Exploring cardioprotective potential of esculetin against isoproterenol induced myocardial toxicity in rats: in vivo and in vitro evidence

Abstract Background Esculetin is a natural coumarin derivative from various plants with multiple pharmacological effects. Hence, the present study was undertaken to explore the cardio protective potential of esculetin against isoproterenol induced myocardial toxicity in rats. Methods The treatment schedule was fixed for 28 days and the rats were divided into five groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100 mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively once a day per oral for 28 days along with ISO for last two consecutive days of the study. Cardiac biomarkers such as CK-MB and LDH, membrane bound Na+ /K+ ATPases activity, myocardial lysosomal enzymes activity and tissue antioxidants status were estimated in the heart tissue samples. The histopathological changes in the myocardium were also assessed. Further, DPPH assay was done to evaluate the free radicals scavenging potential of esculetin. Cytoxicity assay, intracellular ROS levels by DCFDA assay and m-RNA expression of TNF-α, IL-6 and NF-κB by quantitative RT-PCR in H9c2 cell lines. Results The increased levels of CK-MB, LDH, LPO, myocardial lysosomal enzymes and membrane bound Na+ /K+ ATPase levels by ISO administration was significantly increased with concomitant decrease in tissue antioxidant enzymes such as GSH, Catalase, and SOD. Pre-treatment with esculetin for 28 days has significantly decreased the levels of cardiac bio-markers, lysosomal enzymes, membrane bound Na+ /K+ ATPase levels as well as Lipid peroxides which is in contrary to the ISO group. Amelioration of the antioxidant levels were also found in esculetin treated groups. Histopathological examination of heart reveals that myocardial degeneration, mononuclear cell infiltration was noticed in ISO treated rats, whereas the same was restored with esculetin treatment. In H9C2 cell lines esculetin could effectively reduced intracellular ROS inhibition and m-RNA expression of pro-inflammatory cytokines including TNF-α, IL-6 and NF-κB to prevent apoptosis or cell necrosis. Conclusion The study provides the evidence of cardioprotective potentials of esculetin against isoproterenol induced myocardial infarction by antioxidant and myocardial membrane stabilization along with in vitro protection from arsenic induced ROS cell necrosis or apoptosis in H9C2 cells.

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Exploring Cardioprotective Potential of Esculetin Against Isoproterenol Induced Myocardial Infarction in Rats: In Vivo and in Vitro Evidence

10.21203/rs.3.rs-289373/v1 ◽

2021 ◽

Author(s):

Pullaiah Chitikela P ◽

Vinod K Nelson ◽

Sushma R ◽

Narasimha Kumar GV ◽

Thyagaraju K

Keyword(s):

Myocardial Infarction ◽

Body Weight ◽

Lysosomal Enzymes ◽

H9c2 Cell ◽

Rna Expression ◽

Cell Necrosis ◽

Tnf Α ◽

Intracellular Ros ◽

Membrane Bound

Abstract BackgroundEsculetin is a natural coumarin derivative from various plants with multiple pharmacological effects. Hence, the present study was undertaken to explore the cardio protective potential of esculetin against isoproterenol induced myocardial toxicity in rats. Methods The treatment schedule was fixed for 28 days and the rats were divided into five groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively once a day per oral for 28 days along with ISO for last two consecutive days of the study. Cardiac biomarkers such as CK-MB and LDH, membrane bound Na+ /K+ ATPases activity, myocardial lysosomal enzymes activity and tissue antioxidants status were estimated in the heart tissue samples. The histopathological changes in the myocardium were also assessed. Further, DPPH assay was done to evaluate the free radicals scavenging potential of esculetin. Cytoxicity assay, intracellular ROS levels by DCFDA assay and m-RNA expression of TNF-α, IL-6 and NF-κB by quantitative RT-PCR in H9c2 cell lines.ResultsThe increased levels of CK-MB, LDH, LPO, myocardial lysosomal enzymes and membrane bound Na+ /K+ ATPase levels by ISO administration was significantly increased with concomitant decrease in tissue antioxidant enzymes such as GSH, Catalase, and SOD. Pre-treatment with esculetin for 28 days has significantly decreased the levels of cardiac bio-markers, lysosomal enzymes, membrane bound Na+ /K+ ATPase levels as well as Lipid peroxides which is in contrary to the ISO group. Amelioration of the antioxidant levels were also found in esculetin treated groups. Histopathological examination of heart reveals that myocardial degeneration, mononuclear cell infiltration was noticed in ISO treated rats, whereas the same was restored with esculetin treatment. In H9C2 cell lines esculetin could effectively reduced intracellular ROS inhibition and m-RNA expression of pro-inflammatory cytokines including TNF-α, IL-6 and NF-κB to prevent apoptosis or cell necrosis. Conclusion The study provides the evidence of cardioprotective potentials of esculetin against isoproterenol induced myocardial infarction by antioxidant and myocardial membrane stabilization along with in vitro protection from arsenic induced ROS cell necrosis or apoptosis in H9C2 cells.

