bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biological and Therapeutic Significance of Thioredoxin-1 (Trx-1) In Diffuse Large B-Cell Lymphomas
Authors: Pham Lan V , Thompson Michael A. , Tamayo Archito , Li Changping , Powis Garth , Ford Richard J. 
Published: October 2019

Abstract
Abstract 2004
Diffuse large B cell lymphomas (DLBCL) are the most common non-Hodgkin (NHL-B) lymphoma. Current treatment is fairly successfully (∼70-80% remission with R-CHOP frontline chemotherapy), but relapse is common (∼50% after 2–3yrs) with poor salvage therapy options and short survival in relapsed/refractory (r/r) DLBCL. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, which we currently have very little understanding of the basis. Elucidation of molecular pathways and tumor-encoded genes whose expression contribute to the intrinsic resistance and rapid cell growth of lymphoma cells could yield immediate clinical benefits and reveal new therapeutic targets for effective control and treatment of r/r lymphomas. Thioredoxin (Trx) family members play critical roles in the regulation of cellular redox homeostasis. Cancer cells exist in a stressed environment and rely on the Trxs for protection against stress-disregulated redox signaling. The most extensively studied member of the family is Trx-1 whose levels are increased in many human cancers, most likely in direct response to stress. Trx-1 contributes to many of the hallmarks of cancer including increased proliferation, resistance to cell death and increased angiogenesis. Trx-1 is a validated cancer drug target associated with aggressive tumor growth, resistance to standard therapy and decreased patient survival. In this study we showed that the majority of the DLBCL-derived cell lines express high basal levels of thioredoxin-1 (Trx-1) when compared to normal B cells by both Western blotting and real-time PCR. Tissue microarray (TMA) and immunohistochemistry analysis of 100 cases of DLBCL showed that 77% of the cases were positive for Trx-1 and 40% of the cases over-expressed Trx-1. Based on these observations, we hypothesize that aberrant expression of Trx-1 contributes to the development of drug resistance and cell growth and survival phenotype in DLBCL. Inhibition of Trx-1 by small interfering RNA (siRNA) inhibited DLBCL cell line growth in vitro. Down-regulation of Trx-1 also sensitized lymphoma cells to doxorubicin-induced cell growth inhibition. A doxorubicin-resistant human DLBCL cell line (McA), that was highly sensitive to doxorubicin, has been selected by continuously exposing cells to gradually increasing doses of doxorubicin. The resistant phenotype has been retained for over 6 months despite growth in drug-free medium. Western blot analysis detected increased expression of the Trx-1 in the resistant subline. These results indicate that Trx-1 plays a key role in growth and survival, as well as chemoresistance in the pathophysiology of DLBCL. Based on these data, we plan to use small molecule inhibitors in vitro as a proof of principle for possible future human phase I studies.

Disclosures:
Ford: Cell Point: Research Funding.
Protein Kinase C Delta Mediates the Activation of Protein Kinase D In Platelets
Authors: Bhavanasi Dheeraj , Kim Soochong , Goldfinger Lawrence E , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 2021
Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCm, PKD2 and PKCn in humans). They are part of alternate DAG receptors along with RasGRPs, Munc 13s, chimerins and DAG kinases. Members of the novel class of PKC isoforms such as PKCd, PKCh, and PKCe associate with PKD in smooth muscle cells and COS-7 cells. The mechanism of activation of PKD and the specific PKC isoforms required for its activation are not known to date. This study is aimed at investigating the pathways involved in the activation of PKD in platelets. We show that human as well as murine platelets express PKD. PKD could be activated with PAR4 agonist AYPGKF, PAR1 agonist SFLLRN and GPVI agonist convulxin. AYPGKF and SFLLRN induced PKD phosphorylation as early as 30 sec and convulxin induced PKD phosphorylation at 1 minute. PKD phosphorylation induced by AYPGKF and convulxin were sustained for 5 minutes but phosphorylation induced by SFLLRN was attenuated after 2 minutes of stimulation. AYPGKF-induced PKD phosphorylation was reduced with a calcium chelator, dimethyl BAPTA, indicating that calcium-mediated signals might play a role in activation of PKD. PKD phosphorylation in response to AYPGKF was abolished with a Gq inhibitor, YM-254890, but was not affected by Gi-coupled P2Y12 receptor antagonist ARC-69931MX, indicating that PKD phosphorylation is Gq-, but not Gi- or G 12/13-dependent. PKD phosphorylation was abolished with pan-PKC inhibitors, GF109203X and Ro31-8220, indicating that PKCs are required for PKD activation in platelets. PKD phosphorylation was significantly inhibited with a PKC delta inhibitor, rottlerin, but was not affected by the classical PKC inhibitor, Go6976, suggesting that novel PKC isoforms are important for PKD activation. In addition, 2MeSADP that fails to activate PKCd did not induce phosphorylation of PKD in platelets. Furthermore, phosphorylation of PKD induced by AYPGKF was significantly reduced in PKCd-deficient platelets compared to that of wild type platelets. Hence, we conclude that PKD is a common signaling target downstream of various agonist receptors in platelets, and Gq-mediated signals and novel PKC isoforms, in particular PKCd is required for activation of PKD.

