Smoking influences the yield of dendritic cells for cancer immunotherapy

Background: Dendritic cell (DC)-based vaccination is considered to be a potentially effective therapeutic strategy against advanced cancer. The aim of this study was to address the smoking history that might affect the preparation of DC vaccines in validated instructional manufacture. Materials and Methods: Data on mature DCs generated from 102 sessions of leukapheresis performed on 92 patients with advanced cancer or sarcoma were retrospectively evaluated and compared in relation to the data between their smoking history and the generation of DCs from these patients. 61 patients with adenocarcinoma, including 7 with lung, 10 with breast, 8 with stomach, 12 with colorectal, and 23 with pancreatic adenocarcinoma were enrolled. Results: The average yield of autologous DCs (15.5 ± 8.3x107) was thought to be dependent on the number of monocytes (124.2 ± 74.1x107) collected by leukapheresis. The average ratio of DCs/apheresed monocytes (DC/aM ratio) was lower in the smoker group (11.1 ± 7.2%) than that in the non-smoker group (17.2 ± 9.3%, p=0.001). The number of DCs and the DC/aM ratio were lower in the patients with gastric and pancreatic cancer than in those with adenocarcinoma of other sites. Conclusions: As cancer therapy moves forward into the field of personaArticlePharmaceutical Regulatory Affairs. 4(1):133 (2015)journal articl

Rheb is a critical regulator of autophagy during myocardial ischemia: pathophysiological implications in obesity and metabolic syndrome

BACKGROUND: Rheb is a GTP-binding protein that promotes cell survival and mediates the cellular response to energy deprivation (ED). The role of Rheb in the regulation of cell survival during ED has not been investigated in the heart. METHODS AND RESULTS: Rheb is inactivated during cardiomyocyte (CM) glucose deprivation (GD) in vitro, and during acute myocardial ischemia in vivo. Rheb inhibition causes mTORC1 inhibition, because forced activation of Rheb, through Rheb overexpression in vitro and through inducible cardiac-specific Rheb overexpression in vivo, restored mTORC1 activity. Restoration of mTORC1 activity reduced CM survival during GD and increased infarct size after ischemia, both of which were accompanied by inhibition of autophagy, whereas Rheb knockdown increased autophagy and CM survival. Rheb inhibits autophagy mostly through Atg7 depletion. Restoration of autophagy, through Atg7 reexpression and inhibition of mTORC1, increased cellular ATP content and reduced endoplasmic reticulum stress, thereby reducing CM death induced by Rheb activation. Mice with high-fat diet-induced obesity and metabolic syndrome (HFD mice) exhibited deregulated cardiac activation of Rheb and mTORC1, particularly during ischemia. HFD mice presented inhibition of cardiac autophagy and displayed increased ischemic injury. Pharmacological and genetic inhibition of mTORC1 restored autophagy and abrogated the increase in infarct size observed in HFD mice, but they failed to protect HFD mice in the presence of genetic disruption of autophagy. CONCLUSIONS: Inactivation of Rheb protects CMs during ED through activation of autophagy. Rheb and mTORC1 may represent therapeutic targets to reduce myocardial damage during ischemia, particularly in obese patient

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics “biotin-switch” approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis

Oscillatory Fractional Brownian Motion and Hierarchical Random Walks

We introduce oscillatory analogues of fractional Brownian motion, sub-fractional Brownian motion and other related long range dependent Gaussian processes, we discuss their properties, and we show how they arise from particle systems with or without branching and with different types of initial conditions, where the individual particle motion is the so-called c-random walk on a hierarchical group. The oscillations are caused by the discrete and ultrametric structure of the hierarchical group, and they become slower as time tends to infinity and faster as time approaches zero. We also give other results to provide an overall picture of the behavior of this kind of systems, emphasizing the new phenomena that are caused by the ultrametric structure as compared with results for analogous models on Euclidean space

Endocannabinoids Generated by Ca2+ or by Metabotropic Glutamate Receptors Appear to Arise from Different Pools of Diacylglycerol Lipase

The identity and subcellular sources of endocannabinoids (eCBs) will shape their ability to affect synaptic transmission and, ultimately, behavior. Recent discoveries support the conclusion that 2-arachidonoyl glycerol, 2-AG, is the major signaling eCB, however, some important issues remain open. 2-AG can be synthesized by a mechanism that is strictly Ca2+-dependent, and another that is initiated by G-protein coupled receptors (GPCRs) and facilitated by Ca2+. An important question is whether or not the 2-AG in these cases is synthesized by the same pool of diacylglycerol lipase alpha (DAGLα). Using whole-cell voltage-clamp techniques in CA1 pyramidal cells in acute in vitro rat hippocampal slices, we investigated two mechanistically distinct eCB-mediated responses to address this issue. We now report that pharmacological inhibitors of DGLα have quantitatively different effects on eCB-mediated responses triggered by different stimuli, suggesting that functional, and perhaps physical, distinctions among pools of DAGLα exist

