bluuebunny/crossref_metadata_2025_split huggingface dataset:
HLA-A*0201 Restricted Killing Activity of WT1-Specific CTL Generated From the Peripheral Blood Lymphocytes of Normal Healthy Donors.
Authors: Krishnadas Deepa Kolaseri , Stamer Mindy , Dunham Kim , Bao Lei , Lucas Kenneth 
Published: October 2019

Abstract
Abstract 3026
Poster Board II-1002
The Wilms' tumor antigen (WT1) is over-expressed on several human leukemia and solid tumors, and thus is considered as a potential target for cancer immunotherapy. Combating leukemia by targeting WT1 expressing leukemic cells using in vitro generated WT1-specific CTL is one potential approach, but it is difficult to generate an immune response against WT1 due to low T cell precursor frequency in normal healthy individuals. Earlier studies have shown the generation of WT1-A*0201 peptide specific CTL from CD8+ T cells by cloning. Another study reported the production of IFN- γ by WT-1 specific CD8+ T cells. However, the cytolytic killing ability of these IFN- γ producing cells was not further characterized. Here, we demonstrate the generation of WT1-A*0201 specific CTL from the peripheral blood lymphocytes (PBL) of normal healthy donors using CD137 selection. The PBL were stimulated once with RMFPNAPYL (WT1-A*0201 peptide) pulsed autologous dendritic cells and twice with WT1-A*0201 peptide pulsed irradiated peripheral blood mononuclear cells (PBMC). Following three stimulations, the PBL were selected for CD137+ expression and rapidly expanded with OKT3 and IL-2. The WT1-A*0201 specific CTL showed killing of target cells and production of IFN-γ in an antigen-specific manner. The percent killing of WT1-A*0201 peptide pulsed T2 cells (TAP−, HLA- A2+) and autologous B blast (BB) were significantly higher when compared with their control targets. T2 cells and BB either pulsed with an irrelevant A*0201 peptide or un-pulsed served as the control. We have observed similar results with WT1-A*0201 specific CTL generated from normal donor CD8+ cells. However, the efficiency of WT1-A*0201 CTL generated from PBL to kill target cells and produce IFN- γ was higher than CTL from CD8+ cells. The CTL generated from PBL killed BA25, a WT1 expressing A2+ leukemia cell line but failed to kill Molt-4, a WT1 expressing A2− cell line, clearly indicating HLA-A2 restricted CTL activity. The specificity of the generated CTL were further confirmed by staining with WT1-HLA-A*0201 tetramer. The percentage of WT1-specific CD3+CD8+Tetramer+ cells either remained same or higher in CTL generated from PBL when compared with those generated from CD8+ cells. CD137 selection leads to the generation of significant number of CTL in a shorter time when compared to conventional cloning methods. In addition, generation of WT1-A*0201 specific CTL from PBL avoids CD8+ selection. Currently, we are aiming to generate WT1-specific CTL using an overlapping WT1 peptide-mix in order to widen our ability to treat patients with different HLA types. This study has implications for cellular immunotherapy in leukemia patients who relapse following allogeneic stem cell transplantation.

Disclosures
No relevant conflicts of interest to declare.
IL-7 as a Membrane-Bound Molecule for the Costimulation of Tumor-Specific T Cells.
Authors: Hurton Lenka V. , Singh Harjeet , Olivares Simon , Ang Sonny O. , Kelly Susan Staba , Kebriaei Partow , Lee Dean A. , Champlin Richard E. , Cooper Laurence J.N. 
Published: October 2019