Disclosures:
No relevant conflicts of interest to declare.
Transcriptional Regulation of Transferrin Receptor by Heme in Erythroid Cells
Authors: Schranzhofer Matthias , Lok Nam-Chun , Schifrer Manfred , Muellner Ernst W , Ponka Prem 
Published: October 2019

Abstract
Abstract 2049
Erythroid cells are the largest consumers of iron which is delivered to them by tansferrin (Tf) by its cognate receptor (TfR). In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression accompanied by increased iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:1084-91; 2005; Galy et al., Blood 106:2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis.

Disclosures:
No relevant conflicts of interest to declare.
CD137 Identifies a Population of Regulatory T Cells That Inhibit Anti-Tumor Immune Responses In Adoptive Immunotherapy
Authors: Goldstein Matthew J , Kohrt Holbrook E , Houot Roch , Varghese Bindu , Lin Jack T , Swanson Erica , Levy Ronald 
Published: October 2019

Abstract
Abstract 2093

Background:
Adoptive immunotherapy is a promising novel approach to the treatment of cancer. However, clinical translation of adoptively transferred CD4 T cells is limited by cotransfer of an inhibitory population of regulatory CD4 T cells (Tregs). We identified a method of isolating viable antitumor CD4 T cells while excluding Tregs based on two surface markers—CD44 and CD137.

Methods:
We have developed a model for adoptive cell therapy of lymphoma whereby anti-tumor T cells are generated in vivo through vaccination with a CpG-loaded whole cell vaccine (CpG/H11). These vaccine-induced cells can protect from lethal tumor challenge when isolated and transferred into lethally irradiated, syngeneic recipient mice. We investigated the subsets of T cells involved in the anti-tumor response through a combination of in vitro and in vivo assays.

Results:
Adoptive transfer of CD137negCD44hi CD4 T cells, but not other sub-populations, provided protection from B cell lymphoma. We demonstrate that the population of CD137posCD44hi CD4 T cells consists primarily of activated Tregs. In vitro, these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner. We observed that this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, the addition of CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. In the presence of CD137posCD44hi CD4 T cells, homing of other T cell populations to tumor sites was disrupted. These results suggest that CD137 expression on CD4 T cells defines a population of activated Tregs that prevent antitumor immune responses.

Conclusions:
Our findings identify two surface markers that can be used to facilitate the enrichment of anti-tumor CD4 T cells while depleting an inhibitory Treg population. This approach has direct applicability to clinical trials for patients with lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
Normal Cleavage of Von Willebrand Factor by ADAMTS13 In the Absence of Factor VIII In Patients with Severe Hemophilia A
Authors: Chen Junmei , Chung Dominic W. , Le Jennie , Konkle Barbara A. , López José A. 
Published: October 2019

