SummaryPhenol-soluble modulin (PSM) is a peptide complex produced by the nosocomial pathogen Staphylococcus epidermidis that has a strong capacity to activate the human innate immune response. We developed a novel method based on liquid chromatography-mass spectrometry (LC-MS) to quantify the production of the individual PSM components. Each PSM peptide was abundant in most of the 76 S epidermidis strains tested. Importantly, none of the PSM components were secreted by an agr mutant strain, indicating that PSM synthesis is regulated strictly by the agr quorum-sensing system. Furthermore, the agr mutant strain failed to elicit production of TNF a a a a by human myeloid cells and induced significantly less neutrophil chemotaxis compared with the wild-type strain. Thus, quorum-sensing in S. epidermidis dramatically influenced activation of human host defence. We propose that an agr quorum-sensing mechanism facilitates growth and survival in infected hosts by adapting production of the pro-inflammatory PSMs to the stage of infection.

IntroductionGranulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be important in the development of inflammatory models of rheumatoid arthritis and there is encouraging data that its blockade may have clinical relevance in patients with rheumatoid arthritis. The aims of the current study were to determine whether GM-CSF may also be important for disease and pain development in a model of osteoarthritis.MethodsThe role of GM-CSF was investigated using the collagenase-induced instability model of osteoarthritis. We studied both GM-CSF-/- mice and wild-type (C57BL/6) mice treated prophylactically or therapeutically with a monoclonal antibody to GM-CSF. Disease development (both early and late) was evaluated by histology and knee pain development was measured by assessment of weight distribution.ResultsIn the absence of GM-CSF, there was less synovitis and matrix metalloproteinase-mediated neoepitope expression at week 2 post disease induction, and less cartilage damage at week 6. GM-CSF was absolutely required for pain development. Therapeutic neutralization of GM-CSF not only abolished the pain within 3 days but also led to significantly reduced cartilage damage.ConclusionsGM-CSF is key to the development of experimental osteoarthritis and its associated pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abolished existing arthritic pain and suppressed the degree of arthritis development. Our results suggest that it would be worth exploring the importance of GM-CSF for pain and disease in other osteoarthritis models and perhaps clinically for this form of arthritis.

GM-CSF is key to the development of inflammatory and arthritic pain, suggesting that pain alleviation could result from trials evaluating its role in inflammatory/autoimmune conditions.

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus ␦-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of ␦-toxin deformylation. We observed that during exponential and early postexponential growth, ␦-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated ␦-toxin accumulated after the early postexponential phase. The transition from producing both species of ␦-toxin to producing only formyl-methionine-containing ␦-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of ␦-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that ␦-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated ␦-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated ␦-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.Staphylococcus aureus uses a complex regulatory network to control the expression of virulence factors. Central to this regulatory network is the accessory gene regulatory (agr) locus (15,19). This locus consists of two divergently transcribed RNAs, encoding a density-dependent autoinducing system and an RNA effector molecule (RNAIII) (16). RNAIII acts primarily at the level of transcription but also has been shown to posttranscriptionally regulate alpha-toxin (hla) production; however, the exact mechanism of regulation is not known (17). In addition to its regulatory role, RNAIII is the mRNA for ␦-toxin (hld) (11). The mature form of ␦-toxin is a heat-stabile, 26-amino-acid peptide that inserts into and disrupts membranes (4). Additionally, it has been implicated in the prevention of biofilm formation and in stimulation of the oxidative burst of human neutrophils (22, 28). However, the precise function of ␦-toxin in S. aureus pathogenesis has yet to be elucidated.Recently, workers in our laboratory reported construction and characterization of an S. aureus aconitase (acnA) mutant (24). During the course of that study, it was observe...

Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumorlocalized, HER2-dependent 4-1BB clustering and activation. Experimental Design: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFa, and IFNg). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. Conclusions: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule. See related commentary by Su et al., p. 5732