A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development

2001 ◽  
Vol 211 (12) ◽  
pp. 603-610 ◽  
Author(s):  
Stefan Pauls ◽  
Benedikt Geldmacher-Voss ◽  
José A. Campos-Ortega
Keyword(s):  
Embryonic Development ◽  
Fusion Protein ◽  
Histone Variant ◽  
In Vivo Studies

scholarly journals 206 The development of ‘chimeric CD3e fusion protein’ and ‘anti-CD3-based bispecific T cell activating element’ engineered T (CAB-T) cells for the treatment of solid malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A217-A217
Author(s):  
Andy Tsun ◽  
Zhiyuan Li ◽  
Zhenqing Zhang ◽  
Weifeng Huang ◽  
Shaogang Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vivo Studies ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

BackgroundCancer immunotherapy has achieved unprecedented success in the complete remission of hematological tumors. However, serious or even fatal clinical side-effects have been associated with CAR-T therapies to solid tumors, which mainly include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome, etc. Furthermore, CAR-T therapies have not yet demonstrated significant clinical efficacy for the treatment of solid tumors. Here, we present a novel T cell therapeutic platform: a Chimeric CD3e fusion protein and anti-CD3-based bispecific T cell activating element (BiTA) engineered T (CAB-T) cells, which target tumor antigens via the secretion of BiTAs that act independently of MHC interactions. Upon BiTA secretion, CAB-T cells can simultaneously achieve anti-tumor cytotoxic effects from the CAB-T cells and simultaneously activate bystander T cells.MethodsCAB-T cells were generated by co-expressing a chimeric CD3e fusion protein and an anti-CD3-based bispecific T cell activating element. The chimeric CD3e contains the extracellular domain of CD3e, a CD8 transmembrane domain, 4-1BB costimulatory domain, CD3z T cell activation domain and a FLAG tag, while the BiTA element includes a tumor antigen targeting domain fused with an anti-CD3 scFv domain and a 6x His-tag. CAR-T cells were generated as a control. Cytokine release activity, T cell activation and exhaustion markers, T cell killing activity and T cell differentiation stages were analysed. We also tested their tumor growth inhibition activity, peripheral and tumor tissue distribution, and their safety-profiles in humanized mouse models.ResultsCAB-T cells have similar or better in vitro killing activity compared with their CAR-T counterparts, with lower levels of cytokine release (IL-2 and IFNγ). CAB-T cells also showed lower levels of exhaustion markers (PD-1, LAG-3 and TIM-3), and higher ratios of naive/Tscm and Tcm T cell populations, after co-culture with their target tumor cells (48h). In in vivo studies, CAIX CAB-T and HER2 CAB-T showed superior anti-tumor efficacy and tumor tissue infiltration activity over their corresponding CAR-T cells. For CLDN18.2 CAB-T cells, similar in vivo anti-tumor efficacy was observed compared to CAR-T after T cell infusion, but blood glucose reduction and animal mortality was observed in the mice administered with CAR-T cells.ConclusionsThe advantages of CAB-T in in vitro and in vivo studies may result from TCR signal activation of both the engineered CAB-T cells and the non-engineered bystander T cells via cross-bridging by the secreted BiTA molecules, thus offering superior anti-tumor efficacy with a potential better safety-profile compared to conventional CAR-T platforms.

scholarly journals Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

2018 ◽  
Vol 48 (6) ◽  
pp. 2286-2301 ◽  
Author(s):  
Dijiong  Wu ◽  
Keding Shao ◽  
Qihao Zhou ◽  
Jie Sun ◽  
Ziqi Wang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Cell Lines ◽  
Mrna Level ◽  
In Vivo Studies ◽  
Proteolytic Degradation ◽  
Induced Differentiation ◽  
Fluorescent Substrate

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.

scholarly journals Circulation Index as a Quantitative Indicator of Angiogenesis in Chorioallantoic Membrane of Chicken Broilers