Abstract
Abstract 2114
The size of von Willebrand factor (VWF), a carrier protein for factor VIII (FVIII), is regulated by plasma metalloprotease ADAMTS13 proteolytic activity. Recently studies by Cao et al. (PNAS, 2008; 105: 7416–7421) found that under shear stress, exogenous FVIII enhanced ADAMTS13 cleavage of VWF, especially the high molecular weight multimers, in a system using purified proteins. Based on this result, the authors suggested that in the absence of FVIII, such as in patients with severe hemophilia A, VWF will have ultra-large multimers due to defects in ADAMTS13 proteolytic process, which can be corrected by infusion of FVIII. Here, we assessed VWF multimers, antigen, and ADAMTS13 activity in citrated plasma from seven patients with severe hemophilia A. The FVIII levels in six patients were less than 1% and in one was 4%. Plasma from two patients was available both pre- and post-FVIII replacement therapy (recombinant FVIII). All patients displayed VWF multimer patterns similar to those in pooled normal plasma (PNP), and the two patients receiving FVIII infusions displayed no change in VWF multimer size or pattern between their pre- and post-infusion samples. In all patients, the VWF antigen level (0.32–0.76) was below the PNP value (designated as 1), and all had increased ADAMTS13 activity (measured by the ability of plasma to cleave a small A2 peptide substrate) (1.09–1.79, PNP designated as 1), yielding an increased ratio of ADAMTS13 activity to VWF antigen in these patients (1.4–5.2 compared to PNP). We also examined cleavage of endogenous VWF by ADAMTS13 in the plasmas of the two patients studied pre- and post-infusion, yielding different FVIII levels. In this assay, we first diluted plasma 10-fold with a buffer containing 10 mM HEPES, 6.5 mM BaCl2, and 1.5 M urea, incubated at 37°C, and ADAMTS13 cleavage was stopped at different time points with EDTA. VWF multimer patterns were examined on a 1.5% agarose gel. We found that ADAMTS13 cleaved VWF efficiently in patient plasma deficient in FVIII, similar to that in PNP. The extent of cleavage was correlated with the ratio of ADAMTS13 activity to VWF antigen, rather than with the FVIII levels. In conclusion, patients with severe hemophilia A appear to have normal ADAMTS13 processing of VWF multimers in vivo and ex vivo. Further studies of the effect of FVIII and VWF levels on ADAMTS13 cleavage of VWF and clinical correlation are needed.

Disclosures:
No relevant conflicts of interest to declare.
Factor X/Xa Elicits Protective Signaling Responses In Endothelial Cells Directly Via PAR-2 and Indirectly Via EPCR-Dependent Recruitment of PAR-1
Authors: Rezaie Alireza R. , Bae Jong-Sup , Yang Likui 
Published: October 2019

Abstract
Abstract 2115
We recently demonstrated that the Gla-domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier protective response if endothelial cells were pre-incubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells, also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a crosstalk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla-domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Disclosures:
No relevant conflicts of interest to declare.
To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line
Authors: Xing Hongyun , Gong Yuping , Liu Ting 
Published: October 2019

Abstract
Abstract 2135

Objective
To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied.

Methods
Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line.

Results
We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line.

Conclusion
In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib.

Disclosures:
No relevant conflicts of interest to declare.
The Synonymous V107V Mutation In Factor IX Is Not So Silent and May Cause Hemophilia B In Patients
Authors: Kimchi-Sarfaty Chava , Simhadri Vijaya L , Kopelman David , Friedman Adam , Edwards Nathan , Javaid Atiq , Okunji Chinyere , Komar Anton , Sauna Zuben , Katagiri Nobuko 
Published: October 2019

Abstract
Abstract 2197
Hemophilia B is characterized by structural and functional defects in coagulation factor IX (FIX) caused by mutations in the F9 gene. Various mutations (nonsense, missense, etc.) are known to be associated with the disease, including a synonymous V107V mutation reported recently by Knobe and colleagues (Knobe et al., Hemophilia, 2008). However the mechanism by which this synonymous mutation contributes to the disease has not yet been elucidated. Earlier we have shown that synonymous codon substitutions in the mRNA of the multidrug resistance protein (MDR1) may change the conformation of the protein and result in altered functionality (Kimchi-Sarfaty et al., Science, 2008). Here we have performed in silico analyses of the synonymous codon substitution (GTGàGTA) leading to the V107V polymorphism and found that it may change the mRNA structure, stability, codon usage, and 3D structure of the encoded protein. We hypothesize that changes in codon usage might affect the rhythm of protein translation and thus result in slightly altered FIX conformation. In vitro analyses of FIX mRNA and protein expression supported our in silico analyses. The GTGàGTA (V107V) synonymous mutation results in reduced expression levels as well as an encoded protein with a slightly different conformation compared to wild-type FIX. These results show that the V107V polymorphism is not silent and might cause mild hemophilia B. This work sheds further light on ways in which synonymous mutations impact disease.
The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy

Disclosures:
No relevant conflicts of interest to declare.
Ideal Amphipathic Peptdes Coupled to Nanofibrous Micropheres Reduce Hemorrhage In Vivo
Authors: Lemarie Catherine , Wu Jianqiu , Barralet Jake , Blostein Mark 
Published: October 2019