Abstract
Abstract 3035
Poster Board II-1011
Redirecting specificity to a selected cell surface tumor-associated antigen can be accomplished by the genetic modification of T cells to express a chimeric antigen receptor (CAR). Despite systematic modifications to the CAR endodomains to provide T cells with competent signaling, CAR-dependent T-cell activation may remain incomplete resulting in inferior in vivo persistence leading to a suboptimal therapeutic response. To improve T-cell survival and therefore an anti-tumor response, investigators have co-infused soluble recombinant cytokines such as IL-2 and IL-7. IL-7 is a homeostatic cytokine for T cells and supports survival of memory T cells. To provide IL-7 mediated signaling to T cells in the tumor microenvironment and thus enhance the proliferation and survival of CAR+ T cells, we constructed a version of IL-7 as a novel membrane-bound molecule (mIL7) designed to stimulate T cells in cis and trans. The mIL7 construct was electro-transferred with a CD19-specific CAR (on day 0) into primary T cells via multiple transposition of Sleeping Beauty DNA plasmids. These genetically modified T cells could be numerically expanded ex vivo without additional soluble cytokine supplementation on CD19+ artificial antigen presenting cells (aAPC) derived from K562. This resulted in the preferential outgrowth of T cells expressing both mIL7 and CAR (Figure A and B) while CAR+ T cells receiving no soluble cytokine supplementation did not sustain proliferation (Figure B). These mIL7+CAR+ T cells exhibited redirected specific lysis of CD19+ tumor targets. Significantly, the kinetics of propagation of mIL7+CAR+ T cells in the absence of exogenous cytokine was comparable to CAR+ T cells that were numerically expanded in the presence of soluble IL-2. Signaling by IL-7 receptor signal induction in mIL7+CAR+ T cells appeared comparable to signaling by soluble IL-7 in CAR+ T cells, as assessed by phosphorylation of signal transducer and activator of transcription 5 (STAT5). These data demonstrate that mIL7 can be expressed by CAR+ tumor-redirected T cells to enhance their proliferation without the need for additional cytokine support. These results have implications for the design of clinical trials to evaluate whether mIL7+CAR+ T cells can exhibit enhanced persistence and thus improved therapeutic potential.

Disclosures
No relevant conflicts of interest to declare.
Antiphospholipid Antibodies Promote Leukocyte-Endothelial Cell Adhesion by Antagonizing Endothelial NO Synthase Via b2GPI and ApoER2.
Authors: Tarango Cristina , Ramesh Sangeetha , Morrell Craig N. , Thomas Gail D. , Girardi Guillermina , Salmon Jane , Yuhanna Ivan S. , Herz Joachim , Thorpe Philip , Shaul Philip , Mineo Chieko 
Published: October 2019

Abstract
Abstract 3039
Poster Board II-1015
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL), with pathogenic aPL directed primarily against the cell surface protein β2 glycoprotein I (β2GPI), increased risk for thrombosis and cardiovascular events, and pregnancy morbidity. The endothelium is a critical direct target of aPL, which upregulate adhesion molecule expression and procoagulant activity. However, the molecular basis for aPL actions on endothelium is unknown. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) has potent antiadhesive and antithrombotic properties. In this study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and polyclonal aPL were obtained from healthy adults and APS patients, respectively. We found that aPL prevented acetylcholine (Ach)- or VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this was reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG did not affect adhesion. Whereas NHIgG did not alter eNOS activation, stimulation of eNOS by VEGF and other agonists was fully antagonized by aPL. In mice, NO-dependent, Ach-induced increases in carotid vascular conductance were unaffected by NHIgG but impaired by aPL, indicating that these processes are operative in vivo. Additional studies in culture showed that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation, deprivation of β2GPI prevented aPL inhibition of eNOS, and monoclonal antibody against β2GPI mimicked the effects of aPL. Furthermore, receptor-associated protein (RAP) antagonism of LDL receptors, which are known to bind dimerized β2GPI, prevented aPL inhibition of eNOS in culture, and mice null for the LDL receptor apolipoprotein receptor 2 (apoER2) were protected from aPL inhibition of eNOS in vivo. Moreover, aPL-stimulated increases in leukocyte-endothelial adhesion were absent in both eNOS-/- and apoER2-/- mice. Thus, aPL-induced increases in leukocyte-endothelial adhesion are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI and apoER2. Novel therapies for APS can now be developed targeting these mechanisms.

