bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antibody Development in Pediatric Sickle Cell Patients Undergoing Erythrocytapheresis: A 10 Year Review
Authors: Lockwood William B , Godfrey Gwendolyn J , Bertolone Salvatore , Raj Ashok 
Published: April 2020

Abstract
Background: The formation of alloantibodies and/or autoantibodies in multitransfused pediatric sickle cell disease patients undergoing monthly erythrocytapheresis (RBCX) during a 10-year period was evaluated utilizing a retrospective patient chart review.
MATERIALS AND METHODS: Clinical charts and transfusion services serological computer records of 32 patients were reviewed under an IRB approved protocol. All patients were seen in the Pediatric Sickle Cell Clinic, Kosair Children’s Hospital (KCH) between 1998 and 2008. Included in the data review were patient gender, ABO/Rh blood type, interval between RBCX start date and last RBCX, number of RBC components transfused, identity of existing antibody(ies) prior and subsequent to RBCX, and time interval for development of new antibody. All patients received C, E, and K antigen negative RBCs based on their phenotype at the start of their RBCX.
RESULTS: Thirty-two patients received between 8 to 555 RBC transfusions (Table 1). An allo- or autoantibody developed in 5/15 patients (33.3%) receiving C-, E-, K- RBCX; 3/10 patients (33.3%) receiving E-, K- RBCX; 2/4 patients (50%) receiving C-K- RBCX. An autoantibody developed in 3/32 patients (9.4%) as did a low frequency antibody (Low) not otherwise identified. One patient each developed an anti-Kpa and anti-M. In the two patients receiving K- RBCX, no new antibody was identified. Three patients developed anti-E and one patient developed anti-K after receiving partially matched RBCs on an emergency basis.
Table 1. Antibody (Ab) development following erythrocyapheresis (RBCX) of patients with sickle cell disease (n=32)
Transfused Phenotypes Number Patients New Ab Ab ID #RBC to New Ab (mean±SD) Second Ab Ab ID #RBC to Second Ab (mean±SD) *NA=Not applicable C- E- K- 15 5 E, K, M, Auto, Low 34±67 0 NA* NA C- K- 4 2 Auto, Low 114±43 1 Low 203±0 E- K- 11 3 E, Kpa, Auto 22±17 1 Auto 69±0 K- 2 0 NA NA 0 NA NA
Conclusions: Performing RBCX using C, E, K antigen phenotypically matched RBCs prevented development of new alloantibodies in 75% of multitransfused patients. Development of autoantibody or antibody to a low frequency RBC antigen can still occur in any patient receiving RBCX. In this series, all patients, including those who developed RBC antibodies, were able to continue long term RBCX without additional complications. Routine use of C-, E-, K- RBCs is recommended in RBCX to prevent formation of clinically significant antibodies.
Lenalidomide and Rituximab Are a Promising Combination in Vitro, in Vivo Preclinically and in a Phase I/II Clinical Trial in Relapsed/Refractory Mantle Cell Lymphoma
Authors: Wang Michael , Zhang Liang , Fayad Luis , Hagemeister Fredrick , Neelapu Sattva , Samaniego Felipe , Pro Barbara , Younes Anas , Miller Scott , Knight Robert D. , Zeldis Jerome B. , Yi Qing , Romaguera Jorge 
Published: April 2020

