scholarly journals On the biochemical associations of FoxO3a and SirT1 with the stress resistance of cells from the slow senescing Snell dwarf Mouse

2019 ◽  
Author(s):  
Oge Arum
Keyword(s):  
Stress Resistance ◽  
Ex Vivo ◽  
Cellular Stress ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Dwarf Mouse ◽  
Cerebral Neurons ◽  
Snell Dwarf

Tailskin fibroblasts from multiple genotypes of slow aging mice have been shown to be resistant to a broad spectrum of toxicants. The molecular determinants for this in vitro effect, as well as for the delayed/ decelerated senescence of these mice, are uncertain. Here, we have extended this phenomenon of in vitro cellular stress resistance to neurons derived from the cerebral cortex of the Snell Dwarf Mouse. We further investigated the role of the transcription factor FoxO3a and the protein deacetylase SirT1, proteins known to positively mediate cellular stress-resistance, in this paradigm. We found that Snell Dwarfs have a greater proportion of nuclear-localized FoxO3a within their cerebrums than their littermate controls and that the same is true for their unstressed fibroblasts in vitro; yet, Snell Dwarf fibroblasts did not differ in FoxO3a properties in response to the application of three different concentrations of two disparate stresses. Similar results were obtained for SirT1, although SirT1 content did increase under the mild cellular stress of serum deprivation. Taken together, these results depict stress resistance in non-fibroblast cell types of incontrovertible physiological import explanted from slow aging mice. Also, these results strongly suggest that neither FoxO3a nor SirT1 robustly regulate the stress-resistance of Snell Dwarf Mouse cells in vitro, and thus might not play a role in other slow aging mammalian in vitro models in which stress resistance has been documented. That cerebral neurons ex vivo and unstressed fibroblasts in vitro display FoxO3a concentrations suggestive of increased activity introduce the possibility that FoxO3a might partially mediate the in vivo retardation of senescence of these mice.

scholarly journals On the biochemical associations of FoxO3a and SirT1 with the stress resistance of cells from the slow senescing Snell dwarf Mouse

2019 ◽  
Author(s):  
Oge Arum
Keyword(s):  
Stress Resistance ◽  
Ex Vivo ◽  
Cellular Stress ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Dwarf Mouse ◽  
Cerebral Neurons ◽  
Snell Dwarf

Tailskin fibroblasts from multiple genotypes of slow aging mice have been shown to be resistant to a broad spectrum of toxicants. The molecular determinants for this in vitro effect, as well as for the delayed/ decelerated senescence of these mice, are uncertain. Here, we have extended this phenomenon of in vitro cellular stress resistance to neurons derived from the cerebral cortex of the Snell Dwarf Mouse. We further investigated the role of the transcription factor FoxO3a and the protein deacetylase SirT1, proteins known to positively mediate cellular stress-resistance, in this paradigm. We found that Snell Dwarfs have a greater proportion of nuclear-localized FoxO3a within their cerebrums than their littermate controls and that the same is true for their unstressed fibroblasts in vitro; yet, Snell Dwarf fibroblasts did not differ in FoxO3a properties in response to the application of three different concentrations of two disparate stresses. Similar results were obtained for SirT1, although SirT1 content did increase under the mild cellular stress of serum deprivation. Taken together, these results depict stress resistance in non-fibroblast cell types of incontrovertible physiological import explanted from slow aging mice. Also, these results strongly suggest that neither FoxO3a nor SirT1 robustly regulate the stress-resistance of Snell Dwarf Mouse cells in vitro, and thus might not play a role in other slow aging mammalian in vitro models in which stress resistance has been documented. That cerebral neurons ex vivo and unstressed fibroblasts in vitro display FoxO3a concentrations suggestive of increased activity introduce the possibility that FoxO3a might partially mediate the in vivo retardation of senescence of these mice.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1483
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Pre-Clinical Biocompatibility Testing of Peritoneal Dialysis Solutions

