Sickle Cells Abolish Melanoma Tumorigenesis in Hemoglobin SS Knockin Mice and Augment the Tumoricidal Effect of Oncolytic Virus In Vivo

AbstractRabies virus (RV) is the most widely used vector for mapping neural circuits. Previous studies have shown that the RV glycoprotein can be a target to improve the retrograde transsynaptic tracing efficiency. However, the current versions still label only a small portion of all presynaptic neurons. Here, we reshuffled the oG sequence, a chimeric glycoprotein, with positive codon pair bias score (CPBS) based on bioinformatic analysis of mouse codon pair bias, generating ooG, a further optimized glycoprotein. Our experimental data reveal that the ooG has a higher expression level than the oG in vivo, which significantly increases the tracing efficiency by up to 12.6 and 62.1-fold compared to oG and B19G, respectively. The new tool can be used for labeling neural circuits Therefore, the approach reported here provides a convenient, efficient and universal strategy to improve protein expression for various application scenarios such as trans-synaptic tracing efficiency, cell engineering, and vaccine and oncolytic virus designs.

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Cation transport and volume regulation in sickle red blood cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.2.c251 ◽

1993 ◽

Vol 264 (2) ◽

pp. C251-C270 ◽

Cited By ~ 113

Author(s):

C. H. Joiner

Keyword(s):

Sickle Cell ◽

Vascular Occlusion ◽

Cell Swelling ◽

K Channel ◽

Transport Pathways ◽

Root Cause ◽

Sickle Cells ◽

Cellular Dehydration ◽

Mechanism Of Dehydration

Cellular dehydration is one of several pathological features of the sickle cell. Cation depletion is quite severe in certain populations of sickle cells and contributes to the rheological dysfunction that is the root cause of vascular occlusion in this disease. The mechanism of dehydration of sickle cells in vivo has not been ascertained, but three transport pathways may play important roles in this process. These include the deoxygenation-induced pathway that permits passive K+ loss and entry of Na+ and Ca2+; the K(+)-Cl- cotransport pathway, activated by acidification or cell swelling; and the Ca(2+)-activated K+ channel, or Gardos pathway, presumably activated by deoxygenation-induced Ca2+ influx. Recent evidence suggests that these pathways may interact in vivo. Heterogeneity exists among sickle cells as to the rate at which they become dense, suggesting that other factors may affect the activity or interactions of these pathways. Understanding the mechanism of dehydration of sickle cells may provide opportunities for pharmacological manipulation of cell volume to mitigate some of the symptoms of sickle cell disease.

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Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽

10.1182/blood.v71.3.636.636 ◽

1988 ◽

Vol 71 (3) ◽

pp. 636-639 ◽

Cited By ~ 12

Author(s):

GA Green ◽

VK Kalra

Keyword(s):

Flow Cytometry ◽

Gradient Centrifugation ◽

Protein A ◽

Fluorescein Isothiocyanate ◽

Low Density ◽

Autologous Plasma ◽

Sickle Cells ◽

Sickle Erythrocytes

Abstract Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

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IMMU-14. ONCOLYTIC VIRUS EXPRESSING A POSITIVE IMMUNE CHECKPOINT MODULATOR AS A THERAPEUTIC APPROACH FOR DIPG

Neuro-Oncology ◽

10.1093/neuonc/noz175.508 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi122-vi122

Author(s):

Virginia Laspidea ◽

Montse Puigdelloses ◽

Ignacio Iñigo-Marco ◽

Marc Garcia-Moure ◽

Iker Ausejo ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Cell Death ◽

Cell Lines ◽

Oncolytic Virus ◽

Murine Models ◽

Antitumoral Effect ◽

Infected Cells

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

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Demonstration of endothelial adhesion of sickle cells in vivo: a distinct role for deformable sickle cell discocytes

Blood ◽

10.1182/blood.v79.6.1602.1602 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1602-1611 ◽

Cited By ~ 37

Author(s):

ME Fabry ◽

E Fine ◽

V Rajanayagam ◽

SM Factor ◽

J Gore ◽

...