2018 ◽  
Vol 67 (2) ◽  
pp. 164-170
Author(s):  
Zoran Ružić ◽  
Zdenko Kanački ◽  
Dragan Žikić ◽  
Gordana Ušćebrka ◽  
Jovan Mirčeta
Keyword(s):  
Embryonic Development ◽  
Blood Vessels ◽  
Chorioallantoic Membrane ◽  
New Method ◽  
In Vivo Studies ◽  
Circulation Index ◽  
Fast Method ◽  
Angiogenic Response ◽  
Embryonic Life

Summary Chorioallantoic membrane (CAM) is an extraembryonic membrane very frequently used for in vivo studies in various researches. Since researches require a fast method for quantifying the CAM angiogenic response, there is a need to develop a new precise and unbiased method of quantification of angiogenesis in CAM, which would be easy to perform and suitable for analysis of a large number of samples. The objective of this paper is to apply a new method of quantification of angiogenesis in investigation of the development of blood vessels in the CAM, in particular days of embryonic life considered essential for CAM development. The present research included 75 fertilized eggs of heavy hybrid Ross 308. CAM sampling for stereological analyses was in key phases of embryonic development, namely on the 12th, 15th and 19th day. The results of the present investigation show that the increase in embryonic age results in increase in circulation index, which is also an indicator of angiogenic processes developing in CAM. The lowest value of circulation index (0.1952) was recorded on the first sampling day (E12), while the highest value (0.2666) was recorded on the last sampling day (E19). This method may be applied in researching different factors which affect angiogenesis in CAM.

Inhibition of Tumor Growth by RGD Peptide Directed Delivery of Truncated Tissue Factor to the Tumor Vasculature.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1934-1934
Author(s):  
Torsten Kessler ◽  
Ralf Bieker ◽  
Teresa Padro ◽  
Federico Herrera ◽  
Sandra Ruiz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fusion Protein ◽  
Tumor Growth ◽  
Tissue Factor ◽  
Tumor Vasculature ◽  
Binding Activity ◽  
In Vivo Studies ◽  
Binding Assays ◽  
Tumor Vessels

Abstract Selective activation of blood coagulation in tumor vessels with subsequent tumor infarction is a promising anticancer strategy. To this end, a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) was fused to the peptide GRGDSP selectively targeting avb3 and avb5 integrins on tumor endothelial cells. The fusion protein tTF-RGD retained its thrombogenic and integrin binding activity as demonstrated by coagulation assays and binding assays with purified avb3 and endothelial cells. In vivo studies in mice bearing established human adenocarcinomas (CCL185), human melanoma (M21) and human fibrosarcoma (HT1080) revealed that i.v. administration of tTF-RGD induced partial or complete thrombotic occlusion of tumor vessels as indicated by histological analysis. Furthermore, treatment studies showed that tTF-RGD but not untargeted tTF induced significant tumor growth retardation or regression in all three types of solid tumors in mice without apparent side effects such as thrombosis in liver, kidney, heart or lung. Thus, selective thrombosis in the tumor vasculature induced by tTF-RGD may be a promising strategy for the treatment of cancer.

scholarly journals Emerging Roles of MTG16 in Cell-Fate Control of Hematopoietic Stem Cells and Cancer

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Nickolas Steinauer ◽  
Chun Guo ◽  
Jinsong Zhang
Keyword(s):  
Stem Cells ◽  
Fusion Protein ◽  
Cell Fate ◽  
Lymphoblastic Leukemia ◽  
In Vivo Studies ◽  
Hematopoietic Stem ◽  
Regulatory Pathways ◽  
Domain Structures ◽  
Proliferation And Differentiation