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Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Mediators of Inflammation ◽

10.1155/2021/6665028 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Tanzeela Awan ◽

Aaron Babendreyer ◽

Justyna Wozniak ◽

Abid Mahmood Alvi ◽

Viktor Sterzer ◽

...

Keyword(s):

Cell Lines ◽

Inflammatory Mediators ◽

Liver Tissue ◽

Stellate Cell ◽

Hepatoma Cell ◽

Cell Types ◽

Chronic Liver ◽

Liver Inflammation ◽

Tnf Α

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

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Ricin binding and protein synthesis inhibition in human hematopoietic cell lines

Blood ◽

10.1182/blood.v72.4.1357.1357 ◽

1988 ◽

Vol 72 (4) ◽

pp. 1357-1363 ◽

Cited By ~ 2

Author(s):

JE Leonard ◽

CD Grothaus ◽

R Taetle

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Close Correlation ◽

Hematopoietic Cell ◽

Protein Synthesis Inhibition ◽

Human Blood Cells ◽

Kd Values ◽

Membrane Bound ◽

Hematopoietic Cell Lines

Abstract Previous studies showed that human blood cells exhibited varying sensitivities to ricin. To investigate the basis for these differences, ricin binding to human hematopoietic cell lines was assessed and correlated with in vitro ricin sensitivities. Resistant mutants were also isolated and characterized. Ricin binding to CEM cells was rapid, time-dependent, and blocked by unlabeled ricin, but not albumin; ricin binding approached saturation at 3 mumol/L. Scatchard analyses showed multiple classes of binding sites, with maximum and minimum Kd values estimated at 1.5 x 10(-8) mol/L and 2.5 x 10(-7) mol/L. At 4 degrees C, membrane-bound ricin dissociated slowly from the cell surface in the presence of unlabeled ricin, but greater than 95% of the surface-bound ricin was removed with 0.1 mol/L lactose. At 37 degrees C, ricin dissociated from the cell surface with biphasic kinetics. Ricin uptake at 37 degrees C increased linearly for 15 to 30 minutes and plateaued at levels representing 12% to 29% of the amount of ricin bound at 4 degrees C, depending on the cell line. Ricin binding at 4 degrees C varied two- to fivefold among hematopoietic cell lines and was reduced approximately tenfold by incubation with lactose. When compared with parent CEM cells, ricin-resistant CEM variants showed a greater than 95% reduction in ricin binding and showed no detectable binding with lactose added. However, these cells were as sensitive as parent CEM cells to an anti-T-cell ricin immunoconjugate. For all cells examined, there was a close correlation (r = +.9) between ricin bound per cell and in vitro ricin sensitivity. Human hematopoietic cells show widely varying ricin binding, indicating major differences in the carbohydrate content or structure of surface glycoproteins and glycolipids. These variations are probably the major determinant of nonspecific toxicity of ricin immunoconjugates.