2000 ◽  
Vol 20 (5_suppl) ◽  
pp. 5-9 ◽  
Author(s):  
C.J. Holmes
Keyword(s):  
Peritoneal Dialysis ◽  
Screening Program ◽  
Ex Vivo ◽  
Biological Response ◽  
Cell Types ◽  
Hierarchical Level ◽  
Clinical Phase ◽  
Biocompatibility Testing

Pre-clinical biocompatibility testing of peritoneal dialysis (PD) solutions has become an integral part of new solution development. The construction of a pre-clinical screening program for solution biocompatibility should take a hierarchical approach, starting with in vitro cell viability and function assays. The selection of cell types and assay systems for the in vitro studies should be broad enough to permit a balanced interpretation. Whenever possible, animal models are recommended for the next hierarchical level of testing, followed by human ex vivo study designs. Designs of the latter sort provide evidence that a new solution formulation is exerting an altered biological response in vivo; the response is not purely an in vitro artifact or restricted to a given animal species. This article discusses the various approaches available for biocompatibility testing during the pre-clinical phase of solution development, with an emphasis on the advantages and drawbacks of each method.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

2021 ◽  
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.

EXTH-30. HARNESSING CELLULAR STRESS FOR IMMUNE TARGETING OF DIPGS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi169-vi170
Author(s):  
Vidya Gopalakrishnan ◽  
Ajay Sharma ◽  
Sreepradha Sridharan ◽  
Donghang Cheng ◽  
Juan Bournat ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Clinical Investigation ◽  
Cellular Stress ◽  
Single Agent ◽  
Pediatric Brain Tumor ◽  
Cytolytic Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutation

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor. An almost ubiquitous dominant negative mutation at lysine (K)-27 in genes encoding histone genes HIST1H3B and H3F3A found in patient tumors is a driver of DIPG development. ONC201, a small molecule DRD2 antagonist and ClpP agonist developed by Chimerix Inc, targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling. It is under clinical investigation in patients with recurrent H3K27M DMGs. In adults, single agent studies have shown durable objective responses when administered orally. A multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) for pediatric patients with H3K27M DMGs is open and accruing. Preliminary results suggest that the drug has a favorable safety profile and holds promise for patients with DIPGs and other midline gliomas. However, the mechanism of action of ONC201 against DIPGs warrants further study. Here, we show that ONC201 is cytotoxic to DIPGs in vitro and in vivo. RNA Seq analyses revealed cell context specific deployment of PERK-activated UPR and calcium signaling-associated RON tyrosine kinase-macrophage stimulating protein (MSP) signaling in DIPGs. Single cell proteomic assays revealed substantial heterogeneity in the sensitivity of DIPG cells to ONC201, and identified stem-like sub-populations of H3K27M DIPGs with intrinsic insensitivity to the drug. ONC201 treatment, which induces cellular stress, also sensitized DIPG cells to cytolytic activity by ex-vivo expanded and activated innate immune cells, in vitro. Ongoing in vivo experiments are expected to support a novel investigational study in patients with midline gliomas.

Expression of Cardiac Specific Cell Marker in Ex Vivo Differentiated Canine iPSC

2019 ◽  
Author(s):  
Purnima Singh ◽  
Tanmay Mondal ◽  
Kuldeep Kumar ◽  
Kinsuk Das ◽  
N Mahalakshmi ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Ex Vivo ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
Specific Cell ◽  
Specific Marker ◽  
Cell Based Therapy

Induced Pluripotent stem cells (iPSC) have a high ability to renew and differentiate themselves into various lineages and as vehicles of cell based therapy. Stem cell can differentiate under appropriate in vitro and in vivo conditions into different cell types. This study described the establishment of condition for in vitro expression of alpha MHC gene in cardiac differentiated canine iPSC (ciPSC). In vitro differentiation of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB’s were differentiated by using IMDM differentiation media: FBS – 10%, NEAA (100X) – 0.5%, Â-Mercaptoethanol- 100mM, Gentamycin- 5µg/ml supplemented with Azacytidine- 0.5µM. During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 for characterization of cardiomyocytes specific marker expression. Total RNA from EBs were extracted by using Trizol method and subsequently cDNA were synthesized. The differentiated cells expressed cardiac specific gene (Alpha MHC) which started from day 6 of differentiation upto day 24 Immunocytochemistry and relative expression of cardiac specific genes revealed that ciPSC have the potential to differentiate into cardiomyocytes which can be used for cardiac tissue regeneration and as disease models for pharmaceutical testing.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

