bluuebunny/crossref_metadata_2025 huggingface dataset:
Acyl-CoA Turnover in RBC.
Authors: Soupene Eric , Siliakus Melvin , Kuypers Frans A.
Published: October 2019

The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). While formation and utilization of acyl-CoA takes place at the membrane, Acyl-CoA Binding Domain containing proteins (ACBD) in RBC cytosol bind acyl-CoA, limiting product feedback inhibition on ACSL and distribute Acyl-CoA to the various acyl-utilizing enzymes while protecting the cells against its potent detergent character.

We have identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC, a protein with several isoforms that acts as a dimer. To relate its structure to activity, we report the expression of different modified forms in E. coli. Our data indicate that, despite the observed activity, enzyme studies of these mammalian membrane proteins in the host E. coli are strongly hampered by their aggregation into inclusion bodies. While activity can be measured, data on enzyme kinetics and specificity are questionable. Oleoyl-CoA formation from oleic acid, CoA and ATP reveled that the two transmembrane spanning segments predicted at the amino-terminus of the protein are not essential for its activity. Moreover, they are not essential for dimer formation and strong association with membranes. ACSL6 appears to be an integral membrane protein. One of the five spliced isoforms of ACSL6 reported, lacks the so-called fatty acid Gate-domain, and appears to be unable to activate long chain fatty acids. We hypothesize that this form modulates activity of the other active isoforms of ACSL6 through hetero-dimer formation. An EST clone of erythroid precursor cells identified ACBD6 as a potential AcylCoA binding protein in RBC. This modular protein contains an Acyl-CoA binding domain at the amino-terminus and two Ankyrin-repeat motifs (ANK) at the C-terminus. Both the full-length protein and the N-terminus domain were soluble when expressed and purified in E. coli. Expression of the C-terminus domain by itself rendered an insoluble protein. We report that ACBD6 binds long-chain acyl-CoA with a preference for C18:1-CoA over C20:4 and C16:0-CoA and does not bind fatty acid. Truncation of the ANK domain had no effect on the binding activity of the N-terminus domain. Together our findings implicate ACBD6 as part of the Acyl-CoA turnover mechanism in RBC, its actual role on the kinetics of ACSL and/or LPLAT activity needs to be established. The description of these proteins involved in Acyl-CoA turnover in RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.
A Facilitated Tracking and Transcription Mechanism of the HS2 Enhancer in the β-Globin Locus Control Region.
Authors: Tuan Dorothy , Zhu Xingguo , Ling Jianhua , Zhang Ling , Pi Wenhu , Wu Min
Published: October 2019

The 100 kb human β-globin gene locus contains the embryonic ε-, the fetal Gγ- and Aγ-, and the adult δ- and β-globin genes arranged in the transcriptional order of 5′ ε-Gγ-Aγ-δ-β 3′. The β-globin Locus Control Region (LCR), defined by DNase I hypersensitive sites HS1, 2, 3, 4 and 5 located far upstream of the globin genes, is absolutely required for transcriptional activation of the globin genes in erythroid cells. The HS2 site, located respectively 11 and 55 kb upstream of the ε- and β-globin genes, possesses prominent enhancer activity and is able to activate transcription of the globin genes over the long distance. How the HS2 enhancer acts over the long distance is not fully understood. Three mechanisms have been proposed to explain long-range enhancer function: The looping model, the protein tracking/linking model and the facilitated tracking model. We have shown earlier that a transcription mechanism of the RNA polymerase II (pol II)-complex assembled by the HS2 enhancer mediates long-range HS2 enhancer function. In the present study, we used the chicken HS4 insulator to further study the mechanism of long-range HS2 enhancer function. We created the LCR(+I) and LCR(−I) lines in human erythroid K562 cells using Cre-loxP mediated in situ recombination and in zebrafish. The integrated LCR(+I) and LCR(−I) plasmids spanned the natural 11 kb human ε-globin gene locus either with or without the chicken HS4 insulator inserted between the LCR HS2 enhancer and the ε-globin promoter. Analysis of enhancer function in the integrated plasmids and the endogenous ε-globin gene locus provided evidence for a novel facilitated tracking and transcription mechanism of long-range enhancer function: The HS2 enhancer complex containing both the enhancer DNA and the associated pol II tracked and transcribed through the 10 kb intervening DNA to loop with and activate the ε-globin promoter. The interposed insulator interrupted this facilitated tracking and transcription mechanism of the enhancer complex through the intervening DNA, thereby blocking long-range enhancer function.
The Natural History of the Neutrophil Elastase and Coagulation Factor Response to E.coli in a Baboon Model of E.coli/Sepsis Suggests That Elevated Neutrophil Elastase Activity Is Sustained Regardless of Outcome in Clinical Sepsis.
Authors: Samis John A. , Stewart Kenneth A. , Taylor Fletcher B. , Fournier Daniel J. , Nesheim Michael E.
Published: October 2019