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Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3′UTR part of transcripts

AJP Cell Physiology ◽

10.1152/ajpcell.00595.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1240-C1250 ◽

Cited By ~ 26

Author(s):

Maryvonne Baudouin-Legros ◽

Alexandre Hinzpeter ◽

Amandine Jaulmes ◽

Franck Brouillard ◽

Bruno Costes ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Posttranscriptional Regulation ◽

P38 Map Kinase ◽

Cftr Gene ◽

Control Of Gene Expression ◽

Cytosolic Proteins ◽

Tnf Α ◽

Ht 29

Expression of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, which contains the mutations responsible for CF, is regulated by cytokines (TNF-α and IL-1β) in a cell-specific manner. TNF-α decreases CFTR mRNA in human colon cell lines (HT-29), but not in pulmonary cell lines (Calu-3), and IL-1β increases it only in Calu-3 cells. We looked for the cytokine-induced posttranscriptional regulation of CFTR gene expression and studied the modulation of CFTR mRNA stability linked to its 3′ untranslated sequence (3′UTR) in HT-29 and Calu-3 cells. The stability of CFTR mRNA was analyzed by Northern blot after in vitro incubation of total RNAs from CFTR-expressing cells with cytosolic proteins extracted from control or cytokine-treated HT-29 and Calu-3 cells. CFTR mRNA was degraded only by extracts of TNF-α-treated HT-29 cells and not by cytosolic proteins from untreated or IL-1β-treated HT-29 cells. In contrast, extracts of untreated Calu-3 cells enhanced CFTR mRNA degradation, and IL-1β treatment inhibited this; TNF-α had no significant effect. The 3′UTR part of CFTR mRNA was found to be required for this posttranscriptional regulation. The 5′ part of the 3′UTR (the 217 first bases), which contains two AUUUA sequences, was implicated in CFTR mRNA destabilization and the following 136 bases, containing several C-repeats in U-rich environment, in its protection. The proteins, which reacted with the U- and C-repeats of CFTR mRNA 3′UTR, were mainly controlled by stimulation of the p42/p44 and p38 MAP kinase cascades with interaction between these pathways. This posttranscriptional control of gene expression is a common feature of CFTR and many proteins of inflammation.

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Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.2893.2893 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2893-2893

Author(s):

Marc Weinkauf ◽

Grit Hutter ◽

Yvonne Zimmermann ◽

Malte Rieken ◽

Alessandro Pastore ◽

...

Keyword(s):

Cell Cycle ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Signal Pathways ◽

Rna Expression ◽

Cellular Functions ◽

Mantle Cell ◽

Canonical Pathways

Abstract Abstract 2893 Background: The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods: Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results: Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion: In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures: Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

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FSH-Sensitive Murine Sertoli Cell Lines Immortalized by Human Telomerase Gene hTERT Express the Androgen Receptor in Response to TNF-α Stimulation

Bioscience Reports ◽

10.1007/s10540-007-9063-y ◽

2007 ◽

Vol 27 (6) ◽

pp. 403-411 ◽

Cited By ~ 4

Author(s):

Chin-kai Chuang ◽

Kun-Hsiung Lee ◽

Chio-Tin Fan ◽

Yu-Show Su

Keyword(s):

Androgen Receptor ◽

Sertoli Cell ◽

Cell Lines ◽

Sertoli Cells ◽

T Antigen ◽

Immunological Methods ◽

Sv40 Large T Antigen ◽

Tnf Α ◽

Human Telomerase

Sertoli cells are regulated by follicular stimulating hormone (FSH) and testosterone secreted by the pituitary gland and Leydig cells, respectively. However, the expression of the FSH receptor and androgen receptor were undetectable in both primary cultured Sertoli cells and Sertoli cell lines immortalized by SV40 large T antigen. Two Sertoli cell lines, B6Sc-2 and B6Sc-3, were established from the testis of 19-day-old C57BL/6 mice testis by immortalization with human telomere reverse transcriptase. These Sertoli cell lines expressed FSH receptors and the total phosphoprotein patterns were converted after FSH treatment. Additionally, immunological methods demonstrated that these cell lines expressed characteristic Sertoli cell proteins, such as tyrosine-tubulin, vimentin and stem cell factor (SCF). Reverse transcription-polymerase chain reaction (RT-PCR) also indicates that they express Sertoli specific mRNAs, such as Amh, claudin11 and ZO-1. The expression of the androgen receptor in both B6Sc-2 and B6Sc-3 cells could be induced by TNF-α treatment. The present results indicate that these Sertoli cell lines are more native than others and may thus provide useful tools for in vitro studies.

