scholarly journals N-Linked Glycosylation Is Required for C1 Inhibitor-Mediated Protection from Endotoxin Shock in Mice

2004 ◽  
Vol 72 (4) ◽  
pp. 1946-1955 ◽  
Author(s):  
Dongxu Liu ◽  
Xiaogang Gu ◽  
Jennifer Scafidi ◽  
Alvin E. Davis
Keyword(s):  
Lipid A ◽  
Direct Interaction ◽  
Fluorescein Isothiocyanate ◽  
Endotoxin Shock ◽  
Enzyme Linked Immunosorbent Assay ◽  
C1 Inhibitor ◽  
Necrosis Factor Alpha ◽  
Amino Terminal ◽  
Monophosphoryl Lipid A ◽  
Lps Binding

ABSTRACT C1 inhibitor (C1INH) prevents endotoxin shock in mice via a direct interaction with lipopolysaccharide (LPS). This interaction requires the heavily glycosylated amino-terminal domain of C1INH. C1INH in which N-linked carbohydrate was removed by using N-glycosidase F was markedly less effective in protecting mice from LPS-induced lethal septic shock. N-deglycosylated C1INH also failed to suppress fluorescein isothiocyanate (FITC)-LPS binding to and LPS-induced tumor necrosis factor alpha mRNA expression by the murine macrophage-like cell line, RAW 264.7, and cells in human whole blood. In an enzyme linked immunosorbent assay, the N-deglycosylated C1INH bound to LPS very poorly. In addition, C1INH was shown to bind to diphosphoryl lipid A (dLPA) but only weakly to monophosphoryl lipid A (mLPA). As with intact LPS, binding of N-deglycosylated C1INH to dLPA and mLPA was diminished in comparison with the native protein. Removal of O-linked carbohydrate had no effect on any of these activities. Neither detoxified LPS, dLPA, nor mLPA had any effect on the rate or extent of C1INH complex formation with C1s or on cleavage of the reactive center loop by trypsin. These data demonstrate that N-linked glycosylation of C1INH is essential to mediate its interaction with the LPA moiety of LPS and to protect mice from endotoxin shock.

scholarly journals N-Linked Glycosylation at Asn3 and the Positively Charged Residues within the Amino-Terminal Domain of the C1 Inhibitor Are Required for Interaction of the C1 Inhibitor with Salmonella enterica Serovar Typhimurium Lipopolysaccharide and Lipid A

2005 ◽  
Vol 73 (8) ◽  
pp. 4478-4487 ◽  
Author(s):  
Dongxu Liu ◽  
Cort C. Cramer ◽  
Jennifer Scafidi ◽  
Alvin E. Davis
Keyword(s):  
Amino Acid ◽  
Lipid A ◽  
Amino Acid Residues ◽  
C1 Inhibitor ◽  
Gram Negative ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain ◽  
Positively Charged ◽  
Lps Binding

ABSTRACT The C1 inhibitor (C1INH), a plasma complement regulatory protein, prevents endotoxin shock, at least partially via the direct interaction of its amino-terminal heavily glycosylated nonserpin region with gram-negative bacterial lipopolysaccharide (LPS). To further characterize the potential LPS-binding site(s) within the amino-terminal domain, mutations were introduced into C1INH at the three N-linked glycosylation sites and at the four positively charged amino acid residues. A mutant in which Asn3 was replaced with Ala was markedly less effective in its binding to LPS, while substitution of Asn47 or Asn59 had little effect on binding. The mutation of C1INH at all four positively charged amino acid residues (Arg18, Lys22, Lys30, and Lys55) resulted in near-complete failure to interact with LPS. The C1INH mutants that did not bind to LPS also did not suppress LPS binding or LPS-induced up-regulation of tumor necrosis factor alpha mRNA expression in RAW 264.7 macrophages. In addition, the binding of C1INH mutants to diphosphoryl lipid A was decreased in comparison with that of recombinant wild-type C1INH. Therefore, the interaction of C1INH with gram-negative bacterial LPS is dependent both on the N-linked carbohydrate at Asn3 and on the positively charged residues within the amino-terminal domain.

scholarly journals C1 Inhibitor Prevents Endotoxin Shock Via a Direct Interaction with Lipopolysaccharide

2003 ◽  
Vol 171 (5) ◽  
pp. 2594-2601 ◽  
Author(s):  
Dongxu Liu ◽  
Shenghe Cai ◽  
Xiaogang Gu ◽  
Jennifer Scafidi ◽  
Xiao Wu ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Endotoxin Shock ◽  
C1 Inhibitor

scholarly journals Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) ofPlasmodium falciparumin BALB/c Mice

2019 ◽  
Vol 87 (6) ◽  
Author(s):  
Sakineh Pirahmadi ◽  
Sedigheh Zakeri ◽  
Akram A. Mehrizi ◽  
Navid D. Djadid ◽  
Abbas-Ali Raz ◽  
...  
Keyword(s):  
Lipid A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vaccine Adjuvants ◽  
Content Type ◽  
Quillaja Saponaria ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Cpg Oligodeoxynucleotide ◽  
Membrane Feeding ◽  
Functional Antibodies

