Identifying the initiating events of anti-Listeria responses using mice with conditional loss of IFN-γ receptor subunit 1 (IFNGR1).

Although IFN-γ is required for resolution of Listeria monocytogenes infection, the identities of the IFN-γ-responsive cells that initiate the process remain unclear. We addressed this question using novel mice with conditional loss of IFN-γR (IFNGR1). Itgax-cre(+)Ifngr1(f/f) mice with selective IFN-γ unresponsiveness in CD8α(+) dendritic cells displayed increased susceptibility to infection. This phenotype was due to the inability of IFN-γ-unresponsive CD8α(+) dendritic cells to produce the initial burst of IL-12 induced by IFN-γ from TNF-α-activated NK/NKT cells. The defect in early IL-12 production resulted in increased IL-4 production that established a myeloid cell environment favoring Listeria growth. Neutralization of IL-4 restored Listeria resistance in Itgax-cre(+)Ifngr1(f/f) mice. We also found that Itgax-cre(+)Ifngr1(f/f) mice survived infection with low-dose Listeria as the result of a second wave of IL-12 produced by Ly6C(hi) monocytes. Thus, an IFN-γ-driven cascade involving CD8α(+) dendritic cells and NK/NKT cells induces the rapid production of IL-12 that initiates the anti-Listeria response.

Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV.

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.

Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection.

Herein we report the generation of mouse monoclonal antibodies (mAbs) specific for the IFNAR-1 subunit of the mouse interferon-alpha/beta (IFN-alpha/beta) receptor (MAR1 mAbs) that block type I IFN receptor signaling and biologic response induction in vitro and in vivo. These mAbs were generated from Ifnar1 (/) mice immunized by in vivo hydrodynamic transfection with a plasmid encoding the extracellular domain (ECD) of murine IFNAR-1. All MAR1 mAbs bound native receptor expressed on cell surfaces and immunoprecipitated IFNAR-1 from solubilized cells, and two mAbs also detected IFNAR-1 by Western blot analysis. in vitro, the mAbs prevented ligand-induced intracellular signaling and induction of a variety of type I IFN-induced biologic responses but had no effect on IFN-gamma-induced responses. The most effective in vitro blocker, MAR1-5A3, also blocked type I IFN-induced antiviral, antimicrobial, and antitumor responses in vivo. We also explored whether murine IFNAR-1 surface expression required the presence of Tyk2. In contrast to Tyk2-deficient human cell lines, comparable IFNAR-1 expression was found on primary cells derived either from wild-type or Tyk2 (/) mice. These mAbs represent much needed tools to more clearly elucidate the biochemistry, cell biology, and physiologic function of the type I IFNs and their receptor in mediating host-protective immunity and immunopathology.

Multiplatform genome-wide identification and modeling of functional human estrogen receptor binding sites.

BACKGROUND: Transcription factor binding sites (TFBS) impart specificity to cellular transcriptional responses and have largely been defined by consensus motifs derived from a handful of validated sites. The low specificity of the computational predictions of TFBSs has been attributed to ubiquity of the motifs and the relaxed sequence requirements for binding. We posited that the inadequacy is due to limited input of empirically verified sites, and demonstrated a multiplatform approach to constructing a robust model. RESULTS: Using the TFBS for the estrogen receptor (ER)alpha (estrogen response element [ERE]) as a model system, we extracted EREs from multiple molecular and genomic platforms whose binding to ERalpha has been experimentally confirmed or rejected. In silico analyses revealed significant sequence information flanking the standard binding consensus, discriminating ERE-like sequences that bind ERalpha from those that are nonbinders. We extended the ERE consensus by three bases, bearing a terminal G at the third position 3' and an initiator C at the third position 5', which were further validated using surface plasmon resonance spectroscopy. Our functional human ERE prediction algorithm (h-ERE) outperformed existing predictive algorithms and produced fewer than 5% false negatives upon experimental validation. CONCLUSION: Building upon a larger experimentally validated ERE set, the h-ERE algorithm is able to demarcate better the universe of ERE-like sequences that are potential ER binders. Only 14% of the predicted optimal binding sites were utilized under the experimental conditions employed, pointing to other selective criteria not related to EREs. Other factors, in addition to primary nucleotide sequence, will ultimately determine binding site selection.