Neutrophil elastase (NE) functions in the destruction of gram negative bacteria. NE consumption/inactivation of coagulation factors may contribute to the coagulopathies that accompany infections upon insuffcient regulation by its primary plasma inhibitor, alpha-1-protease inhibitor (A1PI). The temporal relationships between NE levels, A1PI activity and antigen, and the activity of Factor (F) II, V, VII, VIII, IX, X, and fibrinogen were studied in three baboons after infusion of 2.40 x 106 cfu/kg (low/sublethal), 1.05 x 108 cfu/kg (medium/sublethal), and 6.49 x 108 cfu/kg (lethal) of E.coli. Immunoblotting for A1PI under native conditions indicated that the A1PI/NE complex levels increased by 5- to 93-fold after 24hrs and remained 3- to 580-fold above baseline at 72hrs and 53hrs following sublethal and lethal E.coli challenge, respectively. The A1PI specific activity (activity/antigen) decreased by 55% and 30% after 48–72hrs and 48–53hrs following the medium and lethal dose, respectively. Increased amounts of 2 forms of A1PI resolved by native PAGE/immunoblotting correlated with decreased inhibitor specific activity. At the lethal dose of E.coli, decreases (%) in fibrinogen (96%), FV (60%), and FVIII (60%) occurred within 2–6hrs. By 53hrs, FV and FVIII levels returned to normal and fibrinogen was twice normal. Similar changes of smaller magnitudes occurred at the lower doses of E.coli. At the lethal dose of E.coli, decreases in FII (50%), FVII (80%), FIX (80%), and FX (60%) occurred over the first 24hrs and these levels were sustained through 53hrs. Similar changes of smaller magnitudes occurred at the lower doses. In conclusion, there is a temporal association between coagulation factor consumption/inactivation, appearance of the A1PI/NE complex, and A1PI inactivation during E.coli/sepsis in baboons. The marked elevation of the A1PI/NE complex beyond 24hrs, even after sublethal challenge, indicates for the first time that increased neutrophil and NE activities persist beyond the initial inflammatory and coagulopathic stages into the subsequent cell injury and degeneration stages that terminate in either recovery or death from E.coli/sepsis.
Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.
Authors: Patel Sanjay , Berry Leslie R. , Smith Lesley , Chan Anthony
Published: October 2019

The rate of prothrombin to thrombin conversion by factor Xa (Xa) is enhanced when Xa is incorporated into the surface-bound prothrombinase complex. However, in comparison to the free state, Xa within the prothrombinase complex is afforded protection from antithrombin + heparin (AT+H) inactivation. We have shown that, unlike AT+H, a covalent conjugate of AT and H (ATH) can neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa +/− prothrombinase complex. Mixtures of CaCl2, phospholipid vesicles, factor Va (Va) and prothrombin in TSP buffer, were combined with Xa. Following addition of either AT+H or ATH, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to corresponding experiments with Xa alone. AT+H inactivation of Xa in prothrombinase occurred at a k2 (x 107 M−1min−1) of 2.34 +/− 0.09. In contrast, neutralization of free Xa by AT+H was significantly faster (k2 = 8.34 +/− 0.18, p = 0.03). Reaction with ATH showed no significant rate difference for Xa inhibition in either the complexed or free states (18.5 +/− 3.3 and 16.3 +/− 3.7, respectively). Intriguingly, the rates achieved for ATH inhibition of complexed and free Xa were significantly greater than that for AT+H with free Xa (p=0.03 and p=0.02, respectively). We conclude that covalent complexes of AT and H do not encounter resistance in the inhibition of Xa in prothrombinase, as seen for non-covalent AT+H mixtures. Thus, it is possible for ATH to effectively inhibit the propagation phase of thrombin generation and thus dampen thrombin production via neutralization of Xa in prothrombinase.
Inhibition of Heparinase I by Defibrotide with Potential Clinical Implications.
Authors: Fareed Jawed , Sonevytsky Adrian , Iqbal Omer , Jeske Walter P. , Iacobelli Massimo , Hoppensteadt Debra
Published: October 2019