2021 ◽  
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.

scholarly journals PERK inhibition mitigates restenosis and thrombosis - a potential low-thrombogenic anti-restenotic paradigm

2019 ◽  
Author(s):  
Bowen Wang ◽  
Mengxue Zhang ◽  
Go Urabe ◽  
Guojun Chen ◽  
Debra Wheeler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Cell Types ◽  
Brdu Incorporation ◽  
Main Stream ◽  
Stream Management ◽  
Drug Eluting ◽  
Normal Blood Flow

AbstractBackgroundDrug-eluting stents (DES) represent the main-stream management of restenosis following treatments of occlusive cardiovascular diseases. However, DES cannot eliminate instent restenosis yet exacerbate thrombogenic risks. To achieve dual inhibition of restenotic smooth muscle cell (SMC) de-differentiation/proliferation and thrombogenic endothelial cell (EC) dysfunction, a common target in both cell types, has been long-sought after. We evaluated the potential of protein kinase RNA-like endoplasmic reticulum kinase (PERK) as such a target for low-thrombogenic anti-restenotic intervention.Methods and ResultsWe used a rat angioplasty model of restenosis and a FeCl3-induced mouse model of thrombosis. Loss-or gain-of-function was achieved by PERK inhibition (GSK2606414, siRNA) or overexpression (adenovirus). Restenosis was robustly mitigated by GSK2606414 administered either via injected (i.v.) lesion-homing platelet membrane-coated nanoclusters or a perivascular hydrogel; it was enhanced by PERK transgene. Whereas PERK inhibition blocked, its overexpression exacerbated PDGF-induced human aortic SMC de-differentiation (reduced smooth muscle α-actin or αSMA) and proliferation (BrdU incorporation). Further, PERK activity promoted STAT3 activation but inhibited SRF transcriptional (luciferase) activity; its protein co-immunoprecipitated with STAT3 and also MRTF-A, the SRF activator for αSMA transcription. Importantly, PERK inhibition also prevented TNFα-induced impairment of human EC growth and upregulation of thrombogenic tissue factor, both in vitro and ex vivo. In vivo, oral gavage of GSK2606414 preserved ~50% of the normal blood flow 60 min after FeCl3-induced vascular injury.ConclusionsPERK inhibition is dual beneficial in mitigating restenosis and thrombosis, thus implicating a potential design for anti-restenotic intervention to overcome the thrombogenicity of DES.

scholarly journals PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway

Blood ◽  
2011 ◽  
Vol 118 (19) ◽  
pp. 5355-5364 ◽  
Author(s):  
Yushan Zhang ◽  
Yehia Daaka
Keyword(s):  
Glycogen Synthase ◽  
Ex Vivo ◽  
Cell Types ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Selective Agonists ◽  
Interfering Rna ◽  
Subtype Selective

Abstract Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3β that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.

scholarly journals OP0001 BARICITINIB, TOFACITINIB, UPADACITINIB, FILGOTINIB, AND CYTOKINE SIGNALING IN HUMAN LEUKOCYTE SUBPOPULATIONS: AN UPDATED EX-VIVO COMPARISON

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1-2
Author(s):  
I. Mcinnes ◽  
G. Rocha ◽  
R. E. Higgs ◽  
D. Dairaghi ◽  
T. Wehrman ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Leukocyte ◽  
Parent Drug ◽  
Cell Types ◽  
Vertical Line ◽  
Cytokine Signaling ◽  
Cell Type ◽  
Research Support

