Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas doublestranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2␣ (eIF2␣). Here we have shown that p38 and eIF2␣ phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gramnegative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the CNS. Bacteriophage have the capacity to penetrate the CNS when administered intranasally. Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine. Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes.

Cell density-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). 1 N-acyl L-homoserine lactones (AHLs) are produced by over 70 species of Gram-negative bacteria, and structural differences within AHLs occur in the length and oxidation state of the acyl side chain (Figure 1). Upon reaching a critical threshold concentration, AHLs bind to their cognate receptor proteins, triggering the expression of target genes. AHLs have been shown to play an important role in the establishment and course of bacterial infections. One example of a Gram-negative pathogen that employs AHL-based QS to regulate the expression of its pathogenicity factors is Pseudomonas aeruginosa. This common environmental microorganism has acquired the ability to take advantage of weaknesses in the host defenses to become an opportunistic pathogen in humans. Most prominent is the role of P. aeruginosa in patients suffering from cystic fibrosis (CF). Over the last 15 years, much progress has been made in elucidating the molecular mechanisms underlying P. aeruginosa pathogenicity. 2 Two different AHLs, N-(3-oxododecanoyl) homoserine lactone (3-oxo-C 12 -AHL) and N-butyryl homoserine lactone (C 4 -AHL), have been identified as QS signaling molecules in P. aeruginosa. Genes regulated by this QS mechanism encode enzymes such as elastases A and B, catalase, and superoxide dismutase as well as exotoxins. 2Interference with QS signaling has been suggested as a new approach for anti-infective therapy. 3 In fact, this strategy has yielded interesting results using AHL analogs as QS antagonists in P. aeruginosa. 4 Alternatively, we have embarked on a program utilizing antibodies to inhibit AHL-mediated quorum sensing signaling in P. aeruginosa. AHL-based QS systems represent an ideal target for antibody-based anti-infective therapy given the highly conserved molecular scaffold and extracellular distribution of AHLs.Our initial hapten design for the elicitation of anti-AHL antibodies focused on synthesizing a set of molecules highly congruent in structure to AHLs while also possessing a pendant carboxylic acid functionality that would enable carrier proteins BSA or KLH conjugates to be easily accessed. However, we were aware that such molecules might also be prone to hydrolysis (ring opened products) under conditions required for chemical coupling and also immunization. Thus, to guide our hapten design the stability of several AHLs under physiological conditions was investigated; we synthesized a number of AHL analogs and their corresponding ring opened hydrolysis products, both of which contained a 4-methoxyphenyl amide group that allowed for a detailed investigation into the rate of hydrolysis of the AHL analogues using HPLC with UV detection (Figure 2 Each compound was assayed for its hydrolysis rate in phosphate buffer saline (PBS), pH 7.2, at a concentration of 200 μM at 37 °C. The half lives of each of the lactones varied from 13.7 to 18.1 hours. Interestingly, the oxidation state and chain length did not influence...

Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such lifethreatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu H95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11⅐AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.