Keyword(s):

Animal Model ◽

Intact Animal ◽

Ex Vivo ◽

Sickle Cells ◽

Endothelial Monolayers ◽

Quantitative Studies ◽

Tissue Edema ◽

In Vivo Animal Model ◽

Von Willebrand

Abstract Different morphologic and density classes of sickle cells (SS) may play distinct roles in the generation of vasoocclusion, explaining the complexity of this phenomena. The densest SS red blood cells (RBCs) (SS4) can induce vasoocculsion in ex vivo microcirculatory preparations as well as in an intact animal model. Previous studies of the interaction of SS deformable discocytes with endothelial monolayers or the rat ex vivo mesocecum preparation have shown adhesion that is desmopressin (dDAVP)-stimulated, von Willebrand factor (vWF)-mediated, and limited to the small venules. However, in vivo adhesion of SS RBCs to the endothelium has neither been demonstrated nor characterized; and, in particular, the relation of adhesion to vasoocclusion is unknown. Using an intact animal model that involves injecting saline- washed, density-defined SS RBCs into the femoral artery of a rat, we find that: (1) Quantitative studies of RBCs retained in the rat thigh using 99mTc-labeled RBCs and gamma camera imaging showed that dDAVP induces a threefold increase in retention of normal (AA) cells and deformable SS discocytes (SS2). (2) electron microscopy and Microfil injection show that the retention of SS2 cells is due to adhesion to the vascular endothelium with no evidence of obstruction. (3) H-1 magnetic resonance imaging showed that retention of SS4 cells induced a dose-dependent increase in tissue edema (presumable secondary to tissue hypoxia), while retention of AA or SS2 cells produced no change. We conclude that endothelial adhesion of deformable SS discocytes can be demonstrated in an in vivo animal model, that this adhesion is enhanced by dDAVP (presumably related to, but not necessarily limited to the release of vWF), and that this phenomenon per se does not lead to vasoocclusion. Nevertheless, adhesion of deformable SS discocytes may have consequences. We hypothesize that adhesion of SS discocytes could narrow the lumen of postcapillary venules and facilitate secondary trapping of SS4 cells and lead to subsequent vasoocclusion.

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STEM-12. ZIKA VIRUS TARGETS GLIOBLASTOMA STEM CELLS THROUGH A SOX2-INTEGRIN α vβ 5 AXIS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.829 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii198-ii199

Author(s):

Clark Chen ◽

Sanjay Dhawan ◽

Zhe Zhu ◽

Pinar Mesci ◽

Jeremy Rich

Keyword(s):

Stem Cells ◽

Zika Virus ◽

Oncolytic Virus ◽

Great Promise ◽

Normal Brain ◽

Zikv Infection ◽

Glioblastoma Stem Cells ◽

Interferon Stimulated Genes ◽

Sox2 Expression

Abstract INTRODUCTION Oncolytic virus hold great promise as a platform for glioblastoma therapeutic development. Zika virus (ZIKV) is an oncolytic virus with exquisite selectivity for infecting and killing glioblastoma stem cells (GSCs). Here, we delineate the molecular determinant of this selectivity. METHODS cell-based glioblastoma models, glioblastoma organoid assays, in vivo murine glioblastoma models, ZIKV infectivity assays, gene silencing, ChIP-seq studies. RESULTS In independent models, ZIKV preferentially infected and lysed SOX2+ GSCs. Silencing of SOX2 expression attenuated this preferential infectivity. Of note, ZIKV infection of GSCs was independent of AXL, its putative receptor in normal brain. ChIP-seq experiments revealed that SOX2 bound within the ITGAV locus (encoding the integrin av subunit), and this binding was associated with accumulation of the active chromatin mark H3K27ac. Silencing of SOX2 suppressed ITGAV expression as well as ZIKV infectivity against GSCs, indicating that integrin is required for ZIKV infection. Of integrin b units, only silencing of integrin b5 prevented the killing of GSCs by ZIKV infection, suggesting ZIKV infection required the avb5 integrin. Supporting this hypothesis, blockade of the avb5 integrin substantially reduced ZIKV infection of GSCs in glioblastoma organoid assays and in clinical glioblastoma specimens. Sox2 expression additionally suppress GSC expression of all members of the interferon-stimulated genes (ISG family), thereby suppressing innate anti-viral response to facilitate ZIKV infection. CONCLUSIONS Collectively, our results reveal that ZIKV infection of GSCs is mediated by integrin α vβ 5 leading to SOX2 expression which negatively regulates antiviral immunity thereby facilitating ZIKV infection.