Disclosures
Thomas: NIcox Research Institute: Research Funding. Thorpe:Peregrine Pharm.: Consultancy, sponsored research agreement.
Human ALDH-Bright Bone Marrow Cells Produce Paracrine Factors That Protect Endothelial Cells From Hypoxic and Nutritional Stress.
Authors: Smith Liisa , White Hannah , Gentry Tracy , Balber Andrew 
Published: October 2019

Abstract
Abstract 3056
Poster Board II-1032
Human bone marrow cell populations isolated by sorting cells on the basis of high aldehyde dehydrogenase expression (ALDHbr cells) include endothelial, mesenchymal, and other progenitor cells (Gentry et al, 2007, Cytotherapy 9, 259). These cell populations home to and are retained at ischemic endothelium, induce angiogenesis, and effect restoration of tissue perfusion in a mouse hind limb ischemia model (Cappoccia et al, 2009 Blood 113, 5340.) Critical limb ischemia patients treated with ALDHbr cells in a Phase I/II clinical trial showed increased limb perfusion. We are studying paracrine and contact dependent mechanisms by which ALDHbr cells may repair damaged endothelium by measuring the protective effects of ALDHbr cells on early passage human umbilical vein endothelial cells (HUVEC) exposed to hypoxic and nutritional stress. ALDHbr cells migrated through membrane filters in response to supernatants conditioned by hypoxic HUVECs more rapidly than to normoxic HUVEC supernatants. ALDHbr cells attached to HUVECs that had been induced to form tubular structures on Matrigel®, and more ALDHbr cells decorated HUVEC tubules under hypoxic than normoxic conditions. ALDHbr cells and HUVECs expressed several adhesion molecule-ligand pairs, including VLA-1/VCAM, that are regulated by hypoxia and could mediate these interactions. While HUVEC tubules fell apart under hypoxic conditions, adding ALDHbr cells in a transwell culture system for 24 hours preserved the branching structure of hypoxic HUVEC tubule networks. Gene array studies demonstrated that ALDHbr cells highly express several angiogenic growth factors, cytokines and matrix remodeling molecules. Expression of proteins corresponding to many of these gene products, including members of the ephrin-Eph receptor family that can direct endothelial growth, has been confirmed by flow cytometry. Additionally, 29 angiogenic factors including angiopoietin 2, VEGF-A,C, and D, and MMP2 were upregulated under hypoxic conditions. Gene array and flow cytometry showed that hypoxic HUVECs expressed surface receptors for many of the angiogenic factors expressed by ALDHbr cells. In hypoxic transwell cultures, ALDHbr cells specifically induced HUVECs to express six angiogenic factors that were not induced by hypoxia alone. ALDHbr cells protected HUVECs from apoptosis and necrosis induced by serum starvation when added to cultures at the time of or 24 hours following medium shift. Thus, in addition to providing progenitor cells that can potentially participate in angiogenesis, ALDHbr cell populations can mediate repair of ischemic injury by releasing a variety of angiogenic and protective factors at sites of endothelial damage.

Disclosures
Smith: Aldagen, Inc.: Employment. White:Aldagen, Inc.: Employment. Gentry:Aldagen, Inc.: Employment. Balber:Aldagen, Inc: Employment, Equity Ownership.
Scanning Electron Microscopy Study of Endothelial Injury and Thrombus Formation in WT and VWF Deficient Mice.
Authors: Barr Justin , Barr Jennifer , Meurice Marielle , Motto David 
Published: October 2019