Defibrotide represents a poly-deoxyribonucleotide derived antischemic and antithrombotic agent. Currently this agent is used for the management of transplantation associated with vascular complications. Defibrotide is a polyanionic electrolyte capable of releasing endogenous antithrombotic mediators such as (tissue factor pathway inhibitor (TFPI) and heparans. Co-administration of defibrotide with heparin has been shown to produce an augmentation of the effect of heparin and an increase in the biologic half-life. This may be due to the drug interactions or inhibition of endogenous heparin digesting enzyme by defibrotide. To test the hypothesis that defibrotide may inhibit heparin digesting enzymes such as heparinases (I–III), the effects of defibrotide were investigated on the digestion of unfractionated heparin. Unfractionated heparin was subjected to bacterial heparinase I (flavobacterion heparinicum) in an isolated biochemical systems where unfractionated heparin was used as a substrate at a fixed concentration of 750 μg/ml. Graded amounts of defibrotide in the range of 6.25–250 μg/ml were supplemented to this mixture. The degree of heparinase digestion of unfractionated heparin was determined by using high performance liquid chromatographic methods and computation of oligosaccharide profiles. Heparinase I was found to digest heparin (MW=15.2kDa) converting it to low molecular mass heparins (MW=4.1kDa). At concentrations of >125 μg/ml defibrotide produced an almost complete inhibition of heparinase I digestion. This inhibition of heparinase digestion was dependent on defibrotide concentration and the IC50 was found to be 12.5 μg/ml. These results suggest that polyelectrolytes such as defibrotide are capable of inhibiting heparin/heparan digestion enzymes. The observed potentiation of anticoagulant effect of heparin in patients treated simultaneously with defibrotide may partially be due to the inhibition of endogenous heparin digesting enzymes. Subsequent studies in primates have revealed that the anticoagulant effects of both unfractionated heparin and low-molecular mass heparins are potentiated by defibrotide as determined by AUC measurements for the circulating level of these agents. Thus, these interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.
New Role for ATP P2X1 Receptors in the Control of Neutrophil Apoptosis and Respiratory Burst Activity.
Authors: Faccinetto Celine , Lecut Christelle , Greimers Roland , Bours Vincent , Oury Cecile
Published: October 2019

P2X receptors are membrane non-selective cation channels that open in response to the binding of extracellular ATP. Seven genes encode P2X receptor subunits (P2X1–7) in vertebrates. Channels form homo- or hetero-multimers of several subunits that are highly permeable to Ca2+. P2X receptors are widely distributed. In recent years, increasing attention has been paid to extracellular ATP as a candidate danger signal locally released at the inception of inflammation. One of the most striking features of ATP is its ability to promote P2X7 receptor-mediated massive release of mature IL-1β from LPS-primed mononuclear phagocytes. On neutrophils, ATP rises intracellular Ca2+ concentrations, contributes to degranulation, adhesion, oxidative burst and delays apoptosis, events that may partly depend on the G-protein coupled P2Y2 receptor and on P2X7. In the present work, RT-PCR, Western blotting and immunofluorescence experiments reveal that neutrophils also express P2X1 and P2X5 receptor subtypes. A microarray analysis indicates that a 3-hour treatment of human peripheral blood neutrophils with the selective P2X1 and P2X1/5 receptor agonist, a,β-meATP, changes the expression of genes mainly involved in the control of cell fate. Accordingly, this agonist causes an increase of phosphatidylserine exposure on neutrophil membranes, maximally occurring after 3 hours and lasting until 18 hours of culture. In the presence of the protein synthesis inhibitor cycloheximide, a,β -meATP promotes caspase-3-dependent neutrophil apoptosis after 3 hours, which is correlated with highly reduced Fcγ RIII (CD16) membrane expression. In addition, a 1 min pretreatment of neutrophils with a,β -meATP potently increases tumor necrosis factor-a (TNF-α )-driven priming (30 min) of the respiratory burst induced by the bacterially derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Furthermore, a,β -meATP produces L-selectin (CD62L) shedding by its own and in an additive manner with TNF-α . This agonist also induces rapid and reversible phosphorylation of the survival kinases ERK (starting after 2 min) and Akt (15 min) as well as phosphorylation and degradation (after 2 min) of I-κ Bα , an inhibitor of the anti-apoptotic transcription factor NF-κ B. Hence, neutrophil P2X1 receptors might have a dual role in inflammation; they would both contribute to neutrophil activation and promote cell death of neutrophils that have reached the end of their useful life span. Activation of P2X1 receptors by extracellular ATP may thus represent novel regulatory mechanisms that govern neutrophil function.
Identification and Characterization of Zebrafish Mast Cells.
Authors: Berman Jason N. , Seibert Jake , Crosby Amanda , Fraser Robert , Gritsenko Olga , Pohajdak Bill , Lin Tong-jun
Published: October 2019

Mast cells (MCs) are well-known for their role in allergic reactions and inflammation, but their developmental origin is controversial. In addition, abnormal clonal proliferation of MCs, referred to as systemic mastocytosis (SM), may be associated with acute myeloid leukemia, portending a poor outcome. Mutations in the C-KIT tyrosine kinase have been identified in SM, that can be potentially targeted by small molecule tyrosine kinase inhibitors. The zebrafish is a robust model organism for studying hematopoiesis and leukemogenesis, and has an inherent capacity to accommodate genetic and chemical modifier screens. Thus, this system holds potential for elucidating MC lineage and for use in high-throughput screening of targeted therapeutics in MC diseases. MCs have not been previously described in zebrafish. We have identified putative MCs in adult zebrafish gill and intestine containing eosinophilic granules that stain with peroxidase acid shift (PAS) and toluidine blue, as well as with carboxypeptidase A5 (cpa5), a zebrafish homologue of the mammalian MC specific enzyme, CPA1. Electron microscopic analysis demonstrates a striking morphologic resemblance to mammalian MCs with abundant homogeneous dense granules. Classical functional studies reveal degranulation of these cells and increased histamine production upon stimulation with compound 48/80 and stimulated IgE. Whole mount in situ hybridization experiments on zebrafish embryos demonstrate cpa5 expression in a population of blood cells at 28 hours post-fertilization co-localizing with the early myeloid marker, pu.1, the granulocytic marker, mpo, and monocytic markers, l-plastin and lysozyme C. These data point to the existence of a zebrafish MC equivalent and suggests that this lineage arises at the level of the granulocyte/monocyte progenitor. Ongoing characterization of these cells will provide further insight into the mechanisms underlying MC development. Furthermore, a zebrafish cpa5 promoter element has been cloned and is being used to generate transgenic zebrafish lines. These transgenic zebrafish will provide a valuable tool in identifying new effective therapeutic agents targeting MCs in allergic and immune responses, as well as in their contribution to leukemic progression.
Novel Insights into the Maturation and Enucleation of Primitive Erythroblasts.
Authors: Baron Margaret H. , Fraser Stuart T. , Isern Joan
Published: October 2019

