Evidence of a Hardening in the Cosmic Ray Proton Spectrum at around 166 TeV Observed by the GRAPES-3 Experiment.

We present the measurement of the cosmic ray proton spectrum from 50 TeV to 1.3 PeV using 7.81×10^{6} extensive air shower events recorded by the ground-based GRAPES-3 experiment between 1 January 2014 and 26 October 2015 with a live time of 460 day. Our measurements provide an overlap with direct observations by satellite and balloon-based experiments. The electromagnetic and muon components in the shower were measured by a dense array of plastic scintillator detectors and a tracking muon telescope, respectively. The relative composition of the proton primary from the air shower data containing all primary particles was extracted using the multiplicity distribution of muons which is a sensitive observable for mass composition. The observed proton spectrum suggests a spectral hardening at ∼166 TeV and disfavors a single power law description of the spectrum up to the Knee energy (∼3 PeV).

Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection.

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab')2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

Extreme particle acceleration in the microquasar Cygnus X-3.

Super-massive black holes in active galaxies can accelerate particles to relativistic energies, producing jets with associated gamma-ray emission. Galactic 'microquasars', which are binary systems consisting of a neutron star or stellar-mass black hole accreting gas from a companion star, also produce relativistic jets, generally together with radio flares. Apart from an isolated event detected in Cygnus X-1, there has hitherto been no systematic evidence for the acceleration of particles to gigaelectronvolt or higher energies in a microquasar, with the consequence that we are as yet unsure about the mechanism of jet energization. Here we report four gamma-ray flares with energies above 100 MeV from the microquasar Cygnus X-3 (an exceptional X-ray binary that sporadically produces radio jets). There is a clear pattern of temporal correlations between the gamma-ray flares and transitional spectral states of the radio-frequency and X-ray emission. Particle acceleration occurred a few days before radio-jet ejections for two of the four flares, meaning that the process of jet formation implies the production of very energetic particles. In Cygnus X-3, particle energies during the flares can be thousands of times higher than during quiescent states.

Terrestrial gamma-ray flashes as powerful particle accelerators.

Strong electric discharges associated with thunderstorms can produce terrestrial gamma-ray flashes (TGFs), i.e., intense bursts of x rays and γ rays lasting a few milliseconds or less. We present in this Letter new TGF timing and spectral data based on the observations of the Italian Space Agency AGILE satellite. We determine that the TGF emission above 10 MeV has a significant power-law spectral component reaching energies up to 100 MeV. These results challenge TGF theoretical models based on runaway electron acceleration. The TGF discharge electric field accelerates particles over the large distances for which maximal voltages of hundreds of megavolts can be established. The combination of huge potentials and large electric fields in TGFs can efficiently accelerate particles in large numbers, and we reconsider here the photon spectrum and the neutron production by photonuclear reactions in the atmosphere.

Gamma-ray localization of terrestrial gamma-ray flashes.

Terrestrial gamma-ray flashes (TGFs) are very short bursts of high-energy photons and electrons originating in Earth's atmosphere. We present here a localization study of TGFs carried out at gamma-ray energies above 20 MeV based on an innovative event selection method. We use the AGILE satellite Silicon Tracker data that for the first time have been correlated with TGFs detected by the AGILE Mini-Calorimeter. We detect 8 TGFs with gamma-ray photons of energies above 20 MeV localized by the AGILE gamma-ray imager with an accuracy of ∼5-10° at 50 MeV. Remarkably, all TGF-associated gamma rays are compatible with a terrestrial production site closer to the subsatellite point than 400 km. Considering that our gamma rays reach the AGILE satellite at 540 km altitude with limited scattering or attenuation, our measurements provide the first precise direct localization of TGFs from space.

Combination therapy with the farnesyl protein transferase inhibitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical cancer models.

SCH66336 is a p.o.-active, farnesyl protein transferase inhibitor. SCH66336 inhibits farnesylation of RAS and other proteins in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. SCH58500 is a replication-deficient, recombinant adenovirus, which expresses the human p53 tumor suppressor. In preclinical models, SCH58500 has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53 and enhanced activity in combination with many chemotherapeutic drugs. Here we report that combination therapy with SCH66336 and SCH58500 has synergistic or additive antiproliferative effects on a panel of tumor cells lines in vitro. The efficacy of the three-drug combination of SCH66336, SCH58500, and paclitaxel was also examined in vitro. Each two-drug interaction displayed such marked synergy, the addition of a third drug to the statistical model could only yield additivity. Greater combined efficacy for SCH66336 and SCH58500 was also observed in vivo in the DU-145 human prostate and wap-ras/F transgenic mouse cancer models.

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.

