scholarly journals Glioblastoma stem cell (GSC)-derived PD-L1-containing exosomes activates AMPK/ULK1 pathway mediated autophagy to increase temozolomide-resistance in glioblastoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yong Zheng ◽  
Liang Liu ◽  
Yan Wang ◽  
Shan Xiao ◽  
Rongkang Mai ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Therapeutic Efficacy ◽  
Low Dose ◽  
High Dose ◽  
Glioblastoma Stem Cell ◽  
Compound C ◽  
Programmed Death ◽  
Protein Expressions

AbstractTemozolomide (TMZ)-resistance hampers the therapeutic efficacy of this drug for glioblastoma (GBM) treatment in clinic, and emerging evidences suggested that exosomes from GBM-derived stem cells (GSCs) contributed to this process, but the detailed mechanisms are still largely unknown. In the present study, we reported that GSCs derived programmed death-ligand 1 (PD-L1) containing exosomes activated AMPK/ULK1 pathway mediated protective autophagy enhanced TMZ-resistance in GBM in vitro and in vivo. Specifically, we noticed that continuous low-dose TMZ stimulation promoted GSCs generation and PD-L1 containing exosomes (PD-L1-ex) secretion in GBM cells, and that PD-L1-ex inhibited cell apoptosis and promoted cell autophagy to increased TMZ-resistance in GBM cells, which were reversed by co-treating cells with the autophagy inhibitor 3-methyladenine (3-MA). Consistently, upregulation of PD-L1 also increased TMZ-resistance in TS-GBM cells, and silencing of PD-L1 sensitized TR-GBM cells to TMZ. In addition, PD-L1-ex activated AMPK/ULK1 pathway to induce autophagy in TMZ treated GBM cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) promoted cell apoptosis in GBM cells co-treated with PD-L1-ex and high-dose TMZ. Finally, we evidenced that PD-L1-ex promoted tumor growth and Ki67 protein expressions to increase TMZ-resistance in GBM in vivo. Collectively, we concluded that GSCs-derived PD-L1-ex activated AMPK1/ULK1 signaling cascade mediated autophagy to increase TMZ-resistance in GBM, and this study provided potential strategies to improve the therapeutic efficacy of TMZ in GBM.

scholarly journals 735 Identification of a small molecule that prevents T cell exhaustion using machine learning algorithms paired with high-resolution single cell RNAseq

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A766-A766
Author(s):  
Isabelle Le Mercier ◽  
Sunny Sun ◽  
Dongmei Xiao ◽  
Laura Isacco ◽  
Daniel Treacy ◽  
...  
Keyword(s):  
T Cell ◽  
Low Dose ◽  
Machine Learning Algorithms ◽  
High Dose ◽  
T Cell Exhaustion ◽  
Tcr Signaling ◽  
Compound A ◽  
Cell Exhaustion

BackgroundT cell responses are tightly regulated and require a constant balance of signals during the different stages of their activation, expansion, and differentiation. As a result of chronic antigen exposure, T cells become exhausted in solid tumors, preventing them from controlling tumor growth.MethodsWe identified a transcriptional signature associated with T cell exhaustion in patients with melanoma and used our proprietary machine learning algorithms to predict molecules that would prevent T cell exhaustion and improve T cell function. Among the predictions, an orally available small molecule, Compound A, was highly predicted.ResultsCompound A was tested in an in vitro T cell Exhaustion assay and shown to prevent loss of proliferation and expression of immune checkpoint receptors. Transcriptionally, Compound A-treated cells looked indistinguishable from conventionally expanded, non-exhausted T cells. However, when assessed in a classical T cell activation assay, Compound A demonstrated dose dependent activity. At low dose, Compound A was immuno-stimulatory, allowing cells to divide further by preventing activation induced cell death. At higher doses, Compound A demonstrated immuno-suppressive activity preventing early CD69 upregulation and T cell proliferation. All together, these observations suggest that Compound A prevented exhaustion with a mechanism of action involving TCR signaling inhibition. While cessation of TCR signaling or rest has been recently associated with improved CAR-T efficacy by preventing or reversing exhaustion during the in vitro manufacturing phase, it is unclear if that mechanism would translate in vivo.Compound A was evaluated in the CT26 and MC38 syngeneic mouse models alongside anti-PD1. At low dose Compound A closely recapitulated anti-PD1 mediated cell behavior changes by scRNA-seq and flow cytometry in CT26 mice. At high dose, Compound A led to the accumulation of naive cells in the tumor microenvironment (TME) confirming the proposed mechanism of action. Low dose treatment was ineffective in MC38 mouse model but a pulsed treatment at high dose also recapitulated anti-PD1 activity in most animals. Importantly, we identified a new T cell population responding to anti-PD1 that was particularly increased in the MC38 mouse model; Compound A treatment also impacted this population.ConclusionsThese data confirm that mild TCR inhibition either suboptimal or fractionated can prevent exhaustion in vivo. However, this approach has a very limited window of activity between immuno-modulatory and immuno-suppressive effects, thereby limiting potential clinical benefit. Finally, these results demonstrate that our approach and platform was able to predict molecules that would prevent T cell exhaustion in vivo.