Abstract
Abstract 3062
Poster Board II-1038
VWF is a large plasma glycoprotein required for normal hemostasis, and performs its function through binding to coagulation Factor VIII, and via interactions with both platelet surface glycoproteins and the activated and/or damaged vascular surface. We have developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation in wild-type and VWF-deficient mice. Thrombus formation is initiated by ferric chloride, and subsequently at defined time points, the circulation is rapidly flushed and aldehyde fixed. The carotid artery is removed, externally fixed, sectioned (both longitudinally and in cross-section), processed for SEM, and visualized. With this protocol we have obtained high-quality images (exceeding 100,000x) of FeCl3-induced endothelial damage and thrombus formation in C57BL/6 and VWF-deficient mice at baseline, and at 30, 60, 90, 120, 240, and 300 seconds post-injury (please access http://sites.google.com/site/mottolab/ to view images). Interestingly, we find that FeCl3 induces little, if any, endothelial denudation and collagen exposure at these time points, with the endothelium clearly appearing changed from baseline, but not damaged. Thus, initial platelet adhesion seems to be occurring in the absence of collagen exposure in this model. In wild-type mice, platelets adhere rapidly to the endothelial surface and assume a cross-linked appearance by 90 seconds, with continual inward growth of the thrombus through the 300 second time point. In VWF-deficient mice, platelets also adhere rapidly to the endothelial surface, but in contrast, remain recognizable longer without assuming a highly-activated phenotype. Compared with wild-type, at all time points examined the VWF-deficient thrombus appears smaller with considerably less cross-linking and platelet activation. Interestingly, during the course of these experiments we also have identified what appears to be red blood cells (RBCs) participating in thrombus formation. Similar to platelets, RBCs interact directly with the endothelial surface, and subsequently become elongated in the direction of blood flow. These elongated RBCs are often observed to cluster and bind platelets, with the subsequent formation of large platelet-erythrocyte complexes. Further characterization of these complexes and the role they may play in thrombus formation is currently in progress. Additionally, similar SEM studies are underway with both ADAMTS13-deficient and GPIb alpha-deficient mice, and with mice transiently expressing in vivo biotinylated VWF for visualization of this molecule at high magnification and resolution. These studies should help better define the mechanisms of endothelial activation and thrombus formation as they occur in situ.

Disclosures
No relevant conflicts of interest to declare.
A Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Leukemic NPMc Protein.
Authors: Du Wei , Maynard Suzette , Sipple Jared , Li Jie , Pang Qishen 
Published: October 2019

Abstract
Abstract 3098
Poster Board III-35
In a subset of AML with a normal karyotype, a frame-shift mutation in the extreme C-terminal of the nucleophosmin (NPM) gene results in the creation of a nuclear export signal, generating a mutant NPM protein (NPMc) that is permanently dislocated in the cytoplasm. In the present study, we have analyzed the interaction between NPMc and a cytoplasmic subcomplex of Fanconi anemia (FA) proteins. Sequence analysis of bone marrow samples from 46 FA patients shows that NPMc mutations were excluded from FA genome. NPMc was degraded more rapidly in AML bone marrow cells from FA patients (t1/2 < 30 min) than in AML cell line HL60 (t1/2 > 90 min). Further analysis revealed that inducible knockdown of FANCA or FANCC in leukemic OCI/AML3 cells carrying the NPMc mutation induced degradation of the cytoplasmic NPMc protein. Forced localization of FANCC to the nucleus also caused rapid NPMc degradation. We also show that NPMc degradation was mediated by the proteasome and that correction of mutant lymphoblasts from FA-A or FA-C patients with a functional FANCA or FANCC protein prevented NPMc ubiquitination and consequently degradation. Moreover, we demonstrate that the cytoplasmic FANCA and FANCC interacted with NPMc in the cytosolic fractions of normal human lymphoblaststic cells and that the acidic domains of NPM were required for the cytoplasmic FA-NPMc complex formation. Finally, using patient-derived FANCC mutant, a nuclearized FANCC and a NOG/SGM3 xenotansplant model, we present evidence that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability and biological function. Thus, these findings reveal the potential molecular mechanism involved in the cytoplasmic retention of the leukemic NPMc.