Primitive erythroblasts (EryP), the first blood cell type to form during mammalian embryogenesis, arise in the yolk sac (YS) as a synchronous cohort before the establishment of the embryonic circulation. The large, nucleated EryP begin to circulate around midgestation, when connections between YS and embryo mature. Two to three days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryP within the circulation. Cells of the two erythroid lineages differ not only in size but also in their expression of globin genes. Recently it has been shown that EryP begin to enucleate around E12.5 in the mouse. We have begun to characterize EryP maturation during mouse embryogenesis, with a focus on circulating cells. Analysis of embryonic blood at different stages revealed a stepwise developmental progression within the primitive erythroid lineage: loss of nucleoli (E9.5–10.5); decrease in cell diameter and cross-sectional area (E10.5–11.5); progressive nuclear condensation (E10.5 onwards); and enucleation (E12.5 onwards). Thereafter, EryD are also present in the bloodstream. A major obstacle to the study of primitive erythroid development is the current inability to easily distinguish EryP from EryD and to cleanly separate these populations, particularly given that enucleation can no longer be considered an exclusively definitive erythroid characteristic. No EryP-specific surface antigens have been identified that would permit the detection and isolation of cells of this lineage. We have exploited the restricted expression of the human embryonic beta-like globin gene epsilon to generate EryP lineage-specific transgenic mouse and embryonic stem (ES) cell models in which green fluorescent protein (GFP) reporter expression is driven exclusively in EryP and have used this line to track quantitatively the differentiation and maturation of circulating EryP during embryogenesis. The maturation of primitive erythroblasts was reflected in their dynamic expression of Ter119 and CD71. Up-regulation of a number of nonerythroid-specific cell surface markers, including integrins and other adhesion molecules, was apparent beginning around E12.5, reflecting a previously unrecognized EryP maturation process. Interestingly, enucleation of EryP is accompanied by changes in surface antigen expression. Thus, the phenotype of primitive reticulocytes is distinct from that of their nucleated precursors. In contrast with the prevailing view that EryP are present only transiently in the embryo, we find that they comprise a stable cell population that persists through the end of gestation. The human epsilon-globin::GFP transgenic mouse model provides a novel system for defining, at the cellular and molecular level, the mechanisms involved in development of the primitive erythroid lineage.
Angiopoietin-1 Supports SRC Activity Acquisition of Human CD34-Bone Marrow Cells.
Authors: Ando Kiyoshi , Nakamura Yoshihiko , Yahata Takashi , Muguruma Yukari , Sato Tadayuki , Matsuzawa Hideyuki , Uno Tomoko , Kato Shunichi , Hotta Tomomitsu
Published: October 2019

CD34 negative hematopoietic stem cells (CD34− HSCs) were identified in mice and humans. Human HSCs are evaluated as severe combined immunodeficient mouse (SCID)-repopulating cells (SRCs), originally identified by the ability to reconstitute hematopoiesis in nonobese diabetic (NOD)/SCID mouse. CD34− cord blood (CB) cells have been hard to engraft in NOD/SCID mice until recent report of successlul engraftment by intra-bone marrow transplantation (iBMT). However, CD34− bone marrow (BM) cells have not been analyzed precisely. We prepared lineage negative CD34 negative (Lin-CD34−) cells by negative selection using CD2,3,7,14,16,19,20,33,34,36,41,56,127, and GlyA antibody. Lin-CD34− BM cells did not engraft in NOD/SCID mice even by using iBMT (0/6). In the previous study, we reported that Lin-CD34− BM cells were able to differentiate into CD34+ cells accompanied by the emergence of colony forming activity after 7 days of stroma-dependent culture, while SRC activity was not detected. (BMT 28, 587–595, 2001) Here we cultured Lin-CD34− BM cells on stroma cells transfected with human angiopoietin-1 cDNA (AHESS-5), since we detected Tie-2 expression on Lin-CD34− BM cells. AHESS-5 supported induction of CD34 much better than HESS-5 cells or empty vector transfected control cells (EVHESS-5), and the effect was blocked by anti-Tie-2 antibody (Fig.1). Furtheremore, CD34+ cells produced from CD34− BM cells engrafted in NOD/SCID mice (11/12). As previously reported, CD34− CB cells differentiate CD34+ cells and acquire SRC activity by stroma-dependent culture without angiopoietin-1. These results highlighted the characteristic differences of CD34− HSCs of BM from CB and the unique role of BM niche for CD34− HSCs.