Abstract
Relapsed/refractory mantle cell lymphoma (MCL) is difficult to treat. Rituximab (R) targets CD20 antigen on the surface of MCL cells while lenalidomide (Len) may target the microenvironment of MCL cells and enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of R. To test this hypothesis, we initiated preclinical studies and a phase I/II clinical trial. In the preclinical study we found that Len and R induced growth inhibition and apoptosis of both cultured and fresh primary MCL cells. Len enhanced R-induced apoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases (JNK), Bcl-2, Bad; increasing release of cytochrome-c; enhancing activation of caspase-3, -8, -9 and cleavage of PARP. Daily treatment with Len increased NK cells by 10 times in SCID mice. The combination of Len and R decreased tumor burden and prolonged survival of MCL-bearing SCID mice. In the phase I/II clinical trial, Eligible patients (pts) with MCL had 1–4 lines of prior therapies. Treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks only during the first cycle with the first dose on Day 1 in Cycle 1. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. Detailed toxicity profile in phase I was reported previously (Wang et al, ASH 2007). Two DLT s occurred at 25 mg including 1 grade 3 hypercalcemia and 1 grade 4 non-neutropenic fever during the first cycle. Six patients from phase 1 were at 20 mg dosage level. One patient from phase 1 was initially at 25 mg dosage level and was subsequently reduced to 20 mg dosage level due to DLT. Eight patients have been enrolled in the phase II trial at MTD. In the 14 patients evaluated at 20 mg dosage level in phase II, median age was 68 (51–77); median prior therapies were 2 (1–4); median cycles received to date were 4 (range 2–26). Grade 3/4 hematologic toxic events included neutropenia (35), febrile neutropenia (2), and thrombocytopenia (11). There was no grade 3–4 anemia. Grade 3 non-hematologic toxic events included fatigue (2) and myalgia (1). Fourteen pts at MTD (20 mg) including 7 in phase I plus 7 in phase II were evaluable for response. Eight out of 14 pts achieved responses including 4 CRs, 4 PR s, 2 SD and 4 PD s.
Conclusions: Lenalidomide in combination with rituximab provided a synergistically therapeutic effect on mantle cell lymphoma cells by enhancement of apoptosis and R-dependent NK cell-mediated cytotoxicity preclinically. Lenalidomide plus rituximab showed early evidence of response with a very favorable toxicity profile in a phase I/II clinical trial. Updated information will be presented at the conference.
A FVIII C1 Domain Loop Containing Phe2093 Contributes Affinity to the Binding of Recombinant FVIII C1C2 Proteins to Activated Platelets
Authors: Hsu Ting-Chang , Pratt Kathleen P. , Thompson Arthur R. 
Published: April 2020

Abstract
Factor VIII (FVIII) circulates bound to von Willebrand factor, and upon proteolytic activation it dissociates and attaches to activated membranes, e.g. at the platelet surface, that expose negatively-charged phosphatidylserine. This membrane association is mediated entirely by the FVIIIa light chain (A3-C1-C2), and the C2 domain is known to be a primary contributor to the membrane affinity. We previously demonstrated that the C1 domain, which is homologous to C2, also contributes to the affinity for activated platelets, (Hsu et al., Blood111, 200–208, 2008). Our earlier work showed that the platelet affinity of recombinant C1C2, as well as the total number of binding sites per platelet, were significantly higher than those measured for recombinant C2. Thus C1 either interacts with the platelet surface directly, or it stabilizes a conformation of C2 that promotes membrane binding. In this study, the affinities for activated platelets of a series of mutant C1C2 proteins were evaluated to determine residues involved in FVIII binding to platelets. C1C2 and C2 proteins were generated with a free cysteine residue substituted for the wild-type serine at position 2296 in C2 (S2296C does not disrupt the protein structure or affect membrane binding), to which a sulfyhydryl-linked fluorescein probe was attached covalently (C1C2* and C2*). Single or double alanine substitutions were introduced at the two beta-hairpin turns in C2 that mediate membrane binding (M2199A, F2200A, L2251A, L2252A), and at C1 residue F2093, which aligns with C2 residue L2252. Washed platelets were activated with SFLLRN-amide, incubated for 1 hr with the wild-type or mutant proteins, and analyzed by flow cytometry on a FACSCaliber. The relative binding affinities were estimated by using multiple protein concentrations and comparing the fluorescent signals to the values for saturation binding of C1C2* and C2*. Alanine substitutions at all of these positions resulted in decreased binding of C1C2*, comparable to the difference in affinity of C2* versus C1C2* (figure 1A). C1C2-F2093C was then generated, and the introduced sulfhydryl was blocked by adding free cysteine, thus introducing the bulky, charged cystine residue at this putative membrane-binding site (C1C2-F2093C-C). Binding assays utilizing detection by monoclonal antibody ESH8, which does not interfere with membrane attachment, followed by a PE-labeled secondary antibody showed that C1C2-F2093C-C had markedly reduced binding to activated platelets compared to C1C2 (figure 1B). These results are consistent with the hypothesis that the C1 domain contacts the membrane directly when FVIIIa becomes attached to the activated platelet surface and indicate that F2093 contributes significantly to this interaction.
Figure Figure
Antisense Oligonucletide Mediated Depletion of Factor VII Provides Protection from Ferric Chloride Induced Thrombosis without Increased Bleeding Risk in Mice
Authors: Crosby Jeff , Zhao Chenguang , Gao Dacao , Siwkowski Andy , Zhang Hong , Monia Brett P. 
Published: April 2020

