Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated ...a novel anti-ADAMTS-5 inhibiting Nanobody
(M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX,
= 4), human osteoarthritic - (HEX,
= 8) and healthy-cartilage (hHEX,
= 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (
/
), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1β (10 ng/mL)) with or without M6495 (1000-0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monodonal antibodies. Here we report the isolation and characterization of ...functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Virus Host Interactions Unit, Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
Correspondence ...Peter Vanlandschoot Peter.Vanlandschoot{at}UGent.be
Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.
Hepatitis B virus (HBV) infections cause 500,000 to 700,000 deaths per year as a consequence of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Efficient and safe antivirals to treat ...chronically infected patients and consequently to prevent development of hepatocellular carcinoma are still awaited. We isolated five single‐domain antibodies (VHHs) that recognize the most abundant envelope protein (S) of HBV. VHHs, when expressed and retained in the endoplasmic reticulum as intrabodies, reduced levels of secreted hepatitis B surface antigen (HBsAg) particles in a cellular HBV model. In a hydrodynamics‐based HBV mouse model, these intrabodies caused a marked reduction in HBsAg concentrations and a 10‐ to >100‐fold reduction in the concentration of HBV virions in plasma. Conclusion: VHHs potently inhibited secretion of HBV virions in vivo, showing that this approach might be useful in the treatment of HBV. To our knowledge, this is the first report of intrabody‐mediated inhibition of viral secretion in mammals. (HEPATOLOGY 2009;49:39‐49.)
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium
Correspondence Geert Leroux-Roels geert.lerouxroels{at}ugent.be
Hepatitis B virus (HBV) infections represent a global health ...problem, since these account for 350 million chronic infections worldwide that result in 500 000–700 000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw . These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes.
Four supplementary figures and a supplementary table of primer sequences are available with the online version of this paper.
In recent years there has been an explosion of published research on biomarkers yet with limited translation into clinical practice and back to bench. Classically, biomarker experiments involved the ...assessment of proteins in blood. Many developments in biomarker research have been performed in the past years that make biomarkers an extremely valuable tool in translational medicine. The goal of this chapter is to highlight a number of recent developments that advanced the field and may help to value the sophisticated use of biomarkers in biomedical research.
PDF
