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 Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion

2020 ◽  
Author(s):  
Caroline Lamarche ◽  
German E. Novakovsky ◽  
Christopher N. Qi ◽  
Evan W. Weber ◽  
Crystal L. Mackall ◽  
...  
Keyword(s):  
T Cell ◽  
Regulatory T Cell ◽  
T Cell Exhaustion ◽  
Antigen Receptors ◽  
Chronic Stimulation ◽  
T Cell Therapy ◽  
Cell Exhaustion ◽  
Graft Versus Host

AbstractRegulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how two methods which induce conventional T cell exhaustion – repetitive stimulation or expression of a tonic-signaling chimeric antigen receptor (CAR) – affect human Tregs. With each repetitive polyclonal stimulation Tregs progressively acquired an exhausted phenotype, and became less suppressive in vitro. Tregs expressing a tonic-signaling CAR rapidly acquired an exhausted phenotype and had major changes in their transcriptome and metabolism. Although tonic-signaling CAR-Tregs remained stable and suppressive in vitro, they lost in vivo function, as tested in a model of xenogeneic graft-versus-host disease. The finding that human Tregs are susceptible to exhaustion has important implications for the design of Treg adoptive immunotherapy strategies.

100 Drug-regulatable engineered T cells eliminate CD33+ and CD33ΔE2+ AML

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A111-A111
Author(s):  
Jacob Appelbaum ◽  
Wai-Hang Leung ◽  
Unja Martin ◽  
Kaori Oda ◽  
Giacomo Tampella ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Splice Variants ◽  
Antigen Binding ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T

BackgroundBioengineered T cell treatments for acute myeloid leukemia (AML) are challenged by near universal expression of leukemia antigens on normal hematopoietic stem/progenitor cells:1 2 ‘on target/off tumor‘ activity may cause myelosuppression while sustained antigen exposure can lead to T cell exhaustion.3 In addition, splicing variants may allow antigen escape. We hypothesize that by using a novel CD33-C2-specific single domain VHH antibody as the antigen targeting domain in dimerizing agent-regulated immunoreceptor complex T cells (DARIC T cells), we will enable pharmacologically-controllable targeting of CD33, allowing eradication of leukemia expressing either of the major splice variants of CD33: i.e., full-length CD33 or CD33ΔE2.MethodsWe engineered DARIC-expressing lentiviral vectors containing encoding separated CD33-C2-specific antigen binding and 41BB-CD3zeta signaling chains that heterodimerize following addition of rapamycin via embedded FKBP12 and FRB* domains.4 Peripheral blood mononuclear cells were stimulated with IL-2, anti-CD3, and anti-CD28 antibodies 24h prior to transduction with DARIC33 lentiviral vector. Surface expression of antigen binding or signaling chains was assessed using biotinylated CD33, or antibodies to VHH-domains or FRB* respectively. Rapamycin-dependent in vitro activity was measured by IFNg release. To evaluate in vivo activity, NSG mice injected with 1 × 105 MOLM-14/luc cells were treated 5-7 days later with 1 × 107 DARIC33 T cells in the presence or absence of rapamycin and tumor progression followed by luciferase activity.ResultsDARIC33+ T cells bound biotinylated-CD33, anti-VHH and anti-FRB* antibodies. Rapamycin addition increased expression of both signaling and antigen-recognition chains, suggesting augmented receptor stability in the presence of dimerizing drug. In the presence of rapamycin, coculture of DARIC33 T cells with cell lines expressing either full length or CD33ΔE25 showed equivalent rapamycin-dependent activation, demonstrating DARIC33 responds to both splice variants. Titration experiments showed rapamycin-dependent activation with EC50 = 25pM. Negligible IFNg release was observed in the absence of drug. DARIC33 T cells significantly extended survival of AML-bearing mice, but only when treated with rapamycin. The DARIC33 T cells were activated in vivo by sub-immunosuppressive rapamycin dosing, as weekly or 0.1 mg/kg QOD dosing led to similar levels of tumor suppression.ConclusionsDARIC33 T cells appear to be potent antileukemic agents: they are activated by AML cell lines in vitro as demonstrated by cytokine release and cytotoxicity, and significantly extend survival in an aggressive xenograft model. Temporal control provided by the DARIC architecture promises to enhance safety and potentially efficacy of CAR T therapy for AML, for example by enabling hematopoietic recovery or providing T cell rest.ReferencesPerna F, Berman SH, Soni RK, Mansilla-Soto J, Eyquem J, Hamieh M, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell 2017 Oct 9;32(4):506–519.e5.Haubner S, Perna F, Köhnke T, Schmidt C, Berman S, Augsberger C, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019 Jan;33(1):64.Lamarche C, Novakovsky GE, Qi CN, Weber EW, Mackall CL, Levings MK. Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion. bioRxiv. 2020 Jun 28;2020.06.27.175158.Leung W-H, Gay J, Martin U, Garrett TE, Horton HM, Certo MT, et al. Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization. JCI Insight [Internet]. 2019 Jun 6 [cited 2019 Jun 11];4(11). Available from: https://insight.jci.org/articles/view/124430Pérez-Oliva AB, Martínez-Esparza M, Vicente-Fernández JJ, Corral-San Miguel R, García-Peñarrubia P, Hernández-Caselles T. Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells. Glycobiology 2011;21(6):757–770.