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Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia

Blood ◽

10.1182/blood.v98.5.1577 ◽

2001 ◽

Vol 98 (5) ◽

pp. 1577-1584 ◽

Cited By ~ 80

Author(s):

Kitty de Jong ◽

Renee K. Emerson ◽

James Butler ◽

Jacob Bastacky ◽

Narla Mohandas ◽

...

Keyword(s):

Red Blood Cells ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Blood Cells ◽

Cell Model ◽

Red Cell ◽

Sickle Cells ◽

Red Cell Survival ◽

Transgenic Murine Models

Several transgenic murine models for sickle cell anemia have been developed that closely reproduce the biochemical and physiological disorders in the human disease. A comprehensive characterization is described of hematologic parameters of mature red blood cells, reticulocytes, and red cell precursors in the bone marrow and spleen of a murine sickle cell model in which erythroid cells expressed exclusively human α, γ, and βS globin. Red cell survival was dramatically decreased in these anemic animals, partially compensated by considerable enhancement in erythropoietic activity. As in humans, these murine sickle cells contain a subpopulation of phosphatidylserine-exposing cells that may play a role in their premature removal. Continuous in vivo generation of this phosphatidylserine-exposing subset may have a significant impact on the pathophysiology of sickle cell disease.

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Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin

Blood ◽

10.1182/blood.v88.11.4348.bloodjournal88114348 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4348-4358 ◽

Cited By ~ 54

Author(s):

A Kumar ◽

JR Eckmam ◽

RA Swerlick ◽

TM Wick

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Sickle Cell ◽

Cell Adherence ◽

Inhibitory Antibody ◽

Sickle Cells ◽

Sickle Erythrocyte ◽

Sickle Erythrocytes ◽

Beta 1 Integrin

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular-cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/-17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu- treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.

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Mn Porphyrin-Based Redox Active Drugs As Novel Anti-Adhesive and Anti-Inflammatory Agents Targeting Sickle Red Blood Cell Oxidative Damage In Vivo

Blood ◽

10.1182/blood-2018-99-119066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 270-270

Author(s):

Thamilarasan Madhan ◽

Rodolfo Estupinan ◽

Rahima Zennadi

Keyword(s):