Abstract
Abstract 2204
We have previously shown that ideal amphipathic peptides (IAP) composed simply of leucine and lysine residues can augment the function of the procoagulant enzyme, factor IX in vitro (Biochem J 2008). We have also shown that surface attachment of IAP can promote hemostasis and reduce bleeding in vivo. We extend these observations by coupling IAPs to a nanomaterial called nanofibrousmicrospheres (NFM). NFMs have a large surface area to volume ratio thereby providing greater surface area and better access to bleeding surfaces especially those that are internal and difficult to reach. Initially we demonstrate, by fluorescently labeling IAP with dansyl chloride, that IAP attaches to NFM. When free dansyl IAP is added to NFM, 90% binds to the surface of NFM via a non-covalent linkage. Both a murine liver laceration model and a murine tail transection model were established to evaluate the in vivo hemostatic properties of IAP attached to NFM (IAP-NFM). In a murine liver laceration model, where the liver is cut with a Surgicutt template, application of IAP-NFM significantly reduces bleeding times by 40% as compared to application of NFM alone (p<0.01), or no treatment (p<0.01). As well, comparison of IAP-NFM to QuikClot, a current commercially used product for control of hemorrhagic injuries, show a greater reduction in bleeding times after application of IAP-NFM as compared to QuikClot. In addition, histologic examination of the lacerated liver after treatment reveal that IAP-NFM elicited little tissue injury whereas widespread hepatocyte injury was caused by QuikClot, due to its known exothermic reaction on tissue. The bleeding time in a murine tail transection model was also reduced by approximately 30% by IAP-NFM, as compared to NFM alone (p<0.01), or no treatment (p<0.05). In conclusion, IAP attachment to NFM results in a biocompatible material that is procoagulant and can promote hemostasis in vivo with the advantage that it exhibits little tissue injury after its application.

Disclosures:
No relevant conflicts of interest to declare.
Association of Quantitative and Qualitative Abnormalities of Von Willebrand Factor and Risk of Death In Hemodialysis Patients
Authors: Holden Rachel , Tuttle Angie , MacLeod Francis , Burbidge Toni , Hegadorn Carol , James Paula D. 
Published: October 2019

Abstract
Abstract 2217

In order to evaluate the possible role of abnormalities of von Willebrand factor in the hemostatic defects seen in indivdiuals with chronic kidney disease (CKD), a cohort study was performed evaluating pre- and post-dialysis levels of von Willebrand factor (VWF), VWF multimer profiles and levels of its cleaving protease, ADAMTS-13. There were 57 subjects (31 males, 26 females) enrolled with CKD with a mean age of 75 years (range 60 – 90). Subjects with known vascular disease were recruited; 49 (86%) had documented ischemic heart disease, 16 (29%) had cerebrovascular disease and 17 (31%) had peripheral vascular disease. A little over half had diabetes mellitus (30 subjects or 54%), 37 (67%) were on antiplatelet therapy and 7 (13%) were chronically anticoagulated with warfarin. Blood samples were drawn immediately pre- and again post-dialysis and all results were compared with a group of age-matched normal controls (Table 1). As has been previously reported, VWF antigen levels (VWF:Ag) and VWF functional activity as measured by the ristocetin cofactor assay (VWF:RCo) were higher in the pre-dialysis samples compared with controls, and both levels were increased even further following dialysis. Additionally, the percentage of high molecular weight VWF multimers (% HMWM) were significantly increased in the pre-dialysis samples compared with controls. This is a novel finding, and the level of % HMWM seen in the subjects is similar to what has been reported in individuals with Thrombotic Thrombocytopenic Purpura (TTP). This difference decreased following dialysis, potentially due to the effect of shear stress on VWF and the resultant proteolytic processing, however still remained significantly higher when compared with controls. ADAMTS-13 functional activity was lower in the subjects compared with controls, providing a possibly explanation for the increase in % HMWM. IL-6 levels are higher in subjects compared with controls. IL-6, which is an inflammatory cytokine known to be increased in patients with CKD, has been previously reported as a marker of inactivation of ADAMTS-13. Two years after enrollment, follow up of the subjects revealed that 22 had died, 17 from documented cardiovascular events. Higher VWF levels at the time of enrollment significantly correlated with risk of death (p=0.041) during the study period. The increase in % HMWM suggests that a “TTP-like phenotype” may also be playing a role. Taken together, these data suggest that both quantitative and qualitative abnormalities of VWF contribute to the risk of thrombotic death in chronic kidney disease.

Disclosures:
No relevant conflicts of interest to declare.