769 CAR T cells undergoing epigenetic reprogramming by low-dose decitabine enhances persistent anti-tumor efficacy in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A817-A817
Author(s):  
Yao Wang ◽  
Weidong Han ◽  
Chuan Tong ◽  
Zhiqianag Wu ◽  
Hanren Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Low Dose ◽  
Cytokine Production ◽  
De Novo ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T

BackgroundAnti-CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a resounding effect on the treatment of B-ALL. However, CAR T cells have been less effective against B-cell non-Hodgkin lymphoma (B-NHL), in part because they become a exhausted state triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function.1-4 It has recently been demonstrated that de novo DNA methylation promoted T-cell exhaustion, whereas methylation inhibition enhanced ICB-mediated T-cell rejuvenation in vivo.5 6 FDA-approved DNA demethylating agents, such as decitabine (DAC), may provide a means to modify exhaustion-associated DNA methylation programs that restrict ICB-responsiveness.MethodsWe treated CAR (CAR-CD19-expressing) T cells with low-dose DAC (dCAR T cells), to determine its effects on antitumor activities, exhaustion- and memory-associate cell phenotype change, cell cytokine production, and cell proliferation. Its impact on antitumor activities was evaluated in vitro functional assays and mouse in vivo studies. We also conducted western blot, flow cytometry, methylation analysis, RNA in situ hybridization and high throughput RNA sequencing to determine the underlying mechanisms of dCAR T cell function.ResultsThe low-dose, short-term DAC treatment in vitro enhanced the central memory (Tcm) population and the ration of CD4/CD8, and induced degradation of DNMT3a.CAR T cell treated by DAC developing into less-differention status by enhancing memory. dCAR T cells exhibit enhanced antitumour reactivity and the maintenance of a memory-like phenotype at low effector:target ratios. Especially shown by the ‘stress test’, the dCAR T cells at very low doses could efficiently control tumours with a very large burden, and have effective recall responses upon tumour re-challenge in vivo. Importantly, the dCAR T cells maintained a higher proportion of cells with a memory phenotype than did the CAR T cells under long-term tumour stimulation. Transcription of gene sets involved in memory maintenance, proliferation, cytokine production and anti-inhibitor processes was triggered by antigen-expressing target cells upon DAC exposure before antigen stimulation. dCAR T cells avoided the exhaustion programme induced during tumour cell stimulation; they did not upregulate the expression of genes encoding inhibitory receptors and retained relatively high expression of memory related transcription factors and genes.ConclusionsCAR T cells underwent DNA reprogramming after DAC treatment, which induced significant sustained cell expansion, cytotoxicity, and cytokine production and reduced exhaustion after antigen exposure.AcknowledgementsWe thank Professor Lin Xin of Tsinghua University and Professor Mingzhou Guo of Chinese PLA General Hospital for support of data analysis.ReferencesWherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15:486–499. doi:10.1038/nri3862Wherry EJ, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007;27:670–684. doi:10.1016/j.immuni.2007.09.006Schietinger A, et al. Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 2016;45:389–401. doi:10.1016/j.immuni.2016.07.011Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol 2014;35:51–60. doi:10.1016/j.it.2013.10.001Ghoneim HE, et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 2017;170:142–157.e119. doi:10.1016/j.cell.2017.06.007Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165. doi:10.1126/science.aaf2807

scholarly journals GBP1 Facilitates Indoleamine 2,3-Dioxygenase Extracellular Secretion to Promote the Malignant Progression of Lung Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Yinnan Meng ◽  
Wei Wang ◽  
Meng Chen ◽  
Kuifei Chen ◽  
Xinhang Xia ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cell ◽  
Immune Escape ◽  
In Vitro Study ◽  
Malignant Progression ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Extracellular Secretion