Cell Adhesion ◽

Blood Cell ◽

Red Blood Cell ◽

Intravital Microscopy ◽

Leukocyte Adhesion ◽

Venous Blood ◽

Blood Gases ◽

Leukocyte Rolling ◽

Sickle Cells

Abstract In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by occlusion of the vessels due largely to sickle red blood cell (RBC) adhesion to both the endothelium and adherent leukocytes. RBC oxidative damage caused by continuous endogenous and exogenous oxidative stress may participate in the occurrence of vaso-occlusive crises. We have evaluated the effects of scavenging reactive oxygen species (ROS) in sickle RBCs on cell adhesion and vaso-occlusion in a humanized mouse model of vaso-occlusion in vivo analyzed by intravital microscopy. To scavenge RBC ROS, we used our manganese porphyrin-based superoxide dismutase (SOD) mimics MnTnBuOE-2-PyP5+ (MnBuOE) and MnTE-2-PyP5+ (MnE), powerful catalysts of superoxide dismutation, and reductants of peroxynitrite, peroxide and hypochlorite. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants was performed after the inflammatory trigger of tumor necrosis factor alpha (TNFα) to induce vaso-occlusion in transgenic sickle mice followed by subcutaneous injection of MnBuOE at 0.1, 0.2 or 2 mg/kg, or MnE at 0.5 or 2 mg/kg. Treatment of sickle mice with only one dose of 0.1, 0.2 and 2 mg/kg MnBuOE decreased dose-dependently adhesion of both sickle cells and leukocytes in enflamed vessels by 68±4% (p<0.01), 85±2.3% (p<0.01) and 89±4.3% (p<0.01), respectively, compared with vehicle-treated sickle mice. MnBuOE at 0.1, 0.2 and 2 mg/kg also caused significant and dose-dependent reduction in leukocyte rolling flux (p<0.05). Similar inhibitory benefits were obtained when MnE was administered to TNFa-treated sickle mice. MnE at 0.5 and 2 mg/kg significantly decreased the number of adherent sickle cells and leukocytes by 76±8.6% (p<0.01) and 92±2.5% (p<0.01), respectively, and leukocyte rolling flux (p<0.01) compared to vehicle-treated animals. The effect of these two SOD mimics on sickle RBCs and leukocyte adhesion, and leukocyte rolling flux was rapid, because a decline in cell adhesion and leukocyte rolling flux were already detectable within the first 15 minutes after injection of the compounds. In contrast, cell adhesion and leukocyte rolling flux were already pronounced 15 minutes following vehicle injection. Reduced cell adhesion to the endothelium by the SOD mimics resulted in improved microcirculatory blood flow in sickle mice. These favorable effects on cell adhesion and vaso-occlusion following SOD mimic treatment were indeed due at least to the significant decrease in sickle RBC ROS levels compared to vehicle-treated mice (p<0.001). The long-term anti-adhesive and anti-inflammatory effects of MnBuOE and MnE in sickle mice were next examined. Subcutaneous administration for 28 days of MnBuOE at 0.1 and 0.5 mg/kg inhibited significantly adhesion of RBCs and leukocytes in enflamed venules by 34±13% (p<0.05) and 69±3.5% (p<0.001), respectively, and leukocyte rolling flux (p<0.001) compared to vehicle-treated sickle mice. Subcutaneous injection of MnE at 0.5 and 1 mg/kg for 28 days also had significant effect on sickle cell and leukocyte adhesion (p<0.01), and leukocyte rolling flux (p<0.01). In addition, venous blood gases were significantly improved by the SOD mimics. The levels of partial pressure of Carbon dioxide (pCO2), partial pressure of oxygen (pO2), base excess of the extracellular fluid (BEecf), bicarbonate (HCO3-) concentration, total CO2 (TCO2) concentration, and the indicators of hypoxia, hemoglobin saturation of oxygen (sO2) and lactate, became close to or within the normal ranges (p<0.05) in sickle mice treated with 1 mg/kg MnE. MnBuOE at 0.1 mg/kg showed only a trend toward an increase in venous blood gases, with a significant decrease in lactate (p<0.05). Leukocytosis in sickle mice treated with the SOD mimics was also alleviated. A significant drop in leukocyte (p<0.05), neutrophil (p<0.01), lymphocyte (p<0.05) and monocyte (p<0.05) counts was detected in sickle mice treated with either 0.1 mg/kg MnBuOE or 1 mg/kg MnE. These beneficial therapeutic outcomes induced by the SOD mimics were due at least in part to a decline in RBC ROS levels (p<0.001) and RBC phosphatidylserine surface exposure (p<0.05), an eryptosis marker. These results suggest that our SOD mimics may represent a valuable novel therapeutic intervention for not only vaso-occlusive crises, but inflammation as well, that should be further evaluated in patients with SCD. Disclosures No relevant conflicts of interest to declare.

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