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Dangshen Erling Decoction Ameliorates Myocardial Hypertrophy via Inhibiting Myocardial Inflammation

Frontiers in Pharmacology ◽

10.3389/fphar.2021.725186 ◽

2022 ◽

Vol 12 ◽

Author(s):

Yigang Zhong ◽

Liuying Chen ◽

Miaofu Li ◽

Lian Chen ◽

Yufeng Qian ◽

...

Keyword(s):

Signaling Pathway ◽

Cardiac Tissue ◽

Myocardial Hypertrophy ◽

Cell Model ◽

H9c2 Cell ◽

Myocardial Inflammation ◽

Cross Sectional ◽

Tnf Α

Myocardial hypertrophy plays an essential role in the structural remodeling of the heart and the progression to heart failure (HF). There is an urgent need to understand the mechanisms underlying cardiac hypertrophy and to develop treatments for early intervention. Dangshen Erling decoction (DSELD) is a clinically used formula in Chinese medicine for treating coronary heart disease in patients with HF. However, the mechanism by which DSELD produces its cardioprotective effects remains largely unknown. This study explored the effects of DSELD on myocardial hypotrophy both in vitro and in vivo. In vitro studies indicated that DSELD significantly (p < 0.05) reduced the cross-sectional area of the myocardium and reduced elevated lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels in the induced H9C2 cell model to study inflammation. In vivo experiments revealed that DSELD restores cardiac function and significantly reduces myocardial fibrosis in isoproterenol (ISO)-induced HF mouse model (p < 0.05). In addition, DSELD downregulated the expression of several inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), IL-1α, IL-1β, IL-3, IL-5, IL-7, IL-12, IL-13, and TNF-α in HF (p < 0.05). Further analysis of the cardiac tissue demonstrated that DSELD produces its anti-inflammatory effects via the Toll-like receptor (TLR)4 signaling pathway. The expression of TLR4 downstream proteins such as matrix metalloproteinase-9 (MMP9) and myeloid differentiation factor-88 (MyD88) was among the regulated targets. In conclusion, these observations suggest that DSELD exerts antihypertrophic effects by alleviating the inflammatory injury via the TLR4 signaling pathway in HF and thus holds promising therapeutic potentials.

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A Cellular Study of Inflammatory Responses in Women with Polycystic Ovary Syndrome as: “An In vitro Model of Anti Breast Tumor Response” Anti-Breast Tumor Response

10.21203/rs.3.rs-16155/v1 ◽

2020 ◽

Author(s):

Fatemeh Rezayat ◽

Mehri Hajiaghaei ◽

Nazanin Ghasemi ◽

Mehrnaz Mesdaghi ◽

Fahimeh Ramezani Tehrani ◽

...

