bluuebunny/crossref_metadata_2025_split huggingface dataset:
Formation of Platelet Strings and Microthrombi in the Presence of ADAMTS13 Inhibitor Does Not Require P-Selectin or β3 Integrin.
Authors: Chauhan Anil K. , Wagner Denisa D. 
Published: October 2019

Abstract
VWF is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies respectively. VWF released from the storage granules upon cellular activation or stimulation with secretagogues are extremely large and known as Ultra-Large von Willebrand factor (UL-VWF). Such filaments were first shown by Dong and colleagues (Dong et. al., 2002) and platelets were seen to align as string of beads on these UL-VWF multimers. These platelet strings are then rapidly cleaved to smaller size by the metalloprotease ADAMTS-13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13). Recently, we have similarly shown that in Adamts13−/− mice platelet strings form and remain on stimulated endothelium in veins, where they wave in the blood stream for many minutes (Motto et. al., 2005). We have also shown that antibody to ADAMTS13 induces platelet strings in activated venules and transient thrombi formation in activated microvenules of wild type similar to Adamts13−/− mice (Chauhan et. al., 2006). The obvious question is what anchors the UL-VWF mulimers to the endothelial surface? In a flow chamber model, using polyclonal antibodies to P-selectin or soluble P-selectin, it was proposed that P-selectin anchors the UL-VWF multimers to the endothelial surface. The present study was designed to address the role of P-selectin and that of the platelet and endothelial integrin β3 in platelet string formation in vivo using intravital microscopy and genetically engineered mice. We demonstrate that in histamine activated venules, causing Weibel-Palade bodies secretion, P-selectin is not required for formation or retention of VWF platelet strings on endothelium in vivo. The mean time the platelet strings (~30–100 μm long) anchored onto the endothelium was 45 s. This duration was significantly longer than in P-selectin−/− mice infused with control Ig (P&lt;0.001) and similar to WT mice treated with the inhibitory antibody. Similar to wild type, 45 s after topical superfusion of calcium ionophore A23187, platelet strings forming transient thrombi formation was seen in microvenules (25–30 μm in diameter) of P-selectin−/− mice when the ADAMTS13 activity was inhibited. Platelet strings were either not seen or were very short lived in the β3−/− mice infused with control Ig, whereas in the mice infused with anti- ADAMTS13 antibody platelet strings could be seen that anchored to the endothelium with a mean time of 50 s (P&lt;0.0001). These observations show that platelet strings are not held by β3 integrin and platelets lacking β3 integrin can adhere firmly to the VWF strings in the histamine activated venules. VWF containing platelet strings could also entangle and form emboli in the absence of β3 integrin.
CD34+AC133+VEGFR-2+ Cells Are Primitive Hematopoietic Progenitors, Not Functional Endothelial Progenitor Cells.
Authors: Case Jamie , Mead Laura E. , White Hilary A. , Saadatzadeh Mohammad R. , Yoder Mervin C. , Haneline Laura S. , Ingram David A. 
Published: October 2019

Abstract
Endothelial progenitor cells (EPCs) are currently used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression. However, there is no uniform definition of an EPC, which complicates interpretation of prior EPC studies. EPCs are primarily defined by expression of cell surface antigens. The most widely cited definition of an EPC is a cell which co-expresses CD34, AC133 and VEGFR-2. Importantly, these antigens are also expressed on the most primitive population of hematopoietic progenitor cells (HPCs), including high proliferative potential- (HPP-) and low proliferative potential-colony forming cells (LPP-CFCs). Remarkably, CD34+AC133+VEGFR-2+ cells have never been isolated and plated in endothelial cell (EC) or hematopoietic cell clonogenic assays to determine what cell progeny can be derived from a CD34+AC133+VEGFR-2+ cell. Utilizing human umbilical cord blood (CB), an enriched source of both EPCs and HPCs, we isolated and purified CD34+AC133+VEGFR-2+ cells by FACS and assayed for the presence of clonogenic endothelial CFCs (ECFCs) plus HPP- and LPP-CFCs. Surprisingly, CD34+AC133+VEGFR-2+ cells do not form ECFCs under any culture conditions previously described for outgrowth of EPCs. However, consistent with a HPC phenotype, CD34+AC133+VEGFR-2+ cells formed both HPP- and LPP-CFCs in multiple independent assays. In addition, all CD34+AC133+VEGFR-2+ cells were shown to co-express the specific hematopoietic cell surface antigen, CD45, which is not present on ECs. Based on this information, we plated CD34+CD45+ or CD34+CD45− cells to determine if EPCs could be separated from HPCs on the basis of CD45 expression. In multiple independent assays, CD34+CD45+ cells consistently formed both HPP- and LPP-CFCs but not EC colonies. In contrast, CD34+CD45− cells form EC colonies but not hematopoietic cell colonies. Taken together, these data demonstrate that circulating CD34+AC133+VEGFR-2+ cells are HPCs and the biologic mechanism for their correlation with cardiovascular disease needs to be re-examined. Furthermore, studies focused on determining the angiogenic potential of CD34+CD45− cells are needed given that this cell population harbors ECFCs.
Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.
Authors: Tojo Arinobu , Izawa Kiyoko , Sekine Rieko , Nagamura-Inoue Tokiko , Kobayashi Seiichiro 
Published: October 2019