Fig. 1 CD34 expression on Lin − CD34 − BM cells after 7 days of culture Fig. 1. CD34 expression on Lin−CD34− BM cells after 7 days of culture
Hematopoietic Origin of Hepatic Stellate Cell.
Authors: Miyata Eri , Masuya Masahiro , Ishikawa Fumihiko , Yoshida Shuro , Kato Keizo , Ohishi Kohshi , Katayama Naoyuki
Published: October 2019

Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. They undergo a gradual transition from a quiescent, fat-storing phenotype to an activated myofibroblast-like phenotype and then produce high amount of extracellular matrix such as collagen by liver injury. They express mesenchymal markers such as vimentin and desmin, or neural/neuroectodermal markers such as glial fibrillary acidic protein (GFAP). Based on the characteristic phenotype, the embryonic origin of stellate cells is thought to be the septum transversum mesenchyme or neural crest. However, their origin in the adult liver is still unknown. Recently, several studies have reported that crude bone marrow (BM) cells can give rise to hepatic stellate cells. However, since adult BM cells are thought to contain hematopoietic stem cells and mesenchymal stem cells, it is important to clarify which type of stem cells is the true source of hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of singe enhanced green fluorescent protein (EGFP)-marked hematopoietic stem cells (Lin− Sca-1+ c-kit+ CD34− cells) into lethally irradiated nontransgenic mice and examined the histology of liver tissues obtained from chimeric mice with carbon tetrahydrochloride (CCl4)-induced injury. Following 12 weeks treatment of CCl4, hepatic nodules and bridging fibrosis developed in all livers. We detected EGFP+ cells in the liver and some of them contained intracytoplasmic lipid droplets, which were proved by oil red O staining. Immunohistochemical analysis demonstrated that 60% of EGFP+ cells were negative for leukocyte common antigen (CD45); however, they expressed vimentin, GFAP and ADAMTS-13, which is a circulating zinc metalloproteinase synthesized in hepatic stellate cells. Moreover, nonparenchymal cell populations were isolated from the livers of chimeric mice with CCl4 treatment and were incubated on noncoated glass slides for 3 days. EGFP+ cells were also positive for type I collagen. These phenotypes are consistent with those of hepatic stellate cells. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells upon liver injury.
An Investigation of Factor XIII Binding to Recombinant γ′/γ′ and γ/γ′ Fibrinogen.
Authors: Gersh Kathryn C. , Lord Susan T.
Published: October 2019

Increased levels of the alternatively spliced γ′ chain of fibrinogen have been measured in patients suffering from coronary artery disease (Lovely et al. Thromb. Haemost.2002;88:26–31). Others have suggested that the γ′ chain serves as a binding site for factor XIII (Siebenlist et al. Biochemistry1996;35:10448–10453), thus increasing the amount of cross-linking and therefore the stability of the γ′-containing clots (Falls and Farrell JBC1997;272:14251–14256). We have expressed recombinant γ′/γ′ homodimer and γ/γ′ heterodimer fibrinogen variants in CHO cells and characterized their polymerization by turbidity. Compared to normal recombinant γ/γ fibrinogen, γ′/γ′ fibrinogen displayed a slower rate of polymerization and a lower final absorbance. γ/γ′ Heterodimer fibrinogen had both a polymerization rate and final absorbance that was intermediate between γ/γ and γ′/γ′. These observations suggest that the presence of the γ′ chain slows lateral aggregation and leads to the formation of thinner fibers. These variants were then used to investigate the binding of factor XIII to fibrinogen. We designed an ELISA assay to measure this binding by immobilizing fibrinogen on a 96-well plate, incubating with factor XIII, and measuring bound factor XIII with an anti-factor XIII A-subunit antibody. Normal recombinant γ/γ fibrinogen bound factor XIII zymogen (A2B2) with a KD of 4.1 x 10−8 M +/− 1.3 x 10−8 M, similar to previous data for plasma fibrinogen. Surprisingly, both γ′/γ′ and γ/γ′ fibrinogen variants bound factor XIII zymogen with the same KD as did γ/γ fibrinogen. As shown by ELISAs with antibodies specific for either γ or γ′, the C-termini of both the γ and the γ′ chains are accessible on this immobilized fibrinogen. These findings suggest that the C-terminus of the γ′ chain is not a critical element in the binding of factor XIII zymogen. Our findings differ from previously reported findings. Further studies are in progress to reconcile these differences.
Factor VIII’s C Domains Bind to Platelets.
Authors: Hsu Ting-Chang , Pratt Kathleen P. , Thompson Arthur R.
Published: October 2019

