b-Arrestin 1-GFP or b-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melaninconcentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both b-arrestin 1-GFP and b-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for b-arrestin 2-GFP. b-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of b-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and traf®cking of TAMRA-(5-and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of b-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of b-arrestin 1-GFP from intracellular vesicles. For the melaninconcentrating hormone receptor the bulk of translocated b-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of b-arrestin±GFP interactions and traf®cking for three G protein-coupled receptors for which the natural ligands have only recently been identi®ed and which were thus previously considered as orphan receptors.

SUMMARYThe presentation of extremely low doses of antigen to T cells is enhanced by immunoglobulin E (IgE)-dependent antigen focusing to CD23, the low-af®nity receptor for IgE, expressed on activated B cells. CD23 contains a C-type lectin domain in its extracellular sequence and a targeting signal for coated pits, required for endocytosis, in its cytoplasmic sequence. CD23 is non-covalently associated with the major histocompatibility complex class II antigen, human leucocyte antigen HLA-DR, on the surface of human B cells, but the fate of this complex following endocytosis is unknown. To answer this question we have labelled these proteins on the surface of RPMI 8866 B cells and traced their route through the cytoplasm. Endocytosis mediated by anti-CD23 antibodies (BU38 and MHM6) led to the loss of CD23 from the cells. Endocytosis mediated by an antibody to HLA-DR (CR3/43) or an antigen±IgE complex (NP-BSA±anti-NP IgE), however, led to recycling of the HLA-DR±CD23 complex to the cell surface on a time scale (3±6 hr) consistent with the recycling of HLA-DR in antigen presentation. Along the latter pathway CD23 label was observed in cytoplasmic organelles that resembled the`compartments for peptide loading' or`class II vesicles' described by previous authors. Two features of the recycling process may contribute to the ef®ciency of antigen presentation. Peptide exchange may be facilitated by the proximity of HLA-DR and antigen in peptide loading compartments of the endosomal network. The return of CD23 with HLA-DR to the cell surface may then help to stabilize speci®c B-cell±T-cell interactions, contributing to T-cell activation.

A potent anti-vascular endothelial growth factor (VEGF) biologic and a compatible delivery system were co-evaluated for protection against wet age-related macular degeneration (AMD) over a 6month period following a single intravitreal (IVT) injection. The anti-VEGF molecule is dimeric, containing two different anti-VEGF domain antibodies (dAb) attached to a human IgG1 Fc region: a dual dAb. The delivery system is based on microparticles of PolyActive™ hydrogel co-polymer. The molecule was evaluated both in vitro for potency against VEGF and in ocular VEGF-driven efficacy modelsin vivo. The dual dAb is highly potent, showing a lower IC50 than aflibercept in VEGF receptor binding assays (RBAs) and retaining activity upon release from microparticles over 12 months in vitro. Microparticles released functional dual dAb in rabbit and primate eyes over 6 months at sufficient levels to protect Cynomolgus against laser-induced grade IV choroidal neovascularisation (CNV). This demonstrates proof of concept for delivery of an anti-VEGF molecule within a sustained-release system, showing protection in a pre-clinical primate model of wet AMD over 6 months. Polymer breakdown and movement of microparticles in the eye may limit development of particle-based approaches for sustained release after IVT injection.

The techniques of flow cytometry, scanning and transmission electron microscopy, and confocal scanning laser microscopy were used to study the physiology of Staphy/ococcus aureus in the early stages of surface-attached culture, and to make direct comparisons with planktonic bacteria grown under the same conditions. Attached bacteria growing in nutrient-rich batch culture were found to go through the same growth phases as equivalent planktonic cultures, but with an exponential growth rate of about half that of the planktonic bacteria. Viability of attached bacteria was very high (around 100°/~) throughout the first 24 h of growth. The size and protein content of attached bacteria varied with growth phase, and both measurements were always smaller than in planktonic bacteria at equivalent growth phases. Respiratory activity per bacterium, as measured by flow cytofluorimetry, and corrected for cell volume, peaked very early in attached cultures (before the first cell division) and declined from then on, whereas in planktonic bacteria it peaked in late exponential phase. Attached and planktonic bacteria showed thicker cell walls in stationary phase than in exponential phase. Membrane potentials of planktonic and attached bacteria were similar in stationary phase, but were much lower in exponential-phase attached cells than in the equivalent planktonic cells. It is apparent that a range of significant physiological adaptations occur during the early phases of attached growth.

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