Abstract
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL.
Figure Figure
The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells.
Authors: Khan Naveed I. , Bradstock Kenneth Francis , Bendall Linda J. 
Published: October 2019

Abstract
Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.
BIOV-111 a European Phase II Trial of Clofarabine (Evoltra®) in Refractory and Relapsed Childhood Acute Lymphoblastic Leukemia.
Authors: Kearns Pamela , Michel Gerard , Nelken Brigitte , Joel Simon , Al-Ghazaly Essam , Beishuizen Auke , Conter Valentino , Gadner Helmut , Henze Gunter , Smith Hazel , Baruchel Andre , Saha Vaskar 
Published: October 2019

Abstract
BIOV-111 is a European phase II non-randomised, open-label study of a next generation purine nucleoside analogue, clofarabine (Evoltra®), in pediatric patients with relapsed/refractory ALL. We report on the efficacy and safety data. Eligible patients (pts) had either primary refractory (2 pts) or relapsed/refractory (51 pts with ≥ 2 prior lines of treatment) ALL. Clofarabine 52mg/m2 daily × 5 days was given every 28–42 days (1 course). The primary endpoint is overall response rate (ORR) defined as either a complete response (CR) or CR without platelet recovery (CRp) after 2 courses. Adverse events (AEs) were graded according to NCI CTC (v3). Plasma, urine and intracellular clofarabine pharmacokinetics were also investigated. To date, 96 courses have been administered to 53 pts from 25 centres. The median number of prior treatments was 2 (range 1–5) and 20 pts (38%) had been previously transplanted. 8/29 pts receiving ≥2 courses responded (1CR, 7CRp) giving an ORR of 28%. Responses were observed in 14/53 (26%) pts receiving at least one course of clofarabine (6CR, 7CRp, 1 PR ). Eight (57%) responders had a prior transplant and 1 of these patients was transplanted post clofarabine. One pt with a prior transplant remains in remission at 20+ months. Four pts (1CR, 3CRp) have proceeded to transplantation. Serious adverse events (n=103) included febrile neutropenia (51/103), seizures (4/103), streptococcal septicaemia (3/103), palmo-plantar erythrodysaesthesia (2/103) and bone pain (2/103). Three hepatic events occurred (raised bilirubin, raised ALT/AST), 1 renal failure and 1 cardiac failure. AEs occurred in 4 pts. The renal and cardiac failure AE occurred in a pt with known anthracycline cardiac myopathy and renal impairment at study entry. Median end of infusion plasma clofarabine concentration (n=25) on days 1 and 2 were 1.5 (0.5–3.2) uM vs 2.0 (0.1–5.1) uM respectively (p=0.01) and were not different in pts achieving a response. Urinary clofarabine recovery on days 1, 5 and 6 (drug -free) was 48±18 %, 46±12 % and 5±3 %. Clofarabine TP analyses are ongoing. Response rates in this ongoing BIOV-111 study are consistent with the pivotal clofarabine study (CLO-212) in relapsed/refractory pediatric ALL. Notably, BIOV-111 has a lower incidence of AEs, including hepatic and renal AEs; (4% and 1% respectively vs 10% and 8% in CLO-212), possibly attributable to fewer prior treatments compared to CLO-212 (median 2 vs 3 respectively). Clofarabine achieves a significant response rate in this heavily pre-treated patient population and durable responses have been observed which may confer a survival advantage with longer follow-up.
Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.
Authors: Collins Natalie B. , Tomashevski Andrei , Kupfer Gary M. 
Published: October 2019