The C domains of factor VIII contain the primary binding site for the cofactor, activated factor VIII, to interact with the phospholipid membranes, including those on the platelet surface. Isolated C2 domain has been shown to bind to phosphotidyl-L-serine-rich lipids and platelets; under flow cytometry, binding to activated platelets was confirmed. For comparison, C1C2, expressed in E.coli, was prepared with up to mg quantities isolated. Fresh, gel-filtered platelets were then studied in a flow cytometer either with or without activation by the thrombin receptor peptide, SFLLRN-amide. Depending upon the conditions, up to 80% of the platelets could be stained with a monoclonal antibody to C2 (ESH8) that is known not to compete with lipid or von Willebrand factor binding. The results were confirmed using a S2296C mutant C1C2 where the free suflhydryl group was either biotinylated and detected by fluorescein labeled streptavidin or directly labeled with fluorescein. As shown in the figure, essentially all platelets bound directly fluorescein labeled C1C2. Using standardized, labeled microbeads, it was estimated that there are 7000–10,000 binding sites per platelet. After platelet activation, the number of platelets binding C1C2 increased with all three detecting systems but only by 15–30%. In contrast, binding of isolated C2, as determined either by ESH8 or as a C2296 biotinylated species, was much lower when the same molar amounts were added, and was primarily detectable following platelet activation. C1C2 binding appeared independent of von Willebrand factor as platelets from two unrelated subjects with severe, type 3 von Willebrand disease gave the same patterns on flow cytometry as seen in platelets from normal subjects. ESH4, a monoclonal antibody known to inhibit binding of C2 to lipid membranes effectively competed C1C2 binding to platelets. Although an indirect alteration the C2 domain conformation cannot be excluded, results support a direct role of C1 in enhancing platelet binding.

Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets
The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII.
Authors: Gale Andrew J. , Rozenshteyn Diana , Riceberg Justin
Published: October 2019

Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.
dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.
Authors: Schmidt Manuel , de Cristobal Javier , Sander Astrid , Brzezicha Bernadette , König Merediz Sven A. , Wittig Burghardt
Published: October 2019

Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis of the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.
Allogeneic Environment and Tissue Damage Provide Critical Stimuli for Regulatory T Cell Expansion and Survival In Vivo after Hematopoietic Cell Transplantation.
Authors: Nguyen Vu H. , Chang Daisy , Negrin Robert S.
Published: October 2019

CD4+CD25+ regulatory T cells (Treg) mediate alloresponses in murine models of bone marrow transplantation (BMT), leading to protection from graft-versus-host disease (GvHD). However, in vivo migration and tissue localization of Treg during this inflammatory response remain unclear. We previously demonstrated co-localization of Treg with effector T cells (Tcon) with initial expansion in secondary lymphoid organs prior to migration into inflamed tissues in a major MHC-mismatched BMT model. To explore the stimuli for Treg proliferation, we evaluated the role of the allogeneic environment by transferring FVB donor luciferase-expressing (luc+) Treg into lethally-irradiated syngeneic recipients. Unlike the allogeneic irradiated setting where Treg expand in the presence or absence of Tcon, adoptively transferred luc+ Treg were not detected in secondary lymphoid organs of syngeneic lethally-irradiated BMT recipients by in vivo bioluminescence imaging (BLI). Syngeneic luc+ Tcon also had significantly different in vivo dynamics, with a 4 day delay and only moderate expansion in lymph nodes. Proliferation was not detected in the spleen, unlike their allogeneic Tcon counterparts, nor in the bone marrow compartments, as seen in lymphopenic models. To assess whether irradiation induced the observed in vivo dynamics of Treg in the allogeneic setting, we transferred FVB luc+ Treg or luc+ Tcon into unirradiated Balb/c Rag2−/−gamma chain (γC) −/− recipients, which lack T, B, and NK cells. After adoptive transfer into Rag2−/−γC−/− recipients, robust Tcon proliferation was observed in secondary lymphoid organs and the bone marrow compartments; however, Treg expansion was weak, and specific localization to lymphoid or nonlymphoid tissues was not observed. Treg were stimulated to localize to and expand in secondary lymphoid organs by the co-transfer of Tcon in unirradiated Rag2−/− (γC) −/− or by conditioning Rag2−/− (γC) −/− recipients with irradiation. Exogenous IL2 administration two weeks following luc+ Treg transfer into unirradiated Rag2−/− (γC) −/− recipients similarly led to localization and expansion of Treg in secondary lymphoid organs. These studies indicate the critical role of proinflammatory cytokines, such as IL2, generated either by irradiation-induced tissue damage or donor Tcon, in the expansion and localization of Treg. Differences between Tcon and Treg expansion in syngeneic or unconditioned allogeneic Rag2−/− γC−/− hosts suggest an important role of conditioning with irradiation alone or in concert with the allogeneic environment, in providing distinct signals for Tcon versus Treg activation, proliferation, and localization.
Glucocorticoid Therapy Targets Proliferation, Differentiation and Bcl-2 Dependent Survival Signaling in ALL Blasts.
Authors: Rhein Peter , Scheid Stefanie , Ratei Richard , Hagemeier Christian , Seeger Karl , Kirschner-Schwabe Renate , Schrappe Martin , Spang Rainer , Ludwig Wolf-Dieter , Karawajew Leonid
Published: October 2019