scholarly journals Επικεκαλυμμένες ενδαγγειακές προθέσεις και επαναστένωση μετά από αγγειοπλαστική

2014 ◽  
Author(s):  
Δημήτριος Λυσίτσας
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Low Dose ◽  
High Dose ◽  
Hsp 70

Εισαγωγή: Η υπερπλασία του έσω χιτώνα παίζει μείζων ρόλο στην επαναστένωση (in-stentrestenosis). Στην παρούσα μελέτη αξιολογήσαμε in vitro την επίδραση της D-24851(κυτταροτοξική ουσία που σταματά τον κυτταρικό κύκλο στο στάδιο G2-M) στονπολλαπλασιασμό των λείων μυϊκών κυττάρων και μελετήσαμε την ασφάλεια και τηνδραστικότητα μίας ενδαγγειακής πρόθεσης (stent) επικαλυμμένης με πολυμερή ουσία πουαπελευθερώνει την D-24851, στην αναστολή της υπερπλασίας του έσω χιτώνα χωρίς ναεμποδίζει την αναγεννητική ικανότητα του ενδοθηλίου σε in vivo πειραματικό μοντέλο.Υλικό και Μέθοδοι: Γυμνά μεταλλικά stent (n=6), stent επικαλυμμένα μόνο με πολυμερήουσία (polymer-coated, n=7) και stent επικαλυμμένα με πολυμερή ουσία πουαπελευθερώνουν 31±1μg (low-dose, n=7), 216±8 μg (high-dose, n=6) ή 1774±39 μg(extreme-dose, n=5) της D-24851 εμφυτεύτηκαν στις μηριαίες αρτηρίες λευκών New Zealandκουνελιών. Τα πειραματόζωα θυσιάστηκαν στις 28 ημέρες για ιστομορφομετρική ανάλυση.Για την αξιολόγηση της ενδοθηλιακής αναγέννησης στις 90 ημέρες, 12 πειραματόζωαχρησιμοποιήθηκαν για την τοποθέτηση polymer-coated (n=3), low dose (n=3), high dose(n=3) or extreme dose (n=3) ενδαγγειακών προθέσεων.Αποτελέσματα: In vitro η D-24851 αναστέλλει την υπερπλασία των λείων μυϊκών κυττάρωνκαι επάγει την απόπτωση τους χωρίς να αυξάνει την επαγωγή της heat shock protein 70(HSP-70), μία κυτταροπροστατευτική και αντι-αποπτωτική πρωτεΐνη. Η θεραπεία με lowdoseD-24851 stents συνδυάστηκε με 38% (P=0.029) μείωση της υπερπλαστικής περιοχήςτου έσω χιτώνα και 35% (P=0.003) μείωση της επι τοις εκατό στένωσης του αυλού σεσύγκριση με τα γυμνά μεταλλικά stents. Ο τραυματισμός και η φλεγμονή του αρτηριακού τοιχώματος δεν παρουσίασαν σημαντικές διαφορές μεταξύ των ομάδων. Τα επικαλυμμέναμόνο με πολυμερή ουσία stents εμφάνισαν παρόμοια ανάπτυξη νεοιστού σε σύγκριση με ταγυμνά μεταλλικά stents. Ωστόσο, όλες οι ομάδες των stents με D-24851 παρουσίασαν ατελήενδοθηλιοποίηση συγκρινόμενα με τα polymer-coated stents.Συμπεράσματα: Οι επικεκαλυμμένες ενδαγγειακές προσθέσεις με πολυμερή ουσία καιχαμηλη δόση D-24851 μειώνουν σημαντικά την υπερπλασιά του έσω χιτώνα. Λόγω τηςατελούς ενδοθηλιοποίησης, μακράς διάρκειας μελέτες είναι απαραίτητες για ναπιστοποιήσουν ότι η αναστολή του νεοιστού παραμένει και μετά τις 28 ημέρες.