Abstract
Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.
Assessment of Anti-Asparaginase (ASNase) Antibodies (Ab) and ASNase Activity after Suspected Clinical Allergy.
Authors: Tiwari Prakash N. , Zielinski Monica , Quinn John J. , Siegel Stuart E. , Gaynon Paul S. , Wakamatsu Peter , Seibel Nita L. , Avramis Vassilios I. 
Published: October 2019

Abstract
Background: ASNase is a critical treatment component in childhood acute lymphoblastic leukemia (ALL). As ASNase is a bacterial protein, production of anti-ASNase Ab, often associated with clinical allergy, poses a frequent problem during ASNase therapy. Sixty percent of higher risk ALL patients, who received native ASNase in induction, were Ab(+) by 5 months into therapy (Panosyan et al., 2004). Moreover, 22%–29% of these patients had no clinical allergy (silent hypersensitivity) and were at increased risk for relapse.
Patients and Methods: Physicians from 13 institutions submitted samples for 48 children in first remission, aged 14 to 252 months, on various SR or HR ALL protocols with suspected ASNase allergy. The male/female ratio was 1.4. The serum samples were assessed for ASNase activity and anti-ASNase Ab as reported earlier (Avramis, et al., Blood 2002). Ab titer is expressed as the numeric ratio relative to an Ab negative control.
Results: Allergic symptoms ranged from localized skin rash and swelling to respiratory difficulties and/or anaphylaxis. Neutralizing anti-PEG-ASNase Ab’s were seen in 41 patients (85%). The results were examined with respect to age, gender, ASNase formulation received, time of post-ASNase administration, and the physician’s clinical observations. Higher Ab ratios > 1.1 correlated with low or no ASNase activity post-PEG-ASNase (46/48 patients) or post-Native ASNase dosing (2 patients). Fourteen patients had cutaneous manifestations (rash, hives, urticaria). All had neutralizing Ab and no enzymatic activity. Nine had higher Ab ratios and 5 had lower Ab ratios. Eight additional patients had localized or generalized swelling; all had higher Ab ratios. Similarly, over the entire CCG-1961 study, 526/1100 patients had a clinical reaction; 476/526 had higher Ab ratios (90%).
Conclusions: We found neutralizing Ab in 85% of patients with apparent clinical ASNase allergy. Neutralizing Ab was correlated with lower or absent ASNase activity. Lower Ab ratios may be associated with earlier time points in the anamnestic response. In the remaining 15% of patients with an apparent clinical reaction, we found no Ab and substantial ASNase activity. Monitoring ASNase activity and Ab may be useful for guiding ASNase therapy. Patients with no Ab and substantial activity, might be rechallenged with the same product despite an apparent allergic reaction.
Type II HMG-CoA Reductase Inhibitors (Statins) Provide Acute-Graft-Versus-Host Disease Protection by Th-2 Cytokine Induction While Sparing Graft-Versus-Leukemia Activity.
Authors: Zeiser Robert , Youssef Sawsan , Baker Jeanette , Steinman Lawrence , Negrin Robert 
Published: October 2019

