In this study, an in vitro flow model and a blocking mAb to P-selectin glycoprotein ligand-1 (PSGL-1) were used to define the role of PSGL-1 in monocyte attachment and rolling on E- and P-selectin and in attachment and accumulation on 6-h TNF-alpha-activated HUVEC. KPL1, an adhesion-blocking mAb directed against the tyrosine sulfate motif of PSGL-1, abolished monocyte-adhesive interactions with P-selectin, but only partially blocked monocyte interaction with E-selectin. Further analysis showed that on E-selectin, KPL1 blocked only secondary (i.e., monocyte/monocyte) interactions, but did not block primary (i.e., monocyte/E-selectin) interactions, with secondary adhesion accounting for 90% of the total adhesive interactions on either E- or P-selectin. On cytokine-activated HUVEC, monocytes initially attached and formed linear strings of adherent cells, which involved both primary and secondary adhesion. PSGL-1 or L-selectin mAb reduced string formation, and the combination of PSGL-1 and L-selectin mAb prevented monocyte strings and inhibited 86% of accumulation. Monocyte attachment and rolling on purified adherent monocytes were also critically dependent on PSGL-1 on the adherent monocytes. These studies document that secondary interactions between monocytes, mediated by PSGL-1, are crucial for monocyte initial attachment, rolling, and accumulation on activated endothelium under laminar shear flow.
Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.
SUMMARY Four prototype strains of Mycobacterium paratuberculosis contained the type-specific glycopeptidolipid antigen of serovar 8 of the M avium complex. This glycolipid was distinguished by a 4,6-(1′-carboxyethylidene)-3- O -methyl-β- d -glucopyranosyl terminal unit. Of 59 low-passage, field isolates of M paratuberculosis, 2 contained this antigen, and these 2 isolates were indistinguishable from M avium serovar 8. However, most M paratuberculosis isolates had no characteristic surface glycopeptidolipid. Seemingly, M paratuberculosis, long regarded as a single species and the causative agent of bovine paratuberculosis, is not a homogeneous taxon. Most isolates obtained from infected ruminants may be antigenically defective, variants of M avium and, thereby, more successful pathogens.
A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria.
Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis/sup x/ ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.
Leukocyte recruitment of sites of inflammation and tissue injury involves leukocyte rolling along the endothelial wall, followed by firm adherence of the leukocyte, and finally transmigration of the leukocyte across cell junctions into the underlying tissue. The initial rolling step is mediated by the interaction of leukocyte glycoproteins containing active moieties such as sialyl Lewisx (sLex) with P-selectin expressed on endothelial cells. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of inflammatory diseases such as arthritis. High-throughput screening of the Wyeth chemical library identified the quinoline salicylic acid class of compounds (1) as antagonists of P-selectin, with potency in in vitro and cell-based assays far superior to that of sLex. Through iterative medicinal chemistry, we identified analogues with improved P-selectin activity, decreased inhibition of dihydrooratate dehydrogenase, and acceptable CYP profiles. Lead compound 36 was efficacious in the rat AIA model of rheumatoid arthritis.