IDO1-mediated immune escape can lead to the malignant progression of tumors. However, the precise mechanism of IDO1 remains unclear. This study showed that IDO1 can bind to GBP1 and increase the extracellular secretion of IDO1 with the assistance of GBP1, thereby promoting the malignant proliferation and metastasis of lung cancer. In vitro study showed that the high expression levels of IDO1 and GBP1 in lung cancer cells promoted cell invasion and migration. In vivo study revealed that knock-down of IDO1 and GBP1 inhibited tumor growth and metastasis. In addition, Astragaloside IV reduces the extracellular secretion of IDO1 by blocking the interaction of IDO1 and GBP1, thereby reducing T cell exhaustion and inhibiting tumor progression. These results suggest that blocking the extracellular secretion of IDO1 may prevent T cell exhaustion and thereby enhance the effect of PD-1 inhibitors on cancer treatment.

VEGF-A drives TOX-dependent T cell exhaustion in anti–PD-1–resistant microsatellite stable colorectal cancers

2019 ◽  
Vol 4 (41) ◽  
pp. eaay0555 ◽  
Author(s):  
Chang Gon Kim ◽  
Mi Jang ◽  
Youngun Kim ◽  
Galam Leem ◽  
Kyung Hwan Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Immune Checkpoint Blockade ◽  
T Cell Exhaustion ◽  
Multiple Cancer ◽  
Cell Exhaustion ◽  
Cancer Settings

Although immune checkpoint blockade therapies have demonstrated clinical efficacy in cancer treatment, harnessing this strategy is largely encumbered by resistance in multiple cancer settings. Here, we show that tumor-infiltrating T cells are severely exhausted in the microsatellite stable (MSS) colorectal cancer (CRC), a representative example of PD-1 blockade–resistant tumors. In MSS CRC, we found wound healing signature to be up-regulated and that T cell exhaustion is driven by vascular endothelial growth factor-A (VEGF-A). We report that VEGF-A induces the expression of transcription factor TOX in T cells to drive exhaustion-specific transcription program in T cells. Using a combination of in vitro, ex vivo, and in vivo mouse studies, we demonstrate that combined blockade of PD-1 and VEGF-A restores the antitumor functions of T cells, resulting in better control of MSS CRC tumors.

142 Contextual reprogramming of CAR T cells for the treatment of HER2-expressing cancers

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A156-A156
Author(s):  
Zhifen Yang ◽  
Lingyu Li ◽  
Ahu Turkoz ◽  
Pohan Chen ◽  
Hana Choi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Proliferation In Vitro

