Monoclonal D1 Research Articles

Effects of endotoxins from Bacteroides intermedius and Escherichia coli on human monocytes in vitro.

Endotoxins (lipopolysaccharides, LPSs) from the strictly anaerobic bacterium Bacteroides intermedius and from Escherichia coli were compared with respect to effects on human monocytes. Exposure of monocyte monolayer cultures to the structurally different endotoxins caused morphological changes, demonstrated by large rounded cells containing cytoplasmic granules with the B. intermedius LPS, and smaller cells with several cytoplasmic protrusions with the E. coli LPS after 10 days of culture. The B. intermedius LPS was, at some doses, able to induce an increased level of the lysosomal enzyme acid phosphatase and increased C3b-receptor-mediated phagocytosis, while other doses gave no effect or weak suppression compared to control cultures. The stimulatory effects were donor-dependent. The E. coli LPS had predominantly suppressive effects on acid phosphatase level and C3b phagocytosis. Both endotoxins resulted in enhanced expression of the activation related cell surface antigen, characterized by the mononuclear phagocyte specific monoclonal antibody 1D5. Biphasic dose-response relationships were observed, particularly pronounced with B. intermedius LPS, giving stimulation of enzyme activity with low (1 ng/ml) and high (0.5 microgram/ml) doses, and suppressed enzyme activity with intermediate doses (0.01-0.10 microgram/ml). The results imply that the effects of endotoxins on different aspects of human monocytes vary considerably; they also suggest that the balance between stimulatory and suppressive properties of a particular endotoxin may influence its net effect on mononuclear phagocyte functions.

Lysozyme bound to the D1.3 monoclonal antibody retains enzymatic activity in assays using N-acetylglucosamine oligomers as substrate

An enzymatic assay using Micrococcus lysodeikticus as substrate had shown that hen egg-white lysozyme (HEL), complexed to the specific monoclonal antibody D1.3, was enzymatically inactive. However, a crystallographic study of the HEL-Fab D1.3 complex at 2.8 Å resolution showed that Fab D1.3 is bound to the enzyme at a region remote from the catalytic site, and that no significant conformational change had occured in the bound lysozyme. To ressolve this apparent discrepancy, oligomers of N-acetylglucosamine, containing an average of six residues and which should not be sterically hindered by the monoclonal antibody, were used as substrate in enzyme assays. In these assays lysozyme complexed to antibody D1.3 retained enzymatic activity, thus supporting the results of the crystallographic study and indicating no major conformational change or rigidification of its structure.

Identification of the thrombin receptor on human platelets by chemical crosslinking.

To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximately 200,000 and a minor one of Mr approximately 400,000. Hirudin prevented the formation of both complexes. The radioactivity of the approximately 200,000-Mr complex was always 7-10-fold greater than the radioactivity of the approximately 400,000-Mr complex regardless of the thrombin concentration to which the platelets were exposed (0.1-29 nM). Although 125I-thrombin complexes generated with thrombasthenic platelets (lacking glycoprotein IIb/IIIa) were indistinguishable from normal, no complexes appeared when Bernard-Soulier platelets (lacking glycoprotein Ib [GPIb]) were used. Complex formation was blocked by rabbit antiglycocalicin antiserum, but not by the monoclonal antibody 6D1, which is directed against the site on GPIb where von Willebrand factor (vWf) binds in the presence of ristocetin. Although cross-linking studies suggested that vWf might partially inhibit thrombin binding to platelets, this was not confirmed by equilibrium binding studies in the presence of vWf and ristocetin. The data suggest, therefore, that at all thrombin concentrations binding occurs at the same membrane site, despite evidence from equilibrium studies for high and low affinity classes of receptors, and that the approximately 400,000-Mr complex is simply a dimer of the approximately 200,000-Mr species. We conclude that the membrane site to which thrombin binds is the glycocalicin portion of platelet GPIb at a site remote from the point of ristocetin-dependent vWf binding.

A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside.

Monoclonal antibody D1.1 originally prepared against the B49 cell line derived from a rat brain tumor was shown to react with a ganglioside present in fetal rat brain. We have found that this antigen is also present in human malignant melanoma tumors as well as many melanoma cell lines. The ganglioside from human melanoma cell lines migrates between GM1 and GM2 on one-dimensional thin layer chromatography. Analysis by two-dimensional thin layer chromatography with intermediate ammonia treatment suggests that the ganglioside contains one or more base-labile O-acyl esters. Mild base hydrolysis under conditions known to remove O-acyl esters results in complete loss of antigenic reactivity. Thus, the alkali-labile moiety is a critical component of the epitope recognized by the antibody. Analysis of the sialic acids of total gangliosides from [6-3H]glucosamine-labeled melanoma cells showed that approximately 10% of these molecules are O-acylated. Similar analysis of the purified ganglioside showed that greater than 30% of the sialic acids comigrated with authentic 9-O-acetyl-N-acetylneuraminic acid. The antibody did not cross-react with normal human skin melanocytes nor with any of a large number of normal human adult and fetal tissues. The antibody also did not react with numerous other malignant cell lines studied. These findings suggest that the antigenic epitope defined by antibody D1.1 contains an O-acylated sialic acid and may arise from aberrant O-acetylation occurring in human malignant melanoma cells.