bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.
Authors: Onai Nobuyuki , Onai Aya , Manz Markus G. 
Published: October 2019

Abstract
Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions.
To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity.
In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.
Highly Efficient Neural Differentiation of Human Peripheral Blood Haematopoietic Stem Cells.
Authors: Moebius Jeannette M. , Widera Darius , Kaltschmidt Barbara , Kaltschmidt Christian , Piechaczek Christoph 
Published: October 2019

Abstract
Neural stem cells are a potential source of cells for cell therapy of neurodegenerative diseases or drug screening. Ethical and practical considerations limit the application of neural stem cells derived from human embryonic stem cells or adult brain tissue. Therefore, alternative sources of adult human neural stem cells are of high interest for basic research as well as potential clinical use. These sources have to satisfy the demands of easy accession, rapid expansion in serum-free media and reliable induction to a neural fate.
CD133 positive hematopoietic stem cells (HSCs) isolated from mobilized leukapheresis were cultured and expanded in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2/bFGF) in serum-free medium. After two weeks of culture, neurosphere formation was observed. Subsequent culture in serum-free, EGF and FGF-2 containing medium resulted in large numbers of CD133−, Nestin+ and Sox-2+ neural stem cells. Neural stem cells derived from HSCs (hNSCs) are highly proliferative and are able to migrate in response to chemotactic stimuli described to induce migration of neural stem cells. We used immunocytochemical techniques and PCR to assess neural differentiation. Adherence to poly-D-lysine/laminin and growth factor deprivation resulted in cells of neuronal morphology and high expression of neuronal differentiation markers. We furthermore show that after retinoic acid treatment neuronal induction was greatly enhanced with more than 90% of cells expressing neuronal differentiation markers as beta-III-tubulin and neurofilaments. In addition, hNSCs were able to differentiate into cells of the glial lineage as shown by expression of glial fibrillary acidic protein (GFAP). Our novel method provides nearly limitless numbers of neural stem cells from an easily accessible autologous adult human source, which could be used as a starting point for further experimental studies and potential therapeutic use.
Phenotypic Comparison of Platelet- and Plasma-Derived Factor Va: Tyrosine Phosphorylation Differentiates the Two Cofactor Pools.
Authors: Wood Jeremy P. , Tracy Paula B. 
Published: October 2019

Abstract
Factor Va, the essential cofactor for thrombin formation via the Prothrombinase complex, exists in two pools in whole blood: 75–80% is found in the plasma, while platelet alpha-granules contain the remaining 20–25%. Platelet-derived factor Va possesses several modifications not present in its plasma counterpart, such as an O-glycosylation on Thr402, and is the more procoagulant molecule, exhibiting resistance to inactivation by activated protein C (APC) and plasmin when bound to the activated platelet membrane surface. As the platelet-derived cofactor pool originates from endocytosis of plasma-derived factor V by megakaryocytes, it can be hypothesized that, subsequent to its endocytosis, factor V is post-translationally modified, proteolytically processed, and packaged into alpha-granules to form the unique platelet-derived molecule. Platelet- and plasma-derived factor Va were purified from three individual donors. Platelets were isolated from whole blood and lysed with Triton X-100 in the presence of the tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid to prevent dephosphorylation by any released tyrosine phosphatases. Factor V in both the platelet lysates and the platelet-poor plasma was activated with thrombin, adsorbed onto a resin-coupled monoclonal antibody against human factor V, washed extensively, and eluted using 10% SDS. Western blotting analyses, using a monoclonal antibody against phosphotyrosine residues, revealed the presence of tyrosine phosphorylation on the light chain of plasma-derived, but not platelet-derived, factor Va. No such modification was observed on the heavy chain of either cofactor. Monoclonal antibodies against the heavy and light chains of factor V were used to verify that similar levels of factor V were present in all samples. This differential modification was observed in all three donors. Subsequent MALDI-TOF analyses of trypsin-digested plasma- and platelet-derived factor Va light chains were performed to identify the site(s) of tyrosine phosphorylation. These data identified phosphorylation of the C-terminal tyrosine residue (Tyr2196) in plasma-derived, but not platelet-derived, factor Va. The phosphorylated fragment (residues 2188–2196, m/z = 1152.5) is consistently present in spectra of plasma-derived factor Va, while only the non-phosphorylated fragment (m/z = 1072.5) has been observed in the platelet-derived cofactor. To demonstrate that the phosphatase inhibitors used were effective, the platelets were lysed in the presence of added plasma-derived factor Va. Tyrosine phosphorylation, as determined by immunoblotting, was still present on the light chain, indicating that the dephosphorylation was occurring prior to platelet lysis. Tyrosine phosphorylation of other proteins has been implicated in regulation of endocytosis. Further experiments will need to be performed to determine if the modification has a similar role in megakaryocyte endocytosis of factor V.
Identification of a Plasmin-Interactive Site in the Factor VIII Light Chain Responsible for Proteolytic Cleavage at Lys36 in the A1 Domain.
Authors: Nogami Keiji , Shima Midori , Takeyama Masahiro , Tatsumi Kohei , Fujita Yuri , Saenko Evgueni L. , Tanaka Ichiro , Yoshioka Akira 
Published: October 2019