Disclosures
No relevant conflicts of interest to declare.
Effect of Blood Transfusion On Serum Haptoglobin.
Authors: Gupta Shilpi , Nakhl Fadi , Weiserbs Kera , Forte Frank J. 
Published: October 2019

Abstract
Abstract 3138
Poster Board III-75

Introduction
Haptoglobin, an alpha2 globulin, is an acute phase reactant, which functions to bind the globin portion of free hemoglobin in the blood. The half-life of serum haptoglobin is approximately five days, but in the presence of free hemoglobin, the hemoglobin-haptoglobin complex is rapidly cleared from the system causing a decrease in the measured serum haptoglobin (normal levels 36-195mg/dl). The measurement of serum haptoglobin is used as one of the laboratory markers for the diagnosis of hemolytic anemia. Since stored blood contains a variable amount of free hemoglobin, transfusion of this blood may affect haptoglobin levels and thereby negate the usefulness of haptoglobin measurement when hemolysis is suspected in recently transfused patients.

Patients and methods
Twenty-0ne patients, who were being transfused with more than one unit of packed red blood cells (PRBC), for non-hemolytic indications, were enrolled in the study and their serum haptoglobin levels were recorded before, immediately after, 24 hours after and 48 hours after PRBC transfusion.

Observations and results
A total of twenty-one patients were enrolled in the study and these patients received a total of forty-seven units of PRBC. The most common indication for transfusion was anemia secondary to blood loss. Eighteen patients received two units, one patient received three units, and two patients received four units of PRBC. Four patients received PRBC's that were more than 30 days old. When blood less than 30 days old was transfused there was a decrease to below normal in the haptoglobin level of only one patient, and this patient had a low normal serum haptoglobin level prior to transfusion. However, in four patients who received blood that was more than 30 days old, two (fifty percent) had a decrease in serum haptoglobin levels to below normal.

Conclusion
Serum haptoglobin can be used in the diagnosis of hemolysis in patients receiving multiple units of PRBC transfusions when the age of the transfused blood is less than thirty days. However, when PRBC's more than thirty days old are transfused measurement of serum haptoglobin might not prove to be a reliable indicator of hemolysis. Further studies with larger sample sizes are needed to confirm our findings.

Disclosures
No relevant conflicts of interest to declare.
Apoptotic Microrna Profiling of Packed Red Blood Cells During Storage.
Authors: Kannan Meganathan , Kulkarni Sandhya , Atreya Chintamani D 
Published: October 2019

Abstract
Abstract 3145
Poster Board III-82

Introduction
During storage, RBCs undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo, following transfusion. MicroRNAs (miRs), the negative regulators of cellular mRNAs, control cellular expression of genes relevant to differentiation and apoptosis via mRNA degradation or inhibition of translation. Recent reports indicate that matured red blood cells (RBC) contain diverse population of miRs in abundance. Understanding the role of miRs in RBC during storage would perhaps provide insights into the mechanisms associated with storage lesions.

Methods
In this study, we utilized a membrane-based array to obtain differential miR profiles of 52-apoptosis-associated miRs in packed RBCs during storage. The packed RBCs were obtained from the National Institutes of Health (NIH) blood bank and stored at appropriate storage condition (4-8°C) for up to 40 days. Samples were collected at days 0, 10, 20, 30 and day 40 and subjected to miR analysis. Our rationale is that since miRs are regulators of apoptosis, profiling of apoptosis associated miRs in packed RBCs during storage would provide the first step towards understanding the underlying mechanisms associated with storage lesions.

Results
Our miR analysis identified perturbation of six miRNAs during packed RBC storage. Two miRs remain at high levels throughout the RBC storage period studied while four miRs demonstrated an upward trend from day 0 to day 40 of storage. TarMir bioinformatics-based target gene identification for miR-96 identified CASPN1 mRNA as its target. The presence of CASPN1 mRNA was confirmed by RT-PCR. Although this observation tempts us to speculate that an interaction of CASPN1 with miR-96 in stored RBC is possible, further experimental verification of this interaction is warranted.

Conclusion
The differential microarray analysis presented here suggest that further refinement of miR profiling of stored red blood cells could be of value as predictive markers of ‘cellular status of RBC’ during storage. Future experimental analysis of selected mRNA-miR interactions in packed RBC during storage would provide insights into the mechanisms of storage lesions
The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures
No relevant conflicts of interest to declare.