We have established 51 solid tumor and 10 ALL in vivo models. The models identify vincristine and cyclophosphamide as having broad-spectrum activity. The PPTP tumor panels appear to generally recapitulate the activity of these agents against specific childhood cancers and to have the potential for identifying novel agents having significant clinical activity.

Infection of cells with DNA viruses triggers innate immune responses mediated by DNA sensors. cGMP-AMP synthase (cGAS) is a key DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which binds to and activates stimulator of interferon genes (STING), leading to IFN production and an antiviral response. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. We report that KSHV infection activates the cGAS-STING pathway, and that cGAS and STING also play an important role in regulating KSHV reactivation from latency. We screened KSHV proteins for their ability to inhibit this pathway and identified six viral proteins that block IFN-β activation through this pathway. This study is the first report identifying multiple viral proteins encoded by a human DNA virus that inhibit the cGAS-STING DNA sensing pathway. One such protein, viral interferon regulatory factor 1 (vIRF1), targets STING by preventing it from interacting with TANK binding kinase 1 (TBK1), thereby inhibiting STING's phosphorylation and concomitant activation, resulting in an inhibition of the DNA sensing pathway. Our data provide a unique mechanism for the negative regulation of STING-mediated DNA sensing. Moreover, the depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. The vIRF1-expressing cells also inhibited IFN-β production following infection with DNA pathogens. Collectively, our results demonstrate that gammaherpesviruses encode inhibitors that block cGAS-STING-mediated antiviral immunity, and that modulation of this pathway is important for viral transmission and the lifelong persistence of herpesviruses in the human population.aposi's sarcoma-associated herpesvirus (KSHV/HHV8) is the etiological agent of several human malignancies, including Kaposi's sarcoma (KS), multicentric Castleman's disease, and primary effusion lymphoma (1, 2). Evasion of the host innate immune response is essential for viral infection, replication, latency, transmission, and lifelong persistence.A member of the gammaherpesvirus subfamily, KSHV contains a large dsDNA genome that encodes for more than 80 ORFs. Different pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins, and retinoic acid-inducible gene-I-like receptors (RLRs), are activated upon KSHV infection in different cell types (3-5). As is the case with many pathogens, multiple PRRs can detect an incoming pathogen in the cell. Depending on the cell type, detection of viral DNA may take place in the nucleus (6) or in the cytoplasm due to premature release of the herpesviral DNA into the cytoplasm (7). It is plausible that defective herpesvirus virions release their genomic contents into the cytoplasm rather than the nucleus and that this viral DNA triggers cytosolic DNA sensors to be activated. Additionally, HSV-1 infection induces mitochon...

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80+ macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4−/− mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5−/− mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF−/− mice showed a similar phenotype as TLR4−/− mice in regulating only ΔfliC bacteria, whereas MyD88−/− mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1−/− and IL-1α/β−/− mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.

Pathogenic Gram-negative bacteria use syringe-like type III secretion systems (T3SS) to inject effector proteins directly into targeted host cells. Effector secretion is triggered by host cell contact, and before contact is prevented by a set of conserved regulators. How these regulators interface with the T3SS apparatus to control secretion is unclear. We present evidence that the proton motive force (pmf) drives T3SS secretion in Pseudomonas aeruginosa, and that the cytoplasmic regulator PcrG interacts with distinct components of the T3SS apparatus to control two important aspects of effector secretion: (i) It coassembles with a second regulator (Pcr1) on the inner membrane T3SS component PcrD to prevent effectors from accessing the T3SS, and (ii) In conjunction with PscO, it controls protein secretion activity by modulating the ability of T3SS to convert pmf.

Summary Pseudomonas aeruginosa uses a type III secretion system to inject protein effectors into a targeted host cell. Effector secretion is triggered by host cell contact. How effector secretion is prevented prior to cell contact is not well understood. In all secretion systems studied to date, the needle-tip protein is required for controlling effector secretion, but the mechanism by which needle-tip proteins control effector secretion is unclear. Here we present data that the P. aeruginosa needle-tip protein, PcrV, controls effector secretion by assembling into a functional needle-tip complex. PcrV likely does not simply obstruct the secretion channel since the pore-forming translocator proteins can still be secreted while effector secretion is repressed. This finding suggests that PcrV controls effector secretion by affecting the conformation of the apparatus, shifting it from the default, effector secretion ‘on’ conformation, to the effector secretion ‘off’ conformation. We also present evidence that PcrG, which can bind to PcrV and is also involved in controlling effector export, is cytoplasmic and that the interaction between PcrG and PcrV is not required for effector secretion control by either protein. Taken together, these data allow us to propose a working model for control of effector secretion by PcrG and PcrV.