Characterization of an FcgammaRI-binding peptide selected by phage display.

The high-affinity IgG receptor, Fcgamma receptor I (FcgammaRI), is expressed exclusively on myeloid cells, and there is a great interest in the targeting of vaccine antigens to FcgammaRI using anti-human FcgammaRI antibodies or fragments derived from such molecules. In order to reduce the size and complexity of the targeting reagent, we have searched for FcgammaRI binding peptides in peptide libraries displayed on phage. The human monocytic cell line U937 was used as target. Phages that displayed the consensus peptide CLRSGXGC were selected and revealed increased binding to IFN-gamma stimulated versus non-stimulated U937 cells as well as to FcgammaRI transfected versus non-transfected IIA1.6 cells. Furthermore, they bound the extracellular domains of soluble FcgammaRI, but neither FcgammaRIIA, FcgammaRIIB nor FcgammaRIIIB. Binding was inhibited by a synthetic version of the peptide, whereas neither human IgG nor the FcgammaRI-specific monoclonal antibodies (mAb) mAb22 and 32.2 interfered. Flow-cytometry analysis and internalization studies showed that a synthetic biotin-conjugated peptide ADGACLRSGRGCGAAK-bio was able to target U937 cells and FcgammaRI transfected IIA1.6 cells, and further to promote internalization and vesicular degradation of streptavidin coupled to 1 microm magnetic beads. These peptides may have potential as FcgammaRI targeting reagents.

Selection and characterization of cyclic peptides that bind to a monoclonal antibody against meningococcal L3,7,9 lipopolysaccharides.

There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.

Conjugates of Tremella polysaccharides with microbeads and their TNF-stimulating activity.

A mannan (A) and a heteroglycan (D) were prepared by partial acidic hydrolysis of T3, a major polysaccharide fraction of the fungus Tremella fuciformis Berk. Methylation analysis of A and D showed that they contained a 1-->3 linked mannosyl main chain, to which were linked different side chains at position 2, 4, or 6 of the mannosyl residues. A and D were conjugated to albumin microbeads (AM) by reductive amination. The conjugates showed significant cytokine-stimulating activity in vitro whereas unconjugated AM had no activity. Not conjugated A and D showed cytokine-stimulating activity only in about 100 times higher concentrations.

The tumor necrosis factor-inducing potency of lipopolysaccharide and uronic acid polymers is increased when they are covalently linked to particles.

Lipopolysaccharide (LPS) and polymers of the uronic acid family stimulate monocytes to produce tumor necrosis factor (TNF). The TNF-inducing potency of these polysaccharides may depend on their supramolecular configuration. In this study detoxified LPS and uronic acid polymers have been covalently linked to particles which have been added to monocytes under serum-free conditions. Reducing the size of mannuronan from 350,000 to 5,500 Da (M-blocks) led to a 10- to 100-fold reduction in TNF-inducing potency. However, covalently linking the M-blocks to monodisperse suspensions of magnetic particles increased the TNF-inducing potency by up to 60,000-fold. Also, the TNF-inducing potency of glucuronic acid polymers was increased when they were linked to particles, but no potentiation was observed with guluronic acid blocks covalently attached to particles. Furthermore, O chains of LPS (detoxified LPS) became potent TNF inducers when they were presented to monocytes on a particle surface. No activation of the LPS-responsive SW480 adenocarcinoma cells was found with detoxified LPS or M-block particles, suggesting a preference for cells expressing CD14 and/or other membrane molecules. The potentiating effects were not restricted to polymers attached to aminated magnetic particles. Of particular interest, we found that short blocks of mannuronan induced TNF production also when covalently linked to biodegradable, bovine serum albumin particles.

Cytokine-Stimulating (In Vitro) Effect on Human Monocytes of Conjugates of Soluble Curdlan and Albumin Microbeads.

Four kinds of conjugates (BSA-M-SC-Ra, BSA-M-SC-Re, AP-M-SC-Ra, and AP-M-SC-Re) composed of soluble fragments of [ß](1 ± 3)-D-glucan (SC) and bovine serum albumin (BSA) or amine-modifled polystyrene (AP) microbeads were prepared: the contents of SC on the microbeads were found to be 13.6, 8.0, 3.6, and 2.6 µg per 1 X 10(8) microbeads, respectively. Unconjugated BSA and AP microbeads and SC did not show cytokine-stimulating activity. BSA-M-SC-Re, BSA-M-SC-Ra, and AP-M-SC-Re showed strong cytokine-stimulating activity in vitro, indicating that conjugates composed of inactive fragments of polysaccharides and microbeads are able to induce cytokine release from mononuclear phagocytes. Conjugates with the SC linked to BSA microbeads by the reducing terminal (BSA-M-SC-Re) showed a stronger effect than conjugates in which SC was linked with BSA microbeads at random positions (BSA-M-SC-Ra).

[Injuries and drug use among soccer players]. / Skader og legemiddelbruk hos fotballspillere.

Injuries in sports are well-known through retrospective studies of different kinds of sports, especially soccer. In this study on injury and drug use, the adverse effects of drugs and the use of medical services were registered in a group of male elite soccer players and in a referent group. There was a higher percentage of injuries and a higher use of drugs in the soccer group compared with the referent group, the largest difference being in the use of antiflogistics. Both groups reported low adverse effects of drugs, and similar frequency of such effects. Doctors and physiotherapists were usually consulted, but more frequently by the soccer group than by the referent group. The results of this study indicate that playing top division soccer is associated with higher risk of injury with a concomitant higher use of drugs than experienced in the referent group.