Abstract
The Tissue Factor-Factor VIIa complex is the initiating protease in the extrinsic pathway of the coagulation cascade. Knockout of Factor VII in mice results in embryonic lethality. However, only humans with severe Factor VII deficiency exhibit any coagulopathy. In order to determine the potential of Factor VII as an anticoagulant target and the utility of antisense oligonucleotides (ASOs) as Factor VII inhibitors, ASOs were designed to promote RNaseH mediated degradation of Factor VII RNA and evaluated in mice. Factor VII is predominantly produced in the liver, a tissue that is tractable for antisense mediated target depletion. Following three weeks of treatment, the Factor VII ASO was well tolerated and supported significant reduction of Factor VII RNA and protein in mouse liver (ED50 of 3.5 mg/kg for RNA and 2.8 mg/kg for protein). Greater than 95% reduction in Factor VII RNA levels was observed at a dose of 25 mg/kg, administered twice weekly. Following three weeks exposure to 100 mg/kg/wk, prothrombin times (PT) from platelet poor mouse plasma was more than doubled, compared to untreated animals, while activated partial thromboplastin times (aPTT) was only increased 16%. Antithrombotic activity of Factor VII ASO was determined following ferric chloride injury to the inferior vena cava. ED50 for inhibition of thrombosis was 24 mg/kg/wk. The effects of Factor VII depletion on hemostatic control were also determined using a mouse tail nick model. No increase in blood loss was detected, compared to untreated animals, up to a weekly exposure of 80 mg/kg, the highest dose utilized in the study. Compared to warfarin, ASO mediated depletion of Factor VII promoted equivalent protection from thrombosis following ferric chloride injury while maintaining better hemostatic control. These findings support the development of an ASO drug targeting Factor VII as an anticoagulant.
Endothelial and Hematopoietic Tissue Factor Are Unnecessary in Two Mouse Models of Hemostasis and Thrombosis
Authors: David Tovo H , Sulciner David , Cornelissen Ivo , Coughlin Shaun R 
Published: April 2020

Abstract
Endothelial and hematopoietic tissue factor are unnecessary in two mouse models of hemostasis and thrombosis
Tissue Factor (TF), in combination with Factor VII, activates the extrinsic coagulation pathway leading to thrombin generation and clot formation. TF is expressed by epithelial, smooth muscle and perivascular cells; in addition endothelial and hematopoietic cells can express TF, especially in response to inflammatory stimuli. Despite extensive studies the exact cellular source of TF during clot initiation and propagation has not yet been clearly identified, and the importance of endothelial or hematopoietic TF in hemostasis and thrombosis is unknown. To address the latter, we measured bleeding time and arterial thrombus formation in TF flox/flox: Tie-2p/e Cre conditional mice, in which endothelial and circulating TF expression is absent. We observed that TF deficiency in the endothelial and hematopoietic compartment was not associated with defective hemostasis. In a FeCl3- induced carotid injury model, in contrast to PAR4 null mice, clotting in TF conditional mice did not significantly differ from that of wild type littermates. This study suggests that TF derived from endothelial or blood cells is not necessary for hemostasis and thrombosis in the models studied.
Figure Figure
Procoagulant Activity and Phosphatidylserine of Amniotic Fluid Cells
Authors: Zhou Jin , Liu Shuchuan , Ma Ming , Hou Jinxiao , Yu Hongjuan , Lu Chengfang , Gilbert Gary , Shi Jialan 
Published: April 2020

Abstract
Amniotic fluid (AF) may induce disseminated intravascular coagulation when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, we speculate that AF cells have procoagulant activity due to the externalization of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to tissue factor (TF), the PS that is externalized on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analyzed and normal platelets, neutrophils, and lymphocytes were harvested as controls. Lactadherin, a glycoprotein, binds to membranes containing PS, inhibits prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding. Thus lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of AF cells was first measured in plasma with AF cells serving as thromboplastin. Activity of AF cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Lactadherin, as an agent to block exposed PS, inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for 55% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by mAb that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches and a rim-pattern indicating a diffuse PS exposure. Lactadherin binding sites and TF distributed to discreet, but overlapping regions of the cells. These results indicate that PS exposure parallels procoagulant activity on AF cells and is required for at least 85% of intrinsic and extrinsic factor Xase activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.
Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD
Authors: Wang Miao , Kluin P.M. , Rosati Stefano , Luinge Marjan , Daenen Simon M. G. J. , Visser Lydia 
Published: April 2020