In the multicentric ALL-BFM (Berlin-Frankfurt-Munster) study, all patients are uniformly treated during the first week of induction therapy which uses glucocorticoids (GC) as the principal therapeutic agent. The GC response assessed at day 8 of therapy provides one of the basic parameters for further risk stratification. In spite of the clinical significance, molecular mechanisms of GC action in vivo are largely unknown. Our recent genome-wide analysis of gene expression in blasts persisting during induction therapy identified a common set of genes differentially expressed in blasts at day 8 (d8) and at diagnosis (d0) (n=457, false discovery rate <0.05). Expression changes indicated therapy-induced inhibition of cell cycling, expression shift towards normal mature B cells and downregulation of the apoptosis regulator Bcl-2. In the current study, we validated the key differences between d8 and d0 blasts at protein and cellular levels. DNA distribution and percentage of cycling blasts was determined by flow cytometry in a series of matched d8 and d0 samples (13 pts) and demonstrated the decreased proliferative activity of d8 cells (4.3-fold, p=0.014). Protein expression, investigated by flow cytometry in a total of 84 pts, demonstrated statistically significant expression decrease of the progenitor cell antigenes CD10, CD34 and TdT and expression increase of the B-cell antigene CD20 and the inflammatory response molecules CD11b and IFNGR1 (p<0.05). We were also able to confirm the lower expression values of the Bcl-2 protein in d8 blasts (p<0.05, n=57). Investigation of GC-sensitive B-lineage leukemia cell lines demonstrated that BCL-2 downregulation by GC was a pre-requisite of GC-induced apoptosis. Moreover, we found a considerable cross-correlation between viability, cell cycling and Bcl-2 expression levels. Upregulation of the Bcl-2 expression via IL-7 receptor signaling prevented GC-induced apoptosis in these cell lines. Collectively, GC therapy interferes with differentiation and proliferation programs in leukemic blasts which are closely related to the Bcl-2 specific apoptotic pathway.
Two Decades of Progresses in Adolescents with Acute Lymphoblastic Leukemia (ALL) Treated in the FRALLE Protocols: Adolescence Is No More a Bad Prognostic Feature If an Intensive Chemotherapy Is Applied.
Authors: Baruchel Andre , Auclerc Marie-Françoise , Perel Yves , Mechinaud Françoise , Auvrignon Anne , Schmitt Claudine , Piguet Christophe , Lejars Odile , Berthou Christian , Demeocq François , Schneider Pascale , Gandemer Virginie , Michel Gérard , Cayuela Jean-Michel , Leblanc Thierry , Leverger Guy ,   the FRALLE Group
Published: October 2019

Adolescence has been claimed since the seventies to be associated to a a bad prognosis in childhood ALL. Out of 4658 patients with ALL, 258 adolescents (15–20 year old)(5.5%) were treated in the successive FRALLE 83, FRALLE 87–89, FRALLE 92 (pilot phase), FRALLE 93 and FRALLE 2000 protocols. The main characteristics were: a sex ratio of 1.8 (M/F), a B-lineage in 71% of the cases vs T lineage in 29%, and a median WBC of 12 G/L (9–1000). Translocation and fusion transcripts were searched for in 120 evaluable BCP-ALL: t(9;22)/BCR-ABL, 8 pts (6%); t(1;19)/E2A-PBX1, 12 pts (10%); t(4;11)/MLL-AF4, 4 pts (3%). Out of 75 evaluable pts t(12;21)/TEL-AML1 was found in only 4 pts (3%). 242 out of 258 adolescents were in CR at the end of induction therapy(EOI)(94%) without any significant difference according to the era. Nevertheless a major difference in the 3y and 5y EFS was found:

Number of pts CR at EOI (%) 3y EFS (%) 5y EFS (%) 10y EFS (%) *: p=.04; **: p=.04 Eighties (F83, F87–89) 100 93 42 +/− 5 35 +/− 5 35 +/− 5 Nineties (F92, F93) 84 93 71 +/−5* 67 +/− 5** 67 +/− 5 2000– (F2000) 74 96 86 +/−5* 86 +/− 10** NYA

The main modification introduced in the nineties was the adoption of a double delayed intensification for the good early responders. Autologous BMT or allogenic BMT were indicated in bad early responders (D8 poor prednisone response, D21 marrow M3 response) and/or unfavourable cytogenetics. The better results of the 2000 protocol can mainly be explained by the further intensification of chemotherapy between induction and delayed Intensification 1 and before delayed Intensification 2. These better results were obtained despite decreasing the indications of BMT (6 performed vs 20 in the nineties) and of CNS irradiation (100% in the nineties vs 35% in the current era, including the TBI for BMTs).