Background:everal JAKi are now used for the treatment of RA; approved doses include baricitinib (bari) 2- and/or 4-mg QD, tofacitinib (tofa) 5-mg BID, upadacitinib (upa) 15-mg QD. The JAK selectivity these agents is proposed to vary across the class.Objectives:In vitro cellular pharmacology of bari to tofa, upa, and filgotinib (filgo) were compared.Methods:PBMCs from 6 healthy donors were incubated with the JAKis over a 7- to 8-point concentration range. Following cytokine stimulation, levels of pSTAT were measured and IC50 calculated in gated leukocyte subpopulations. Therapeutic dose relevance was assessed using calculated mean concentration-time (CT) profiles over 24 hours for bari 2- and 4-mg QD; tofa 5- and 10-mg BID; upa 15- and 30-mg QD; filgo 100- and 200-mg QD. Average daily % inhibition of pSTAT (%SI) was calculated for each JAKi, cytokine, and cell type; filgo %SI integrated parent drug + metabolite.Results:The cytokines did not signal in all cell types (Figure 1). When signaling was detected, IC50 and %SI for a particular JAKi were generally similar across cell types, with dose-dependent inhibition (Figures 1 & 2). Based on IC50s, upa was most and filgo/metabolite least potent across JAK2/2 or JAK2/TYK2-dependent (IL-3, GM-CSF, G-CSF), JAK1/3-dependent (IL-2, 4, 15, 21), and JAK1/2/TYK2 dependent (IL-6 & 10, IFN-α & γ) signaling pathways.Figure 1.IC50 values (nM) for baricitinib, tofacitinib, upadacitinib, filgotinib (parent and metabolite) in cytokine-stimulated human PBMC preparations. *p<0.01, **p<0.001, ***p<0.0001 vs. bari.Incorporating CT profiles, no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Comparing bari 4-mg to tofa 5-mg BID, upa 15-mg QD, and filgo 100-mg QD, %SI of JAK2/2 or JAK2/TYK2-dependent cytokines was highest with bari 4-mg and upa. Inhibition of JAK1/2/TYK2 cytokines was highest with bari 4-mg. Inhibition of JAK2/2 or JAK2/TYK2, and of JAK1/3-dependent cytokines was lowest for filgo 100-mg QD. For bari 2-mg QD vs. these other JAKi doses, %SI of JAK2/2 or JAK2/TYK2 was highest with upa followed by bari 2-mg. The highest inhibitors of the JAK1/2/TYK2-dependent cytokines varied by cytokine / cell type though consistently included upa. Inhibition of JAK1/3 was highest for upa and tofa. Across comparisons, filgo 100-mg QD showed the least %SI of JAK2/2 or JAK2/TYK2-dependent, and of JAK1/3-dependent cytokines. Filgo reached the highest levels of %SI among agents only for 200-mg QD vs. lower doses of the other JAKi (for selected JAK1/2/TYK2-dependent cytokines).Conclusion:JAKis display different in vitro pharmacologic profiles which, coupled to their in vivo pharmacokinetics, suggest they modulate distinct cytokine pathways to differing degrees and durations over 24 hours. Ex vivo whole cell assays seem distinct from cell free kinase inhibition assays in determining the overall cytokine modulatory potential of members of the JAKi class.References:[1]McInnes IB, et al. Arthritis Res Ther. 2019 Aug 2;21(1):183Figure 2.Baricitinib, tofacitinib, upadacitinib, filgotinib: calculated average percent daily STAT inhibition for selected cytokines. -p<0.01, --p<0.001, ---p<0.0001 significantly lower compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”). +p<0.01, ++p<0.001, +++p<0.0001 significantly higher compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”).Disclosure of Interests:Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Guilherme Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Richard E Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Daniel Dairaghi Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Thomas Wehrman Shareholder of: An insignificant amount in AbbVie as part of a larger portfolio, Consultant of: Primity Bio Inc. works with many pharmaceutical and biotech companies and provides CRO services., Evan Wang Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Zhang Xin Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jorge Ross Terres Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Terence Rooney Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB

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