scholarly journals Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1007-1013 ◽  
Author(s):  
WJ Wallis ◽  
DD Hickstein ◽  
BR Schwartz ◽  
CH June ◽  
HD Ochs ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Bacterial Infections ◽  
Quaternary Structure ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Glycoprotein Complex ◽  
Dose Dependent Fashion ◽  
Alpha Beta ◽  
Dose Dependent

Abstract We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

scholarly journals Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1007-1013 ◽  
Author(s):  
WJ Wallis ◽  
DD Hickstein ◽  
BR Schwartz ◽  
CH June ◽  
HD Ochs ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Bacterial Infections ◽  
Quaternary Structure ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Glycoprotein Complex ◽  
Dose Dependent Fashion ◽  
Alpha Beta ◽  
Dose Dependent

We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

Epitope mapping and analysis of a growth-enhancing monoclonal antibody by limited tryptic digestion of porcine GH

1995 ◽  
Vol 145 (1) ◽  
pp. 169-174 ◽  
Author(s):  
H-M Shieh ◽  
R T Bass ◽  
B S Wang ◽  
M J Corbett ◽  
B L Buckwalter
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Helical Structure ◽  
Tryptic Digestion ◽  
Amino Acid Residues ◽  
Optimal Sequence ◽  
Epitope Region ◽  
Dose Dependent Fashion ◽  
Growth Promoting ◽  
Dose Dependent

Abstract In this study, the epitope of a murine PS-7·6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70–95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7·6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7·6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70–95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75–90) was the optimal sequence recognized by PS-7·6 mAb. Sequential alanine substitution of each residue of pGH(75–90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7·6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75–95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i+3 (i+7) of the critical amino acids appears consistent with PS-7·6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7·6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals. Journal of Endocrinology (1995) 145, 169–174

scholarly journals Evernimicin (SCH27899) Inhibits both Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureusCells

2000 ◽  
Vol 44 (6) ◽  
pp. 1413-1417 ◽  
Author(s):  
W. Scott Champney ◽  
Craig L. Tober
Keyword(s):  
Bacterial Infections ◽  
Ribosomal Subunit ◽  
Viable Cell ◽  
Cellular Growth ◽  
Antibiotic Resistant ◽  
Ribosomal Subunits ◽  
Dose Dependent Fashion ◽  
Antibiotic Inhibition ◽  
Reduced Rate ◽  
Dose Dependent

ABSTRACT The effects of the everninomicin antibiotic evernimicin (SCH27899) on growing Staphylococcus aureus cells were investigated. Cellular growth rates and viable cell numbers decreased with increasing antibiotic concentrations. The rate of protein synthesis, measured as35S-amino acid incorporation, declined in parallel with the growth rate. Significantly, the formation of the 50S ribosomal subunit was inhibited in a dose-dependent fashion as well. 30S ribosomal subunit synthesis was not affected over the same concentration range. Evernimicin did not stimulate the breakdown of mature ribosomal subunits. Pulse-chase labeling experiments revealed a reduced rate of 50S subunit formation in drug-treated cells. Two erythromycin-resistant strains of S. aureus that carried the ermC gene were as sensitive as wild-type cells to antibiotic inhibition. In addition, two methicillin-resistant S. aureus organisms, one sensitive to erythromycin and one resistant to the macrolide, showed similar sensitivities to evernimicin. These results suggest a use for this novel antimicrobial agent against antibiotic-resistant bacterial infections.

scholarly journals C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.

1988 ◽  
Vol 168 (1) ◽  
pp. 443-448 ◽  
Author(s):  
S Okusawa ◽  
K B Yancey ◽  
J W van der Meer ◽  
S Endres ◽  
G Lonnemann ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Mononuclear Cells ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Human Pbmc ◽  
Human Mononuclear Cells ◽  
Dose Dependent Fashion ◽  
Clinical Conditions ◽  
Dose Dependent

We have demonstrated that purified C5a is a potent stimulus to human PBMC secretion of TNF-alpha, IL-1 beta, and IL-1 alpha, which proceeds in a dose-dependent fashion. At a given concentration of C5a, TNF-alpha and IL-1 beta secretion did not differ significantly; both were secreted in significantly greater quantity than IL-1 alpha. Clinical conditions such as Gram-positive and Gram-negative bacterial infections, trauma, and immune complex diseases activate complement. Through the mediation of TNF and IL-1 secreted in response to C5a, these diverse disorders can share common features of fever, coagulopathy, acute phase protein production, and disordered metabolism.

scholarly journals Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14

1999 ◽  
Vol 67 (9) ◽  
pp. 4668-4672 ◽  
Author(s):  
Domenico Caccavo ◽  
Antonella Afeltra ◽  
Salvatore Pece ◽  
Giuseppe Giuliani ◽  
Marina Freudenberg ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Lipid A ◽  
Solid Phase ◽  
Bactericidal Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Dose Dependent Fashion ◽  
Bactericidal Activities ◽  
Dose Dependent

ABSTRACT Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.

