Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

The primary cosignaling receptors belong to either the Ig CD28-like or TNF receptor (TNFR) superfamilies (1-5). Currently, the CD28 family consists of five lymphoid-specific coreceptors [CD28, inducible T cell costimulator (ICOS), cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and B and T lymphocyte attenuator (BTLA)] (1-3). CD28 and ICOS are single Ig-variable (IgV) domain glycoproteins that promote T cell activation, whereas the structurally related CTLA-4, PD-1, and BTLA receptors function to attenuate T cell activation. To date, all of the ligands that have been described for the CD28-like family members belong to the B7 superfamily (1-3, 6-9). Six B7 family members have been described, all of which have conserved extracellular IgV and Ig-constant domains (3). In the TNFR-TNF superfamily, five receptor-ligand interactions have been described that act as positive regulators. These include OX40-OX40L, 4-1BB-4-1BBL, CD27-CD70, CD30-CD30L, and herpesvirus entry mediator (HVEM)-homologous to lymphotoxin, which shows inducible expression and competes with herpes simplex virus (HSV) glycoprotein D (gD) for HVEM, a receptor expressed by T lymphocytes (HVEM-LIGHT) (4).The most recently identified CD28 family member is the inhibitory coreceptor, BTLA (10-12). BTLA is expressed on developing B and T cells, all mature lymphocytes, splenic macrophages, and mature marrow-derived dendritic cells (10,11). BTLA contains a single IgV domain and two intracellular immunoreceptor tyrosine-based inhibitory motifs that are phosphorylated after BTLA coligation to antigen receptors, resulting in recruitment of protein tyrosine phosphatases SHP-1 and SHP-2 (13). Because of this, and that coligation of BTLA to the TCR inhibits T cell activation, BTLA is implicated as a negative regulator of T cell activation (10, 11). This finding is further supported by the observation that BTLA-deficient T cells show increased proliferation and that BTLA Ϫ͞Ϫ mice have increased Ab response and show increased incidence and severity to an autoimmune disorder (9, 10).Initially, BTLA was proposed to interact with a B7 family member called B7x (10, 14). This conclusion, however, was based on an indirect binding study testing the interaction of B7x-Ig fusion to spleen and lymph node cells from either WT or BTLA-deficient mice. Furthermore, we have been unable to detect any specific binding of BTLA-Fc to B7x-transfected cells (data not shown). Spurred by the inability to confirm the BTLA-B7x interaction, we screened a secreted protein library (15) by using surface plasmon resonance (SPR) and identified HVEM as a coreceptor for human BTLA.Here we show that BTLA and HVEM interact with high affinity and can form a trimeric complex with TNF ligands LIGHT or lymphotoxin ␣ (LT␣). Our binding studies suggest that BTLA interacts with the outer surface of the HVEM͞TNF complex, suggesting structural models for how HVEM might engage BTLA on the cell surface. Finally, we demonstrate that binding of HVEM to BTLA results in the inhibition of T cel...

Exocytotic secretion in neuroendocrine cells is actiNeurotransmitter and peptide hormone secretion are mediated by the fusion of secretory vesicles with the plasma membrane, an exocytotic process that requires ATP and is strongly dependent upon and activated by Ca 2ϩ . Insights into the molecular basis of regulated membrane fusion have been provided by the identification of several required synaptic proteins such as vesicle-associated membrane protein/synaptobrevin, syntaxin and SNAP-25, which are substrates for clostridial neurotoxin proteases (1) and receptors for N-ethylmaleimide-sensitive factor/SNAP proteins (2), and synaptotagmin, a vesicle Ca 2ϩ

Ca2؉ -dependent activator protein for secretion (CAPS) is a cytosolic protein essential for the Ca 2؉ -dependent fusion of dense-core vesicles (DCVs) with the plasma membrane and the regulated secretion of a subset of neurotransmitters. The mechanism by which CAPS functions in exocytosis and the means by which it associates with target membranes are unknown. We identified two domains in CAPS with distinct membrane-binding properties that were each essential for CAPS activity in regulated exocytosis. The first of these, a centrally located pleckstrin homology domain, exhibited three properties: charge-based binding to acidic phospholipids, binding to plasma membrane but not DCV membrane, and stereoselective binding to phosphatidylinositol 4,5-bisphosphate. Mutagenesis studies revealed that the former two properties but not the latter were essential for CAPS function. The central pleckstrin homology domain may mediate transient CAPS interactions with the plasma membrane during Ca 2؉ -triggered exocytosis. The second membrane association domain comprising distal C-terminal sequences mediated CAPS targeting to and association with neuroendocrine DCVs. The CAPS C-terminal domain was also essential for optimal activity in regulated exocytosis. The presence of two membrane association domains with distinct binding specificities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion.

ABSTRACT:The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-␣ monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/ kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentrationtime curve from time 0 to day 7 than the WT (749 ؎ 71.9 and 819 ؎ 81.5 versus 592 ؎ 56.8 g/ml ⅐ day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.