Conclusions:excellent results can now be achieved in adolescents with ALLthis study emphasizes again the need to treat adolescents with ALL according to pediatric intensive protocols and not “adult-type” protocols, as we recently suggested (Boissel et al, J Clin Oncol 2003).Whether this could also be applied to young adults remains to be demonstrated but seems appealing.
Acute Lymphoblastic Leukemia in Adolescents and Young Adults in Finland.
Authors: Usvasalo Anu , Saarinen-Pihkala Ulla , Elonen Erkki , Raty Riikka , Vettenranta Kim
Published: October 2019

Background: Long-term survival in pediatric acute lymphoblastic leukemia has dramatically improved during the past three decades through intensification of therapy. Progress has also been made regarding therapy of adult ALL. Recent reports in the literature suggest better outcome on pediatric protocols, as compared with adult ones. In pediatric ALL, age over 10 years is a high-risk factor in many protocols. The arbitrary age limit of 16 years may not truly reflect the biologic differences in ALL between adolescents vs. young adults. The purpose of the present work was to evaluate the outcome of adolescents with ALL in Finland, treated on pediatric vs. adult ALL protocols.

Materials and methods: All patients aged 10–25 years diagnosed with ALL during 1990–2005 in Finland were included. There were 120 patients (aged 10–16 yrs) treated on pediatric common Nordic (NOPHO) protocols with a total duration of 2 yrs, and 100 patients (aged 17–25 yrs) treated on Finnish National ALL adult protocols with a total duration of 3.5 yrs. In total 15 underwent allo-SCT in first remission. Data had been collected to the pediatric Nordic NOPHO database, as well as to the Finnish Leukemia Group ALL database. Events were defined as death, relapse, or second malignancy.

Results: The 5-year EFS was 68 % for children (n=62) and 55% for adults (n=72) (p=0.04), and the 5-year OS was 75 % for children and 68 % for adults (p=0.075), respectively. When only patients treated with conventional chemotherapy were included (stem cell transplant recipients in 1CR excluded), the 5-year EFS was 65 % for children (n=53) and 55 % for adults (n=66) (p=0.09), and the 5-year OS was 80 % for children and 65 % for adults (p=0.09), respectively. Regardless of protocol, EFS in the age group of 10–15 years was 74 % and in the group 15–25 years 57 % (p=0,06).

Conclusions: EFS regarding all patients was better on pediatric than on adult protocols. All the other comparisons reached statistically only a trend level. The difference in outcome between pediatric and adult protocols in Finland is less clear than reported elsewhere. Age seems to be a prognostic factor also in the adolescent age groups. The non-transplanted patients seemed to have a better outcome, suggesting that those with higher risk probably ended up with transplant. Although therapy is an important risk factor, our further analysis of cytogenetic features including CGH array may reveal other risk factors that may turn out to be even more powerful.

EFS, pediatric vs. adult treatment group EFS, pediatric vs. adult treatment group
Curcumin Inhibits Proliferation of Leukemic Cells Expressing P210BCR-ABL or BCR-ABL-T315I and Prolongs Survival of Mice with B-Cell Acute Lymphoblastic Leukemia (B-ALL).
Authors: William Basem M. , Goodrich Ami , Peng Cong , Li Shaoguang
Published: October 2019

The prognosis of BCR-ABL-induced B-ALL remains dismal with no effective chemotherapies currently available. Curcumin, a natural compound derived from tumeric, has been shown to induce apoptosis in the leukemic cell line K562 and in many solid tumor cells. We tested whether curcumin has an inhibitory effect on B-lymphoid transformation by wild type BCR-ABL or BCR-ABL-T315I mutant. We first used BCR-ABL-expressing pre-B cells isolated from B-ALL mice and BCR-ABL-expressing BaF/3 cells. We treated these cells with different concentrations of curcumin (10, 30, or 50 uM), and found that proliferation of the cells was significantly inhibited at concentrations as low as 10 uM. Curcumin also induced apoptosis of the cells, as shown by increased Annexin V-positive cells detected by FACS. Western blot analysis showed that Curcumin treatment resulted in decreased JNK2 activation and a significant increase in IKKa/b expression. To further investigate the underlying mechanisms for cellular apoptosis induced by curcumin, we screened for apoptosis related transcription factors that were activated or inactivated in curcumin-treated BCR-ABL-expressing pre-B cells isolated from B-ALL mice. To do so, we probed nuclear extracts of these cells onto a commercial DNA oligonucleotide arrays (Panomics) representing the canonical binding sites for 52 transcription factors. We observed decreased expression of NFkB at 48 hours and a significant increase in expression of AP-2, p53, and vitamin D receptor 3 (VDR3) at 24 hours. The p53 induction by curcumin was further demonstrated by Western blotting. To examine the efficacy of curcumin on B-ALL, we induced B-ALL in BALB/c mice by transplanting BCR-ABL-transduced donor bone marrow cells into lethally irradiated recipient mice. The B-ALL mice were treated with intraperitoneally injected curcumin (40 mg/kg, once a day), and the treated mice survived significantly longer than the vehicle-treated B-ALL mice (p=0.04; Peto-Wilcoxon test). Taken together, our results suggest a potential use of curcumin in treating BCR-ABL-induced B-ALL.