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Breast Tumor ◽

Mononuclear Cells ◽

Polycystic Ovary ◽

Healthy Controls ◽

Tumor Cell Lines ◽

Tnf Α ◽

Mcf 7

Abstract Background: Although Polycystic Ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age; is unclear whether PCOS increases the risk of subsequent development of, Gynecologic cancers namely breast cancer. The present study we aimed to compare the antitumoral ability of peripheral blood mononuclear cells (PBMCs) of women with PCOS with that of healthy controls using the co-culture system between effector cells and target tumor cell lines. Materials & Methods: PBMCs were isolated from 25 women with PCOS and 25 non hirsute eumenorrheic healthy controls by density gradient centrifugation ficoll. Breast tumor cell lines (MDA-468, MCF-7) were incubated as the two target cells and were cultured adjacent to PBMCs in the transwell co-culture system. Proliferation rate of the effectors cells evaluated by BrdU cell proliferation assay after 48 and 72 hours and T CD3+ lymphocytes were assessed using flow cytometry. TNF-α cytokine production was evaluated in cell culture supernatant by sandwich ELISA technique. Results: After 48 hours incubation with MDA-468 and MCF-7, the mean proliferation score of PBMCs was significantly higher in women with PCOS compared to that of healthy controls (921.04; P=0.021 vs 287.6; P=0.002, respectively). In PCOS women, after 72 hours of incubation, TNF-α concentration was significantly reduced compared to 48-hour cultures (921.04 ± 271.4 pg/dl vs 545.6 ± 151.1 pg/dl at 48 h and 72 h intervals respectively, P<0.05); it was increased in healthy controls. There was no significant difference in CD3+ CD8+ cells between the PCOS group and healthy controls. Conclusion: The ability of PBMCs to produce of TNF-α in women with PCOS decreased gradually; as a result of which they may lack the ability required to form an in vitro efficient antitumor response to breast tumor cell lines. It is assumed that threshold activation of mononuclear cells is reduced in women with PCOS and a low-grade inflammatory condition may provide a positive background for arising myeloid derived suppressor cells (MDSCs).

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CD123-Specific Chimeric Antigen Receptor Redirected T Cells Exhibit Potent Cytolytic Activity and Multiple Effector Functions Against Acute Myeloid Leukemia without Altering Normal Hematopoietic Colony Formation in Vitro

Blood ◽

10.1182/blood.v120.21.950.950 ◽

2012 ◽

Vol 120 (21) ◽

pp. 950-950

Author(s):

Armen Mardiros ◽

Cedric Dos Santos ◽

Tinisha McDonald ◽

Christine Brown ◽

Xiuli Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Colony Formation ◽

Cytolytic Activity ◽

Car T Cells ◽

Tnf Α ◽

Donor T Cells ◽

Car T

Abstract Abstract 950 Current treatment regimes for acute myeloid leukemia (AML) achieve complete remissions in only a subset of individuals and most adult patients will relapse within 5-years, emphasizing the need for novel treatment alternatives. One such therapy may be the administration of T cells engineered to express chimeric antigen receptors (CARs) specific for AML-associated antigens. CARs are typically composed of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3ζ signaling domain and may contain one or more costimulatory endodomains. When expressed in T cells, CARs redirect T cell specificity to surface antigens on target cells in an MHC-independent manner. The interleukin 3 receptor alpha chain (IL3Rα, CD123) is a cell surface receptor which is aberrantly over-expressed on multiple hematologic malignancies including AML. Previous work has demonstrated that CD123 is not expressed on all CD34+/CD38− hematopoietic stem cells and is restricted to cells of the myeloid lineage, making CD123 an attractive target for CAR T cell therapy. We have therefore generated two novel CD123-specific (CD123R) CARs using scFvs from previously characterized antibodies, designated 26292 and 32716, which bind two distinct epitopes on CD123. Here we demonstrate that T cells expressing CARs derived from either 26292 or 32716 effectively redirect T cell specificity against CD123+ cells. Healthy donor T cells (n=3) engineered to express the CD123R CARs efficiently lysed CD123+ cell lines LCL and KG1a while sparing the CD123− cell line K562 as demonstrated by a 4 hour chromium-51 (51Cr) release assay. Additionally, both of the CD123R CAR T cells produced similar levels of IFN-γ and TNF-α and displayed comparable levels of antigen-dependent proliferation following co-culture with CD123+ cell lines. The potent cytolytic activity and activation of our CD123-targeting T cells was not limited to tumor cell lines. Indeed, CD123R CAR T cells, but not donor-matched CD19-specific (CD19R) CAR T cells, robustly lysed panel of primary AML samples (n=6, 3 – persistent, 1 – relapsed, 2 - untreated) (* p<0.05, ** p<0.001 using the unpaired students' t-test comparing 26292 or 32716 CAR T cells to donor-matched CD19R CAR T cells), and exhibited multiple effector functions for both CD4 and CD8 T cell subsets (ie CD107a degranulation, IFN-γ and TNF-α production, and antigen specific proliferation) when co-cultured with primary AML samples (n=3, 2 – relapsed, 1-persistent). To examine the effect our CD123-specific T cells have on normal and leukemic progenitor cells, we co-cultured CD123R CAR T cells, or donor-matched CD19-targeting T cells, with either CD34-enriched cord blood (CB, n=3) or primary AML samples (n=3, 2 – relapsed, 1 - untreated) for 4 hours (E:T 25:1) prior to plating in semisolid methylcellulose progenitor culture. CD123-targeting T cells did not significantly reduce the number of colony-forming unit granulocyte-macrophage (CFU-GM) or burst-forming unit erythroid (BFU-E) colonies from CB when compared to CD19R CAR T cells. Finally, while CD19-specific T cells had little impact on leukemic colony formation of primary AML samples, CD123-targeting T cells significantly reduced leukemic colony formation in vitro. Collectively, our data demonstrate that CD123-specifc CARs can be expressed in primary healthy donor T cells, distinguish between CD123+ and CD123− cells, and mediate robust anti-leukemic activity against a panel of poor-risk primary AML patient samples. Importantly, we demonstrate that CD123R CAR T cells have little impact on normal progenitor colony formation while significantly reducing the growth of clonogenic myeloid leukemic progentiors in vitro. Thus, CD123R CAR T cells are a promising candidate for future immunotherapy of AML. Disclosures: Bhatia: Novartis: Consultancy, Honoraria. Jensen:ZetaRx: Equity Ownership, Patents & Royalties.