BackgroundCombining checkpoint inhibition (CPI) to adoptive cell therapy (ACT) is a promising strategy to prevent chimeric antigen receptor (CAR)-engineered T cell exhaustion and improve outcomes. However, cumulative toxicities and costs limit this approach. Here, we apply a conditional, antigen-dependent non-editing CRISPR interference-(CRISPRi) modulation circuit that we originally described in yeast and eukaryotes1–3 (RB-340-1) to promote CAR T resilience to checkpoint suppression extending in vivo persistence and effectiveness.MethodsRB-340-1 is an CAR T cell product engineered via synthetic biology approaches to express a combination of molecules to prevent CAR T cell exhaustion and improve solid tumor treatment outcomes. The components include two constructs. The first construct encodes an anti-HER2 (4D5) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a programmed cell death protein 1 (PD1) promoter region-targeting single guide RNA (PD1sg). The second construct encodes a protein, linker for activation of T cells (LAT), complexed to nuclease-deactivated/dead Cas9 (dCas9)-Kruppel-associated box (Krab) via a TEV-cleavable linker. Activation of CAR brings CAR-TEV in close proximity to the LAT-dCas9-Krab complex releasing the enzyme for nuclear localization to the PD1 regulatory region to conditionally and reversibly suppress its expression. RB-340-1 was compared in vitro and in vivo against conventional and control (cRB-340-1, lacking PD1sg) HER2 CAR T cells in combination with CPI with Atezolizumab (5 mg/Kg administered intravenously twice a week).ResultsRB-340-1 consistently induced higher production of homeostatic cytokines such as IL-2 resulting in significantly enhanced proliferation in vitro (figure 1a). Our in vivo data showed significantly enhanced suppression of growth of HER2+ FADU oropharyngeal cancer xenografts upon intra-tumoral (figure 1b) and systemic administration (figure 1c) and prolonged persistence of CAR T cells in vivo.Abstract 142 Figure 1Rb-340-1 performance in vitro and in vivoRB-340-1 (orange) decreased PD-1 expression resulting in enhanced cytokine production and proliferation in vitro (figure 1a) and superior tumor suppression in vivo after intra-tumoral (figure 1b) or intravenous (figure 1c) administration compared to conventional CAR T cells (blue) or cRB-340-1 (green). Conventional CAR T cells or cRB-340-1 CAR T combination treatment with PDL1 blockade (Atezolizumab) is shown as dashed line. Colonization of tumors by human CD45+ cells is shown at the bottom of figure 1b & 1cConclusionsIntrinsic conditional regulation of checkpoint expression in CAR T cells provides a simplified approach to combination therapies that limits systemic toxicities and reduces costs. Since the expression of multiple genes can be simultaneously controlled by CRISPRi, broader applications can be envisioned in the future.Ethics ApprovalNot ApplicableConsentNot applicableReferencesGilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-Scale CRISPR-mediated control of gene repression and activation. Cell 2014;159(3):647-61.Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442–51.Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152(5):1173–83.

γδ T cells for immune therapy of patients with lymphoid malignancies

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 200-206 ◽  
Author(s):  
Martin Wilhelm ◽  
Volker Kunzmann ◽  
Susanne Eckstein ◽  
Peter Reimer ◽  
Florian Weissinger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Low Dose ◽  
Γδ T Cells ◽  
Low Grade ◽  
Treatment Schedule ◽  
Γδ T Cell

Abstract There is increasing evidence that γδ T cells have potent innate antitumor activity. We described previously that synthetic aminobisphosphonates are potent γδ T cell stimulatory compounds that induce cytokine secretion (ie, interferon γ [IFN-γ]) and cell-mediated cytotoxicity against lymphoma and myeloma cell lines in vitro. To evaluate the antitumor activity of γδ T cells in vivo, we initiated a pilot study of low-dose interleukin 2 (IL-2) in combination with pamidronate in 19 patients with relapsed/refractory low-grade non-Hodgkin lymphoma (NHL) or multiple myeloma (MM). The objectives of this trial were to determine toxicity, the most effective dose for in vivo activation/proliferation of γδ T cells, and antilymphoma efficacy of the combination of pamidronate and IL-2. The first 10 patients (cohort A) who entered the study received 90 mg pamidronate intravenously on day 1 followed by increasing dose levels of continuous 24-hour intravenous (IV) infusions of IL-2 (0.25 to 3 × 106 IU/m2) from day 3 to day 8. Even at the highest IL-2 dose level in vivo, γδ T-cell activation/proliferation and response to treatment were disappointing with only 1 patient achieving stable disease. Therefore, the next 9 patients were selected by positive in vitro proliferation of γδ T cells in response to pamidronate/IL-2 and received a modified treatment schedule (6-hour bolus IV IL-2 infusions from day 1-6). In this patient group (cohort B), significant in vivo activation/proliferation of γδ T cells was observed in 5 patients (55%), and objective responses (PR) were achieved in 3 patients (33%). Only patients with significant in vivo proliferation of γδ T cells responded to treatment, indicating that γδ T cells might contribute to this antilymphoma effect. Overall, administration of pamidronate and low-dose IL-2 was well tolerated. In conclusion, this clinical trial demonstrates, for the first time, that γδ T-cell–mediated immunotherapy is feasible and can induce objective tumor responses. (Blood. 2003;102:200-206)

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.

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