scholarly journals Toll-Like Receptor Signaling in Airborne Burkholderia thailandensis Infection

2009 ◽  
Vol 77 (12) ◽  
pp. 5612-5622 ◽  
Author(s):  
T. Eoin West ◽  
Thomas R. Hawn ◽  
Shawn J. Skerrett
Keyword(s):  
Low Dose ◽  
Inflammatory Responses ◽  
High Dose ◽  
Tlr Signaling ◽  
Necrosis Factor Alpha ◽  
Airborne Infection ◽  
Essential Components ◽  
High Doses

ABSTRACT Melioidosis is a tropical disease endemic in southeast Asia and northern Australia caused by the gram-negative soil saprophyte Burkholderia pseudomallei. Although infection is often systemic, the lung is frequently involved. B. thailandensis is a closely related organism that at high doses causes lethal pneumonia in mice. We examined the role of Toll-like receptors (TLRs), essential components of innate immunity, in vitro and in vivo during murine B. thailandensis pneumonia. TLR2, TLR4, and TLR5 mediate NF-κB activation by B. thailandensis in transfected HEK293 or CHO cells. In macrophages, TLR4 and the adaptor molecule MyD88, but not TLR2 or TLR5, are required for tumor necrosis factor alpha production induced by B. thailandensis. In low-dose airborne infection, TLR4 is needed for early, but not late, bacterial containment, and MyD88 is essential for control of infection and host survival. TLR2 and TLR5 are not necessary to contain low-dose infection. In high-dose airborne infection, TLR2 deficiency confers a slight survival advantage. Lung and systemic inflammatory responses are induced by low-dose inhaled B. thailandensis independently of individual TLRs or MyD88. These findings suggest that redundancy in TLR signaling or other MyD88-dependent pathways may be important in pneumonic B. thailandensis infection but that MyD88-independent mechanisms of inflammation are also activated. TLR signaling in B. thailandensis infection is substantially comparable to signaling induced by virulent B. pseudomallei. These studies provide additional insights into the host-pathogen interaction in pneumonic Burkholderia infection.

scholarly journals Concordance between Thioacetamide-Induced Liver Injury in Rat and Human In Vitro Gene Expression Data

2020 ◽  
Vol 21 (11) ◽  
pp. 4017
Author(s):  
Patric Schyman ◽  
Richard L. Printz ◽  
Shanea K. Estes ◽  
Tracy P. O’Brien ◽  
Masakazu Shiota ◽  
...  
Keyword(s):  
Liver Injury ◽  
Low Dose ◽  
Pearson Correlation ◽  
Organ Injury ◽  
High Dose ◽  
Rna Seq ◽  
Bile Duct Proliferation ◽  
Toxicological Studies

The immense resources required and the ethical concerns for animal-based toxicological studies have driven the development of in vitro and in silico approaches. Recently, we validated our approach in which the expression of a set of genes is uniquely associated with an organ-injury phenotype (injury module), by using thioacetamide, a known liver toxicant. Here, we sought to explore whether RNA-seq data obtained from human cells (in vitro) treated with thioacetamide-S-oxide (a toxic intermediate metabolite) would correlate across species with the injury responses found in rat cells (in vitro) after exposure to this metabolite as well as in rats exposed to thioacetamide (in vivo). We treated two human cell types with thioacetamide-S-oxide (primary hepatocytes with 0 (vehicle), 0.125 (low dose), or 0.25 (high dose) mM, and renal tubular epithelial cells with 0 (vehicle), 0.25 (low dose), or 1.00 (high dose) mM) and collected RNA-seq data 9 or 24 h after treatment. We found that the liver-injury modules significantly altered in human hepatocytes 24 h after high-dose treatment involved cellular infiltration and bile duct proliferation, which are linked to fibrosis. For high-dose treatments, our modular approach predicted the rat in vivo and in vitro results from human in vitro RNA-seq data with Pearson correlation coefficients of 0.60 and 0.63, respectively, which was not observed for individual genes or KEGG pathways.

scholarly journals Efficacy of NS-718, a Novel Lipid Nanosphere-Encapsulated Amphotericin B, againstCryptococcus neoformans

1998 ◽  
Vol 42 (7) ◽  
pp. 1722-1725 ◽  
Author(s):  
Mohammad Ashraf Hossain ◽  
Shigefumi Maesaki ◽  
Hiroshi Kakeya ◽  
Tetsuhiro Noda ◽  
Katsunori Yanagihara ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Antifungal Agent ◽  
Low Dose ◽  
Yeast Cells ◽  
High Dose ◽  
Pulmonary Cryptococcosis ◽  
High Dose Therapy ◽  
Dose Therapy