A biophysical model of endocannabinoid-mediated short term depression in hippocampal inhibition

Memories are believed to be represented in the synaptic pathways of vastly interconnected networks of neurons. The plasticity of synapses, that is, their strengthening and weakening depending on neuronal activity, is believed to be the basis of learning and establishing memories. An increasing number of studies indicate that endocannabinoids have a widespread action on brain function through modulation of synap–tic transmission and plasticity. Recent experimental studies have characterised the role of endocannabinoids in mediating both short- and long-term synaptic plasticity in various brain regions including the hippocampus, a brain region strongly associated with cognitive functions, such as learning and memory. Here, we present a biophysically plausible model of cannabinoid retrograde signalling at the synaptic level and investigate how this signalling mediates depolarisation induced suppression of inhibition (DSI), a prominent form of shortterm synaptic depression in inhibitory transmission in hippocampus. The model successfully captures many of the key characteristics of DSI in the hippocampus, as observed experimentally, with a minimal yet sufficient mathematical description of the major signalling molecules and cascades involved. More specifically, this model serves as a framework to test hypotheses on the factors determining the variability of DSI and investigate under which conditions it can be evoked. The model reveals the frequency and duration bands in which the post-synaptic cell can be sufficiently stimulated to elicit DSI. Moreover, the model provides key insights on how the state of the inhibitory cell modulates DSI according to its firing rate and relative timing to the post-synaptic activation. Thus, it provides concrete suggestions to further investigate experimentally how DSI modulates and is modulated by neuronal activity in the brain. Importantly, this model serves as a stepping stone for future deciphering of the role of endocannabinoids in synaptic transmission as a feedback mechanism both at synaptic and network level

Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus Magnaporthe oryzae

In this study we characterize a novel positive and single stranded RNA (ssRNA) mycovirus isolated from the rice field isolate of Magnaporthe oryzae Guy11. The ssRNA contains a single open reading frame (ORF) of 2,373 nucleotides in length and encodes an RNA-dependent RNA polymerase (RdRp) closely related to ourmiaviruses (plant viruses) and ourmia-like mycoviruses. Accordingly, we name this virus Magnaporthe oryzae ourmia-like virus 1 (MOLV1). Although phylogenetic analysis suggests that MOLV1 is closely related to ourmia and ourmia-like viruses, it has some features never reported before within the Ourmiavirus genus. 3' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) and extension poly(A) tests (ePAT) suggest that the MOLV1 genome contains a poly(A) tail whereas the three cytosine and the three guanine residues present in 5' and 3' untranslated regions (UTRs) of ourmia viruses are not observed in the MOLV1 sequence. The discovery of this novel viral genome supports the hypothesis that plant pathogenic fungi may have acquired this type of viruses from their host plants

Differential VLBI observations of two sub-satellites of SELENE (KAGUYA), OKINA and OUNA for lunar gravimetry

The Japanese lunar explorer SELENE (KAGUYA), which has been launched on Sep. 14th, 2007, utilizes VLBI observations in lunar gravimetry investigations. This can particularly improve the accuracy of the low degree gravitational harmonics. Combination of ground based VLBI observations and Doppler measurements of the spacecrafts enable three dimensional orbit determinations and it can improve the knowledge of the gravity field near the limb. Differential VLBI Radio sources called VRAD experiment involves two on-board sub-satellites, Rstar (Okina) and VBtar (Ouna). These will be observed using differential VLBI to measure the trajectories of the satellites with the Japanese network named VERA (VLBI Exploration of Radio Astrometry) and an international VLBI network. Two new techniques, a multi-frequency VLBI method and the same-beam VLBI method, are used to precisely measure the angular distance between the two sub-satellite radio sources Okina and Ouna. The observations are at three frequencies in S-band, 2212, 2218 and 2287 MHz, and one in X-band, 8456 MHz. We have succeeded in making VLBI observations of Okina/Ouna with VERA and the international network, and have also succeeded in correlating of signals from Okina/Ouna, and obtained phase delays with an accuracy of several pico-seconds in S-band