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D3: A Gene Induced During Myeloid Cell Differentiation of Linlo c-Kit+ Sca-1+ Progenitor Cells

Blood ◽

10.1182/blood.v93.2.527.402k32_527_536 ◽

1999 ◽

Vol 93 (2) ◽

pp. 527-536 ◽

Cited By ~ 1

Author(s):

Sarah R. Weiler ◽

John M. Gooya ◽

Mariaestela Ortiz ◽

Schickwann Tsai ◽

Steven J. Collins ◽

...

Keyword(s):

Cell Differentiation ◽

Progenitor Cells ◽

Cell Lines ◽

Marrow Cell ◽

Physiological Role ◽

Myeloid Cell ◽

Lymphoid Cells ◽

Rna Expression ◽

Normal Bone Marrow Cell

In an effort to characterize molecular events contributing to lineage commitment and terminal differentiation of stem/progenitor cells, we have used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and cell lines blocked at two distinct stages of differentiation. The cell lines used were EML, which is representative of normal multipotential primitive progenitors (Sca-1+, CD34+, c-Kit+, Thy-1+) able to differentiate into erythroid, myeloid, and B-lymphoid cells in vitro, and MPRO, which is a more committed progenitor cell line, with characteristics of promyelocytes able to differentiate into granulocytes. One clone isolated by this approach was expressed in MPRO but not in EML cells and contained sequence identical to the 3′ untranslated region of D3, a gene cloned from activated peritoneal macrophages of unknown function. We have observed a novel pattern of D3 gene expression and found that D3 is induced in EML cells under conditions that promote myeloid cell differentiation (interleukin-3 [IL-3], stem cell factor [SCF], and all-trans-retinoic acid [atRA]) starting at 2 days, corresponding to the appearance of promyelocytes. D3 RNA expression reached a maximum after 5 days, corresponding to the appearance of neutrophilic granulocytes and macrophages, and decreased by day 6 with increased numbers of differentiated neutrophils and macrophages in vitro. Induction of D3 RNA in EML was dependent on IL-3 and was not induced in response to SCF or atRA alone or SCF in combination with 15 other hematopoietic growth factors (HGF) tested. Similarly, D3 was not expressed in the normal bone marrow cell (BMC) counterpart of EML cells, Linlo c-Kit+Sca-1+ progenitor cells. D3 RNA expression was induced in these cells when cultured for 7 days in IL-3 plus SCF. A comparison of the expression of D3 RNA in cell lines and normal BMC populations demonstrated that D3 is induced during macrophage and granulocyte differentiation and suggests a potential physiological role for D3 in normal myeloid differentiation.

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