ABSTRACT In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC forCryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.

scholarly journals In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection

2009 ◽  
Vol 90 (3) ◽  
pp. 602-613 ◽  
Author(s):  
Laurent Gillet ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Heparan Sulfate ◽  
Low Dose ◽  
Envelope Glycoproteins ◽  
High Dose ◽  
Cell Binding ◽  
Host Colonization ◽  
Infection Dose ◽  
Herpesvirus 4

Many herpesviruses bind to heparan sulfate (HS). Murid herpesvirus-4 (MuHV-4) does so via its envelope glycoproteins gp70 and gH/gL. MuHV-4 gp150 further regulates an HS-independent interaction to make that HS-dependent too. Cell binding by MuHV-4 virions is consequently strongly HS-dependent. Gp70 and gH/gL show some in vitro redundancy: an antibody-mediated blockade of HS binding by one is well tolerated, whereas a blockade of both severely impairs infection. In order to understand the importance of HS binding for MuHV-4 in vivo, we generated mutants lacking both gL and gp70. As expected, gL−gp70− MuHV-4 showed very poor cell binding. It infected mice at high dose but not at low dose, indicating defective host entry. But once entry occurred, host colonization, which for MuHV-4 is relatively independent of the infection dose, was remarkably normal. The gL−gp70− entry deficit was much greater than that of gL− or gp70− single knockouts. And gp150 disruption, which allows HS-independent cell binding, largely rescued the gL−gp70− cell binding and host entry deficits. Thus, it appeared that MuHV-4 HS binding is important in vivo, principally for efficient host entry.

scholarly journals Kaempferol Protects Mice from D-GalN/LPS-induced Acute Liver Failure by Regulating the Autophagy Pathway

2020 ◽  
Author(s):  
Yuan Tian ◽  
Feng Ren ◽  
Ling Xu ◽  
Weihua Li ◽  
Mei Liu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Liver Function ◽  
Opposite Effect ◽  
Low Dose ◽  
High Dose ◽  
Severe Liver ◽  
Autophagy Pathway ◽  
Different Doses

Abstract Background Kaempferol, a flavonoid compound present in many edible plants, has been used in traditional medicine and has various biological functions. Acute liver failure (ALF) is a lethal clinical syndrome with severe liver function damage. There are currently no effective treatments for ALF except for liver transplantation. The aim of this study is to explored the mechanisms underlying the therapeutic effect of kaempferol in ALF. Methods The ALF mouse model was established using D-galactosamine (D-GalN, 700 mg/kg)/lipopolysaccharide (LPS, 10 µg/kg). Two hours before the administration of D-GalN/LPS, different group of mice were pretreated according different doses of kaempferol, 6 hours after injection of D-GalN/LPS, and then killed. The survival rate, liver function and inflammatory cytokine levels were assessed. It was determined whether kaempferol pretreatment protected hepatocytes from ALF induced by D-GalN/LPS via autophagy pathway in vivo and in vitro. Results Pretreatment with a high dose of kaempferol significantly decreased the survival rate and increased severe liver damage; however, pretreatment with a low dose of kaempferol showed the opposite effect. Furthermore, pretreatment with a high dose of kaempferol augment the levels of proinflammatory cytokines and markers of the MAPK signaling pathway, while pretreatment with a low dose of kaempferol showed the opposite effect. In addition, pretreatment with a high dose of kaempferol decreased autophagy, but pretreatment with a low dose of kaempferol increased autophagy in vivo and in vitro. It was also proved that pretreatment with 3-methyadenine (3- MA) or Atg7 siRNA to inhibit autophagy partially negated the hepatoprotective effect of kaempferol (5 mg/kg) pretreatment in ALF mice induced by D-GalN/LPS. Conclusions Our findings demonstrate that effects of different doses of kaempferol on D-GalN/LPS-induced ALF is remarkably different by regulating the autophagy pathway. Therefore, we should consider selecting the optimal dose of kaempferol as a potential treatment method for patients with ALF.

