SummaryUsing human endothelial cells, we de®ne a mechanism that accounts for the induction of interleukin 8 (IL-8) by protein I/IIf, an adhesin from Streptococcus mutans serotype f. We report that protein I/IIf interactions with endothelial cells increased the tyrosine phosphorylation of three cellular components with relative mass of 145 000, 125 000 and 70 000 in endothelial cells. These proteins were identi®ed as phospholipase Cg (PLCg), focal adhesion kinase (FAK) and paxillin after immunoprecipitation with monoclonal antibodies (mAbs) and immunoblotting with antiphosphotyrosine mAbs. These results suggested that b1 integrins could be one of the components implicated in the modulin activity of protein I/IIf. By incubating protein I/IIf with either puri®ed a5b1 integrins or with a5b1 integrins overexpressing CHO cells, we demonstrated that a5b1 integrins act as cell receptors for protein I/IIf. We also showed that protein I/IIf interactions with a5b1 integrins lead to IL-8 secretion. Using speci®c inhibitors, we demonstrated that protein I/IIf-induced IL-8 release involves mitogenactivated protein kinases (MAPKs), and that PLCg and PKC also seem to contribute to protein I/IIf stimulation. However, PI-3K activation is not involved in IL-8 release. Altogether, these results indicate that, after binding to a5b1 integrins, protein I/IIf induces IL-8 release by activating the MAPKs signalling pathways.

No abstract

Superfolder green fluorescent protein (sfGFP) is a fusion tag which plays a dual role in monitoring and purifying the recombinant fusion proteins using specific binders. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies (HCAbs) occurring in camelids. They are produced as recombinant proteins in E. coli and have different biotechnological applications, including the detection and purification of their specific antigens. To produce anti-sfGFP specific nanobodies, an adult one-humped camel was successfully immunized and immune response was evaluated by ELISA, which showed an active participation of HCAbs in this response. A relatively large nanobody "immune" library of 5 × 10(8) individual transformants, with 87.5 % positivity, was prepared from the blood of the immunized camel. Phage display biopanning on this nanobody library resulted in the isolation of seven anti-sfGFP specific nanobodies, referred to as NbsfGFP01, 02, 03, 04, 06, 07 and 08. These nanobodies were able to recognize sfGFP tag as free or in fusion with growth hormone in ELISA and immuno-blotting. Furthermore, they showed important apparent affinities in the detection and capture of sfGFP by ELISA, and they targeted three different epitopes on the surface of their antigen. The interesting characteristics of these molecular binders make them valuable tools for more in-depth structural and functional studies related to sfGFP fusion proteins.

We have tested the ability of protein I/II, an adhesin from oral streptococci, to stimulate the production of pro-inflammatory cytokines by synovial cells isolated from both rheumatoid arthritis and control patients. Protein I/II triggers synovial fluid cells to produce interleukin (IL)-6 and IL-8 while secretion of tumor necrosis factor-alpha (TNF-alpha) was less enhanced. Using fibroblast-like synoviocytes, we found that protein I/II also exerts an immunomodulatory effect (IL-6 and IL-8 release) on these cells. These findings indicate that, if it gains access to the joint cavity, protein I/II could participate in the initiation and/or perpetuation of rheumatic diseases, by stimulating pro-inflammatory cytokine release from various synovial cells.