ABSTRACTPlasmodium falciparumcell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, andQuillaja saponariaMolina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity ofEscherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in femaleAnopheles stephensimosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development inA. stephensi. However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) inA. stephensi. It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

scholarly journals Modulation of Lipopolysaccharide-Induced Monocyte Activation by Heparin-Binding Protein and Fucoidan

1998 ◽  
Vol 66 (12) ◽  
pp. 5842-5847 ◽  
Author(s):  
Michael Heinzelmann ◽  
Hiram C. Polk ◽  
Frederick N. Miller
Keyword(s):  
Lipid A ◽  
Binding Protein ◽  
Divalent Cations ◽  
Fluorescein Isothiocyanate ◽  
Scavenger Receptors ◽  
Supporting Evidence ◽  
Heparin Binding ◽  
Necrosis Factor Alpha ◽  
Heparin Binding Protein ◽  
Tnf Α

ABSTRACT Activated polymorphonuclear leukocytes release heparin-binding protein (HBP; also known as CAP37 or azurocidin) from azurophilic granules. HBP is a strong chemoattractant for monocytes that also prolongs monocyte survival and potentiates endotoxin (lipopolysaccharide [LPS])-induced production of tumor necrosis factor alpha (TNF-α). We investigated the binding of fluorescein isothiocyanate-conjugated HBP to human monocytes in the presence of EDTA and the polysaccharide fucoidan. EDTA, which chelates divalent cations, has been widely used to study the role of divalent cations in receptor-ligand interactions or enzyme activity. Fucoidan has been used to inhibit the binding of various ligands to scavenger receptors or selectins. Scavenger receptors are multiligand receptors that mediate endocytosis of proteases, protease-inhibitor complexes, lipoproteins, and LPS-lipid A. Fucoidan also interferes with leukocyte rolling by binding to L-selectins (expressed on leukocytes) and P-selectins (expressed on platelets and endothelium). We demonstrate that the binding of the neutrophil-derived protein HBP to monocytes is inhibited in the presence of EDTA and fucoidan. In addition, fucoidan and EDTA abrogate the enhancing effect of HBP on LPS-induced TNF-α production. These data provide supporting evidence that HBP binds to a receptor expressed on monocytes. This receptor is dependent on divalent cations and is possibly related to the scavenger receptor. Furthermore, we demonstrate that fucoidan, by itself, stimulates TNF-α release from isolated monocytes in a CD14-independent fashion. This is an important finding for the interpretation of results from studies that use fucoidan to “block” either scavenger receptors or L- or P-selectins.

scholarly journals Adjuvant Activity of Monophosphoryl Lipid A for Nasal and Oral Immunization with Soluble or Liposome-Associated Antigen

2000 ◽  
Vol 68 (10) ◽  
pp. 5509-5516 ◽  
Author(s):  
Noel K. Childers ◽  
Keri L. Miller ◽  
Giang Tong ◽  
Juan Carlos Llarena ◽  
Terrance Greenway ◽  
...  
Keyword(s):  
Lipid A ◽  
Immunoglobulin A ◽  
Oral Immunization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Soluble Antigen ◽  
Adjuvant Activity ◽  
Anamnestic Response ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid

ABSTRACT The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 μg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.

scholarly journals Immunization with the Haemophilus ducreyi Hemoglobin Receptor HgbA with Adjuvant Monophosphoryl Lipid A Protects Swine from a Homologous but Not a Heterologous Challenge

2010 ◽  
Vol 78 (9) ◽  
pp. 3763-3772 ◽  
Author(s):  
William G. Fusco ◽  
Galyna Afonina ◽  
Igor Nepluev ◽  
Deborah M. Cholon ◽  
Neelima Choudhary ◽  
...  
Keyword(s):  
Lipid A ◽  
Haemophilus Ducreyi ◽  
Class I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Whole Cells ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Heterologous Challenge ◽  
Hemoglobin Binding ◽  
Strict Requirement

ABSTRACTHaemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified fromH. ducreyiclass I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAIdispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge withH. ducreyistrain 35000HP expressing either class I or class II HgbA (35000HPhgbAIand 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologousH. ducreyistrain 35000HPhgbAIbut not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAIIfrom nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAIbut not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAIfrom both studies bound whole cells of 35000HPhgbAIbetter than 35000HPhgbAIIand partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologousH. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

Cerebral metabolism during experimental endotoxin shock and after preconditioning with monophosphoryl lipid A

2014 ◽  
Vol 126 ◽  
pp. 115-122 ◽  
Author(s):  
Claudia Ditz ◽  
Ludger Bahlmann ◽  
Stephan Klaus ◽  
Alexander Keck ◽  
Nils Onken ◽  
...  
Keyword(s):  
Lipid A ◽  
Cerebral Metabolism ◽  
Endotoxin Shock ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid

scholarly journals Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB from Porphyromonas gingivalis

2002 ◽  
Vol 70 (7) ◽  
pp. 3557-3565 ◽  
Author(s):  
Qiu-Bo Yang ◽  
Michael Martin ◽  
Suzanne M. Michalek ◽  
Jannet Katz
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Costimulatory Molecules ◽  
Th2 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Salivary Iga ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Control Serum

ABSTRACT Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 μg) alone or with MPL (25 μg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was ≈1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.

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