Abstract 1079: Modulation of Atherosclerotic Plaque Characteristics by Minocycline: An Evaluation by Molecular Imaging of Matrix Metalloprotainase (MMP) Expression

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Satoru Ohshima ◽  
Shinichiro Fujimoto ◽  
Hironori Nakagami ◽  
Nezam Haider ◽  
Jun Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Molecular Imaging ◽  
Low Dose ◽  
High Cholesterol Diet ◽  
High Dose ◽  
Targeted Interventions ◽  
Mmp Inhibitor ◽  
Atherosclerotic Lesions

MMP activation in atherosclerotic lesions plays an important role in plaque progression and vulnerability, and is a potential target for plaque stabilization. We evaluated the effectiveness of minocycline (MC) in modulation of plaque characteristics using molecular imaging of MMP expression, compared with fluvastatin (FS). 38 NZW rabbits with experimental atherosclerotic lesions were subjected to in vivo micro SPECT/CT imaging for the assessment of MMP activity using 99mTc-labeled broad spectrum MMP inhibitor (MPI). Atherosclerotic lesions were produced by balloon deendotheliazation of abdominal aorta, and high cholesterol diet for 4 months. Of these, 7 received low dose MC (1.5mg/kg), 7 high dose MC (3mg/kg, N=7), 6 FS (1mg/kg), and 6 combination of low dose MC and FS (MC+FS) in the fourth month, 12 received no intervention (Rx-control). 8 unmanipulated normal chow-fed rabbits were used as disease controls (Dz-control). After in vivo imaging, aortas were explanted for %ID/g MPI uptake, histological characterization, and MMP activity assays. Parallel in vitro studies were performed for the effect of MC and FS on NF-κB, VCAM-1 and MMP-9 expression from cultured TNF-α-stimulated smooth muscle cells and macrophages. Target accumulation of MPI was best visualized in atherosclerotic aorta in Rx-control atherosclerotic rabbits. % ID/g MPI in Rx-control (.10±.04%) was significantly higher than Dz-control (.016±.004%), MC-low (.081±.02%) and high (.045±.01%) dose, FS (.056±.011%), and MC+FS (.049±.005%) rabbits, and showed a significant correlation with histologically and biochemically-verified MMP-2, -9 activity. MC-treated plaques showed marked reduction in inflammation and increase in smooth muscle content. In vitro cell culture confirmed that NF-κB and VCAM-1 expression in activated VSMC were reduced by MC, and that MMP-9 expression in activated macrophage was also inhibited by MC. The present study demonstrates that MC is equally effective as FS in modulation of plaque characteristics, and that molecular imaging can characterize various biologic processes in vivo and allows for the study of the efficacy of targeted interventions.

scholarly journals Effect of mini-tyrosyl-tRNA synthetase on ischemic angiogenesis, leukocyte recruitment, and vascular permeability

2008 ◽  
Vol 295 (4) ◽  
pp. R1138-R1146 ◽  
Author(s):  
Gang Cheng ◽  
Hua Zhang ◽  
Xianglei Yang ◽  
Eleni Tzima ◽  
Karla L. Ewalt ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Low Dose ◽  
Trna Synthetase ◽  
Mustard Oil ◽  
Blue Dye ◽  
High Dose ◽  
Mouse Ear ◽  
High Concentrations

Mini-tyrosyl-tRNA synthetase (mini-TyrRS), the N-terminal domain of tyrosyl-tRNA synthetase, is a recently identified protein released by endothelial cells that exhibits angiogenic and leukocyte chemoattractant, ELR-motif (Glu-Leu-Arg)-dependent activities in vitro. We sought to determine whether exogenous mini-TyrRS exerts these and other cytokine-like actions in physiological and pathological settings in vivo. High-dose mini-TyrRS (600 μg·kg−1·day−1) augmented while low-dose mini-TyrRS (3 μg·kg−1·day−1), unexpectedly, inhibited angiogenesis in the ischemic mouse ear. Enhanced angiogenesis was associated with increased CD45- and CD4-positive leukocyte accumulation. Mini-TyrRS also had biphasic actions on both basal and mustard oil-evoked and VEGF-evoked leakage of Evan's blue dye-albumin in nonischemic ear and in endothelial cell monolayers, that is, low-dose inhibited and high-dose augmented leakage. Mutation of the ELR motif of mini-TyrRS abolished the above activities. Mini-TyrRS was reduced (immunoblot) in extracts of ischemic calf muscle and in thoracic aorta explants exposed to hypoxia or VEGF. Inhibition of VEGF with a soluble Flt1 “trap” protein abolished this hypoxic-induced reduction in mini-TyrRS in aorta explants. These data show that mini-TyrRS has dose-dependent biphasic effects on ischemic angiogenesis and vascular permeability in vivo, that is, antiangiogenic and antipermeability activities at low concentration and proangiogenic, propermeability activities at high concentrations.

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