Abstract
Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival.
Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry.
Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases.
Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.
Deregulation of MicroRNA Underlies Severe Congenital Neutropenia Pathogenesis
Authors: Velu ChinavenMeni S. , Baktula Avinash M , Bourdeau Tristan , Grimes H. Leighton 
Published: April 2020

Abstract
The zinc finger protein Growth factor independent-1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cell (HSC) maintenance and granulocytic lineage differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 (encoding GFI1N382S) which generate dominant negative acting proteins. GFI1N382S proteins sequester limiting cofactors to deregulate a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs and suggest that that transcriptional control of microRNA genes is critical for GFI1N382S-associated SCN phenotypes. First, the expression of Gfi1 and miR21 and miR-196 is reciprocal: 1) in wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), 3) during treatment-induced differentiation of human myeloid leukemia cell lines, and 4) upon conditional deletion of Gfi1 in primary sorted murine CMP and GMP. Biochemical analyses reveal that miR21 and miR-196 are direct transcriptional targets of Gfi1. Subsequently, forced expression of wild type Gfi1 rescues expression of microRNA in Gfi1−/− Lin- bone marrow cells, while forced expression of Gfi1N382S in wild type Lin- bone marrow cells significantly deregulates miR-21 and miR-196 expression. Similarly, we demonstrate elevated miR21 and miR196b levels in CD34+ cells from a GFI1N382S SCN patient. Flow cytometric analysis and colony assays reveal that the overexpression or knockdown of either miR induces changes in myeloid development, but that co-expression of both miR (as seen in Gfi1−/− mice and GFI1N382S SCN patients) completely blocks G-CSF-induced granulopoiesis. These data provide a molecular understanding of SCN disease pathogenesis.
Chronic Lymphocytic Leukemia Cells Promote Increased BAFF Expression in Monocytes Partially through TNF-Alpha Leading to Increased CLL Cell Survival
Authors: Messmer Davorka , Chang Hsu-Hsiang , Kipps Thomas J. 
Published: April 2020

Abstract
The tumor microenvironment plays a critical role in growth, metastasis, and survival of tumors. Chronic lymphocytic leukemia (CLL) B cells of many patients can rapidly undergo apoptosis in vitro unless co-cultured with accessory cells, which elaborate factors that promote CLL-cell survival. A major type of such accessory cells is nurse-like cells (NLC), which prior studies demonstrated could differentiate from CD14+ blood mononuclear cells when exposed to CLL B cells in vitro, and presumably in vivo. In the absence of CLL cells, blood mononuclear CD14+ cells typically differentiate into macrophages that have a distinctive morphology from that of NLC. We found that, compared with macrophages, NLCs consistently expressed significantly higher levels of B-cell activating factor (BAFF) (n=11), a tumor-necrosis-factor (TNF)-related ligand that promotes survival of B cells, including CLL B cells. To investigate the influence of CLL cells on the differentiation of NLC in vitro, purified CLL B cells or B cells of healthy donors were cultured for 24h to generate conditioned media. When CD14+ blood mononuclear cells of healthy donors were cultured for 7 days in media conditioned by CLL cells, but not in non-conditioned media or in media conditioned by normal B cells, the cells assumed the morphology of NLC, acquired high-level expression of BAFF, and developed an enhanced capacity to support CLL cell survival in vitro. Similar activity was also found in the sera of CLL patients, but not in the sera of healthy control donors. The capacity of either CLL-conditioned media (n=4) or CLL-patient sera (n=9) to induce NLC differentiation leading to increased expression of BAFF was significantly reduced by a decoy receptor for TNF-a, suggesting that TNF-a contributes to the differentiation of NLC in vitro. However, TNF-a alone was not sufficient to induce high-level expression of BAFF on blood mononuclear CD14+ cells, suggesting that additional factors are involved. The results show a previously unrecognized symbiosis between CLL cells and accessory cells in their microenvironment. Because the survival of neoplastic B cells in vivo potentially depends upon NLC found in the lymphoid tissues of patients with CLL, identification of the factor(s) involved in inducing their differentiation could provide a novel target for therapy of patients with this disease.