Lipopolysaccharide Induces Upregulation of Neutral Endopeptidase 24.11 on Human Neutrophils: Involvement of the CD14 Receptor

1995 ◽  
Vol 89 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Christine Fagny ◽  
Arnaud Marchant ◽  
Eric De Prez ◽  
Michel Goldman ◽  
Monique Deschodt-Lanckman
Keyword(s):  
Monoclonal Antibody ◽  
Whole Blood ◽  
Bacterial Infections ◽  
Direct Interaction ◽  
Neutral Endopeptidase ◽  
Human Neutrophils ◽  
Membrane Expression ◽  
Endopeptidase 24.11 ◽  
Factor Α

1. As lipopolysaccharide is a major stimulator of neutrophil responses during Gram-negative bacterial infections, we studied its effect on the membrane expression of neutral endopeptidase 24.11/CD10 on neutrophils in a model of endotoxaemia in vitro. Lipopolysaccharide added to human whole-blood induced a marked and sustained CD10/neutral endopeptidase upregulation that was already detectable at 0.1 ng/ml and was maximal at a lipopolysaccharide concentration of 10 ng/ml. 2. We observed that neither tumour necrosis factor-α nor any newly synthesized protein was involved in the upregulation observed after 1 h incubation with 10 ng/ml lipopolysaccharide. 3. We further studied whether the lipopolysaccharide-induced CD10/neutral endopeptidase upregulation was mediated by lipopolysaccharide binding to the neutrophil CD14 receptor. Incubation of whole blood with an anti-CD14 monoclonal antibody before the addition of 0.1 ng/ml or 0.5 ng/ml lipopolysaccharide resulted in complete inhibition of CD10/neutral endopeptidase upregulation. In contrast, at a lipopolysaccharide concentration of 10 ng/ml, the anti-CD14 monoclonal antibody had an incomplete blocking effect. 4. The differential requirement for the CD14 receptor, depending on the lipopolysaccharide dose, was confirmed by the study of a patient suffering from paroxysmal nocturnal haemoglobinuria (in whom a complete defect in neutrophil CD14 expression was previously documented). 5. We finally confirmed these results using purified neutrophils, demonstrating that lipopolysaccharide-induced CD10/neutral endopeptidase upregulation depends on direct interaction with neutrophil CD14.

scholarly journals Differential glycosylation of alpha-1-acid glycoprotein (AGP-1) contributes to its functional diversity

2020 ◽  
Author(s):  
Mosale Seetharam Sumanth ◽  
Shancy P Jacob ◽  
Kandahalli Venkataranganayaka Abhilasha ◽  
Bhanu Kanth Manne ◽  
Venkatesha Basrur ◽  
...  
Keyword(s):  
Acute Phase ◽  
Platelet Reactivity ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Peptide Sequence ◽  
Human Neutrophils ◽  
Intracellular Camp ◽  
Acid Glycoprotein ◽  
Dose Dependent Fashion ◽  
Dose Dependent

AbstractAlpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12 to 20 different glycoforms. We isolated a glycoform secreted from stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behaviour, electrophoretic mobility, and glycosylation. The function of these two glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration and NETosis in a dose-dependent fashion, while nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. However, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by Platelet-activating Factor (PAF) or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and were accompanied by phosphorylation of the PKA substrate Vasodialator stimulated phosphoprotein (VASP) and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity while nAGP-1 glycoform also limits pro-inflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on two critical cells of acute inflammation.

scholarly journals Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

1987 ◽  
Vol 105 (1) ◽  
pp. 9-18 ◽  
Author(s):  
E Kubalek ◽  
S Ralston ◽  
J Lindstrom ◽  
N Unwin
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Alpha Subunit ◽  
Torpedo Marmorata ◽  
Fab Fragment ◽  
Alpha Subunits ◽  
Immunogenic Region ◽  
Electron Image ◽  
Alpha Beta ◽  
Tubular Crystals

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

scholarly journals A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation

Blood ◽  
1985 ◽  
Vol 65 (6) ◽  
pp. 1553-1556 ◽  
Author(s):  
BR Schwartz ◽  
HD Ochs ◽  
PG Beatty ◽  
JM Harlan
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Glycoprotein ◽  
Myristate Acetate ◽  
Glycoprotein Complex ◽  
Antigen Complex ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Neutrophil Aggregation ◽  
Dose Dependent

Abstract We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.

scholarly journals Analysis of the Effects of Cigarette Smoke on Staphylococcal Virulence Phenotypes

2015 ◽  
Vol 83 (6) ◽  
pp. 2443-2452 ◽  
Author(s):  
Elisa K. McEachern ◽  
John H. Hwang ◽  
Katherine M. Sladewski ◽  
Shari Nicatia ◽  
Carola Dewitz ◽  
...  
Keyword(s):  
Surface Charge ◽  
Cigarette Smoke ◽  
Bacterial Infections ◽  
Content Type ◽  
Immune Resistance ◽  
Dose Dependent Fashion ◽  
Human Immune ◽  
Macrophage Killing ◽  
Dose Dependent

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen,Staphylococcus aureus, and in particular methicillin-resistantS. aureus(MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival;P< 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold;P= 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% (P< 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% (P< 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation ofmprF, consistent with increased MRSA AMP resistance.S. aureuswithoutmprFhad no change in surface charge upon exposure to CSE.In vivo, CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice (P< 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers.

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