Synergy (or antagonism) between two chemical agents is an in vitro empirical phenomenon, in which the observed effect of the combination is more (or less) than what would be predicted from the effects of each agent working alone. Although mathematical synergy is not directly provable in the clinical setting, it does predict a favorable outcome when the two therapeutics are combined in vivo and strongly suggests the presence of in vivo synergy. In contrast, overt antagonism warns of future problems. Sophisticated three-dimensional statistical modeling was used to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus (Ad)-mediated p53 gene therapy (p53 Ad) and paclitaxel (Taxol) in a panel of human tumor cell lines. Cells were either pretreated with paclitaxel 24 h before p53 Ad or treated with both agents simultaneously. Cell proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 gene therapy. In no case was the interaction antagonistic. Cell cycle analysis demonstrated that p53 Ad arrested cells in G0/G1 prior to apoptotic cell death, whereas paclitaxel arrested cells in G2-M prior to apoptotic cell death. When combined, the relative concentration of each agent determined the dominant cellular response. These results are consistent with the previously reported cell cycle effects of p53 or paclitaxel, respectively; however, these data fail to explain the observed drug synergy. We found that low concentrations of paclitaxel (1-14 nM) increased the number of cells transduced by recombinant Ad 3-35% in a dose-dependent manner, which is one possible mechanism for the observed synergy. Of particular note, the concentrations of paclitaxel responsible for increased Ad transduction were lower than the concentrations required for microtubule condensation. The efficacy of combination therapy was also evaluated in vivo. In the p53null SK-OV-3 xenograft model of ovarian cancer, a dosing schedule of p53 Ad that, by itself, had a relatively minimal effect on tumor burden (16%) caused a much greater decrease in tumor burden (55%) when combined with paclitaxel. Greater combined efficacy was also observed in the p53mut DU-145 prostate, p53mut MDA-MB-468 breast, and p53mut MDA-MB-231 breast cancer xenograft models in vivo. In summary, p53 Ad for cancer shows enhanced efficacy when combined with paclitaxel. This combination is recommended for clinical cancer trials.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

Structure-activity relationship of 3-substituted N-(pyridinylacetyl)-4- (8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene )- piperidine inhibitors of farnesyl-protein transferase: design and synthesis of in vivo active antitumor compounds.

Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.

Adenovirus-mediated p53 gene therapy has greater efficacy when combined with chemotherapy against human head and neck, ovarian, prostate, and breast cancer.

PURPOSE: Adenovirus-mediated p53 gene therapy for cancer is currently undergoing phase I/II clinical trials. The drug used in our clinical trials (p53 Ad; ACN53; SCH58500) consists of a replication-deficient, type 5 adenovirus vector expressing human wildtype p53 tumor suppressor under the control of the cytomegalovirus promoter. In preclinical models, p53 Ad has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53, both in vitro and in vivo. Results from early clinical trials using p53 gene therapy by itself support optimism for the future of this therapeutic approach. However, it is likely that many phase II/III trials will incorporate an arm comparing traditional chemotherapy against chemotherapy combined with p53 gene therapy. Therefore, it is important to study possible interactions between p53 Ad and chemotherapeutic drugs in preclinical models before starting the clinical trials. METHODS: Proliferation of tumor cells was quantitated after incubation with various combinations of p53 Ad and chemotherapeutic drugs. Human tumor xenografts in scid mice were dosed with intraperitoneal or intratumoral p53 Ad with or without chemotherapeutic drugs and the tumor burden after therapy monitored. RESULTS: p53 Ad combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide inhibited cell proliferation more effectively than chemotherapy alone in SCC-9 head and neck, SCC-15 head and neck, SCC-25 head and neck, SK-OV-3 ovarian, DU-145 prostate, MDA-MB-468 breast, and MDA-MB-231 breast tumor cells. No obvious dependence on dosing schedule was observed. Greater anticancer efficacy was also demonstrated in four human tumor xenograft models in vivo. Of particular significance, there was enhanced efficacy using the three drug combination of p53 Ad, cisplatin, and paclitaxel in an ovarian cancer model. CONCLUSION: These results support the combination of p53 gene therapy with chemotherapy in clinical trials.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

Discovery of powerful gamma-ray flares from the Crab Nebula.

The well-known Crab Nebula is at the center of the SN1054 supernova remnant. It consists of a rotationally powered pulsar interacting with a surrounding nebula through a relativistic particle wind. The emissions originating from the pulsar and nebula have been considered to be essentially stable. Here, we report the detection of strong gamma-ray (100 mega-electron volts to 10 giga-electron volts) flares observed by the AGILE satellite in September 2010 and October 2007. In both cases, the total gamma-ray flux increased by a factor of three compared with the non-flaring flux. The flare luminosity and short time scale favor an origin near the pulsar, and we discuss Chandra Observatory x-ray and Hubble Space Telescope optical follow-up observations of the nebula. Our observations challenge standard models of nebular emission and require power-law acceleration by shock-driven plasma wave turbulence within an approximately 1-day time scale.

Detection of gamma-ray emission from the Vela pulsar wind nebula with AGILE.

Pulsars are known to power winds of relativistic particles that can produce bright nebulae by interacting with the surrounding medium. These pulsar wind nebulae are observed by their radio, optical, and x-ray emissions, and in some cases also at TeV (teraelectron volt) energies, but the lack of information in the gamma-ray band precludes drawing a comprehensive multiwavelength picture of their phenomenology and emission mechanisms. Using data from the AGILE satellite, we detected the Vela pulsar wind nebula in the energy range from 100 MeV to 3 GeV. This result constrains the particle population responsible for the GeV emission and establishes a class of gamma-ray emitters that could account for a fraction of the unidentified galactic gamma-ray sources.