Abstract
Recent studies have shown the beneficial impact of 3-hydroxy-3-methyl-CoA (HMG-CoA) reductase inhibitors (statins) on autoimmunity and allograft rejection. To investigate whether statins are capable of protecting from acute graft vs host disease (aGVHD) we utilized an established murine model (FVB/N->Balb/c) across major MHC barriers. Type II statins were potent inducers of a T-helper cell (Th)-2 cytokine profile in the adoptively transferred T cells upon exposure in vitro or in vivo. Expansion of alloreactive luciferase transgenic T cells as measured by total body light emission was significantly reduced by donor pre-treatment (10mg/kg bodyweight) or in vitro T cell treatment (10nM) with atorvastatin (AT vs PBS; p=0.0008) and fluvastatin (FLU vs PBS; p=0.0007). The beneficial effect of statin treatment translated into significantly reduced aGvHD lethality in statin as compared to PBS treated animals. Th-2 biased donor T cells could be tracked until day 15 after BMT by intracellular cytokine staining and FACS analysis. Host treatment prior to transplantation induced down-regulation of the costimulatory molecules CD40, CD80 and CD86 and MHC class II on recipient APCs and enhanced the protective statin effect when donor and recipient were treated (survival AT vs PBS, p=0.0012). Induction of the Th-2 cytokine profile was still permissive for in vivo graft vs leukemia effects by cytolytic effector function of CD8+ T cells against A20 B-cell lymphoma cells as assessed by bioluminescence imaging, FACS, histology and survival. In vitro CD8+ T cells derived from AT treated animals displayed equal cytolytic activity against Yac1 T-cell lymphoma cells as compared to T cells derived from PBS treated animals. Mechanistically we identified a role for the STAT-6 pathway in statin induced Th-2 induction as aGvHD protection was partially reversed with STAT-6−/− donors. The in vivo statin effect could be antagonized by L-mevalonate indicating the relevance of the mevalonate pathway for statin mediated aGvHD protection. Since L-mevalonate produced by the HMG-CoA reductase is the precursor of non steroidal isoprenoid compounds, such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, the statin impact on prenylation status of critical signaling proteins in alloreactive T cells was determined by Western blot. In vitro AT treatment reduced the levels of RAP-1, RhoB and Ras prenylation in allo-antigen exposed T cells and abrogated expression of T-bet which is critical for Th1 induction.
We conclude that type II statins have significant protective impact on aGvHD lethality by Th-2 cytokine induction and inhibition of an uncontrolled Th-1 response, which is of great clinical relevance given the widespread use and well defined toxicity profile of these agents.
Overexpression of CXCR4 in Acute Myeloid Leukemia Predicts Adverse Overall and Progression-Free Survival Independently of FLT3 Gene Mutation.
Authors: Konoplev Sergej , Rassidakis George Z. , Estey Elihu H. , Kantarjian Hagop M. , Huang Xuelin , Xiao Lianchun , Andreeff Michael , Konopleva Marina , Medeiros L. Jeffrey 
Published: October 2019

Abstract
CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. We analyzed the prognostic significance of CXCR4 expression in 122 AML patients with diploid karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy specimens obtained at diagnosis from untreated patients were immunostained with an antibody specific for CXCR4 (Abcam, clone 2074; Cambridge, MA). Statistical analysis included the following variables: CXCR4 expression, age, sex, race, incorporation of cytarabine in the therapeutic regimen, presence of multilineage dysplasia, history of antecedent hematologic disorder, history of prior chemotherapy (for either hematological or non-hematological disorder), performance status, leukocytosis, hemoglobin, platelet count, bilirubin, and creatinine. There were 70 men and 52 women with a median age of 62 years (range, 22–82 years). Median follow-up was 18 months (range, <1–97 months). Seventy-six patients achieved complete remission (CR); 39 relapsed. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (p=0.09). In the univariate Cox model, CXCR4 expression was associated with shorter overall survival (OS), p=0.008, and disease-free survival (DFS), p=0.012, figure1.
Figure Figure
Shorter OS was also associated with failure to achieve CR (p=<0.0001), age (p=0.01), presence of multilineage dysplasia (p=0.006), poorer performance status (p=0.049), higher creatinine level (p=0.00002), and lower hemoglobin level (p=0.042). Shorter DFS was associated with failure to achieve CR (p=0.0002), presence of multilineage dysplasia (p=0.02), history of prior chemotherapy (p=0.04), and higher creatinine level (p=0.0009).
In multivariate analysis, shorter OS was associated with CXCR4 expression (p=0.047), age (p=0.029), presence of multilineage dysplasia (p=0.042), and high creatinine level (p=0.016). Shorter DFS was associated with CXCR4 expression (p=0.036), history of prior chemotherapy (p=0.044), presence of multilineage dysplasia (p=0.007), and high creatinine level (p=0.034). These results suggest that CXCR4 expression is associated with poor prognosis in AML patients and is independent of FLT3 gene mutation.