MTG16 (myeloid translocation gene on chromosome 16) and its related proteins, MTG8 and MTGR1, define a small family of transcriptional corepressors. These corepressors share highly conserved domain structures yet have distinct biological functions and tissue specificity. In vivo studies have shown that, of the three MTG corepressors, MTG16 is uniquely important for the regulation of hematopoietic stem/progenitor cell (HSPC) proliferation and differentiation. Apart from this physiological function, MTG16 is also involved in carcinomas and leukemias, acting as the genetic target of loss of heterozygosity (LOH) aberrations in breast cancer and recurrent translocations in leukemia. The frequent involvement of MTG16 in these disease etiologies implies an important developmental role for this transcriptional corepressor. Furthermore, mounting evidence suggests that MTG16 indirectly alters the disease course of several leukemias via its regulatory interactions with a variety of pathologic fusion proteins. For example, a recent study has shown that MTG16 can repress not only wild-type E2A-mediated transcription, but also leukemia fusion protein E2A-Pbx1-mediated transcription, suggesting that MTG16 may serve as a potential therapeutic target in acute lymphoblastic leukemia expressing the E2A-Pbx1 fusion protein. Given that leukemia stem cells share similar regulatory pathways with normal HSPCs, studies to further understand how MTG16 regulates cell proliferation and differentiation could lead to novel therapeutic approaches for leukemia treatment.

In vitro and in vivo studies of new cationic Zn(II)‐benzonaphthoporphyrazines as photosensitizers for PDT

2001 ◽  
Vol 5 (8) ◽  
pp. 645-651
Author(s):  
M. Peeva ◽  
M. Shopova ◽  
U. Michelsen ◽  
D. Wöhrle ◽  
G. Petrov ◽  
...  
Keyword(s):  
In Vivo Studies

Effect of caffeine on theophyliine on cerebral blood flow response to brain activation: In vitro and in vivo studies

2005 ◽  
Vol 25 (1_suppl) ◽  
pp. S198-S198
Author(s):  
Joseph R Meno ◽  
Thien-son K Nguyen ◽  
Elise M Jensen ◽  
G Alexander West ◽  
Leonid Groysman ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Brain Activation ◽  
In Vivo Studies ◽  
Blood Flow Response ◽  
Flow Response

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

Ex Vivo Inhibition of Platelet Aggregation in Patients with Severe Limb Arteriopathy Treated with BAY U3405, a Specific IX A2 Receptor Antagonist

1994 ◽  
Vol 72 (05) ◽  
pp. 659-662 ◽  
Author(s):  
S Bellucci ◽  
W Kedra ◽  
H Groussin ◽  
N Jaillet ◽  
P Molho-Sabatier ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Randomized Study ◽  
In Vivo Studies ◽  
Walking Distance ◽  
Peripheral Blood Flow ◽  
Walking Ability ◽  
Double Blind ◽  
Platelet Factor

SummaryA double-blind, placebo-controlled randomized study with BAY U3405, a specific thromboxane A2 (TX A2) receptor blocker, was performed in patients suffering from severe stade II limb arteriopathy. BAY U3405 or placebo was administered in 16 patients at 20 mg four times a day (from day 1 to day 3). Hemostatic studies were done before therapy, and on day 2 and day 3 under therapy. On day 3, BAY U3405 was shown to induce a highly statistically significant decrease of the velocity and the intensity of the aggregations mediated by arachidonic acid (56 ± 37% for the velocity, 58 ± 26% for the intensity) or by U46619 endoperoxide analogue (36 ± 35% for the velocity, 37 ± 27% for the intensity). Similar results were already observed on day 2. By contrast, such a decrease was not noticed with ADP mediated platelet aggregation. Furthermore, plasma levels of betathrombo-globulin and platelet factor 4 remained unchanged. Peripheral hemodynamic parameters were also studied. The peripheral blood flow was measured using a Doppler ultrasound; the pain free walking distance and the total walking ability distance were determined under standardized conditions on a treadmill. These last two parameters show a trend to improvement which nevertheless was not statistically significant. All together these results encourage further in vivo studies using BAY U3405 or related compounds on a long-term administration.

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