial infections and exposure to gram-negative bacterial endotoxin. BPI is also a target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA). BPI-ANCA are present in cystic fibrosis, inflammatory bowel disease, vasculitis, and primary sclerosing cholangitis; presence of BPI-ANCA appears associated with a higher inflammatory disease activity and greater organ damage. BPI-ANCA as well as ANCA directed at other neutrophil-granule proteins may exacerbate inflammation by nonspecific effects of extracellular and cell-associated immune complexes. BPI-ANCA may further worsen inflammation by reducing the ability of BPI to promote clearance of gram-negative bacteria and bacterial-associated endotoxin.

CD14(+) cells are necessary for increased survival of eosinophils in response to lipopolysaccharide.

There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness. Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma. Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro. In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria. Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X. In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival. Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS. However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h. These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population. It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type.

The Functional Association of Polymyxin B with Bacterial Lipopolysaccharide Is Stereospecific: Studies on Polymyxin B Nonapeptide

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.