Abstract
Factor VIII is inactivated by limited proteolytic cleavages at Arg336 and Lys36 by plasmin (Abst #1017 and 1018, BLOOD106, 2005). We recently identified a plasmin-interactive site in the A2 domain responsible for cleavage at Arg336, and also reported that the cleavage at Lys36 was selectively regulated by the light chain that might interact with its protease. In the current study, several approaches were employed to examine the localization of plasmin-interactive site(s) responsible for cleavage at Lys36. Rate constant of plasmin-catalyzed factor VIIIa inactivation by the addition of the 1649A3-C1-C2 (intact light chain) or 1690A3-C1-C2 fragment was reduced by ~2-folds in a dose-dependent manner using one-stage clotting assay, but not by the 1722A3-C1-C2. SDS-PAGE analysis revealed that Lys36 cleavage in A1/1722A3-C1-C2 dimer by plasmin was markedly slower than that in the A1/1649A3-C1-C2 or A1/1690A3-C1-C2 dimer. Surface plasmon resonance-based assay using anhydro-plasmin (Ah-plasmin), catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that the 1722A3-C1-C2 bound to Ah-plasmin (Kd; 191 nM) with an ~3-fold lower affinity compared with 1649A3-C1-C2 or 1690A3-C1-C2 (68 or 83 nM, respectively). Recombinant A3 domain, expressed in E.Coli, also bound to Ah-plasmin with similar affinity (Kd; 44 nM), but the C2 failed to bind, suggesting the presence of a plasmin-binding site within N-terminus of the A3 domain. On the other hand, Glu-Gly-Arg-active-site modified factor IXa blocked the light chain binding to Ah-plasmin by ~60% (Ki: 10.5 nM), supporting that another plasmin-binding site juxtaposes or overlaps with a factor IXa-binding site (residues 1804–1818) in the A3 domain. The 6-aminohexanoic acid, a competitor of lysine-binding site of plasmin, blocked by >90% the light chain and Ah-plasmin interaction (Ki; 6.8 microM). Based on the major contribution of lysine-binding site(s) of plasmin for interaction with the light chain, evaluation of the A3 sequence revealed two regions involving clustered lysine residues of 1690–1705 and 1804–1818. Two synthetic peptides corresponding to these regions blocked the light chain binding to Ah-plasmin by ~50% (Ki: ~20 microM). We conclude that an extended surface centered the 1690–1705 and 1804–1818 regions within the A3 domain interacts with plasmin for the cleavage at Lys36 within the A1 domain.
Targeted Depletion and Inhibition of Activated T Lymphocytes with SGN-70, a Humanized Antibody Specific for the Activation Antigen CD70.
Authors: McEarchern Julie A. , Klussman Kerry , Boursalian Tamar E. , Grewal Iqbal S. , Law Che-Leung 
Published: October 2019

Abstract
CD70 (CD27L) is a member of the tumor necrosis factor superfamily that is predominantly expressed on activated lymphocytes and is important for generation of B and T memory and effector responses. High levels of CD70 are also found on chronically activated T cells and lymphocytes in patients with autoimmune disorders. Blockade of CD70 interaction with its receptor has been shown to inhibit the onset of experimental autoimmune encephalomyelitis and cardiac allograft rejection in mice. We have engineered a humanized anti-CD70, SGN-70, that mediates Fc-dependent antibody effector functions and blocks binding of CD70 to its receptor. In this report we show that SGN-70 inhibits co-stimulation of T lymphocytes, and selectively depletes CD70+ activated T cells. Depletion of antigen-specific T cells by SGN-70 was demonstrated using CD8+ T cells specific for a peptide (M58-66) derived from the influenza A matrix protein M1. Stimulation of peripheral blood mononuclear cells with the M1 peptide markedly induced CD70 expression within a discrete expanding of M1 peptide-specific CD8+Vβ17+ T cell subset. Treatment of peptide-stimulated cultures with SGN-70 (0.01–1 μg/mL) decreased the number of CD70+CD8+Vβ17+ cells by >80%. This depletion was dependent on the activity of CD16+ cells within the culture since blocking CD16 completely eliminated the depleting effect of SGN-70. Non-activated Vβ17 negative cells were not affected by SGN-70 and were functionally equivalent to untreated control cultures in subsequent response to mitogenic re-stimulation. Together, these data demonstrate the capacity of SGN-70 to selectively target activated cells and its potential to limit expansion of antigen-specific T lymphocytes. Thus, SGN-70 may have therapeutic potential to target T cell-mediated autoimmune and inflammatory diseases.
CD4+CD25+ Marrow Infiltrating Lymphocytes in Myeloma Patients Display an Activated Phenotype and Lack Suppressive Function.
Authors: Noonan Kimberly A. , Mgebroff Stephanie , Serafini Paolo , Meckel Kristen , Bonyhadi Mark , Borrello Ivan 
Published: October 2019

Abstract
We have previously shown that marrow infiltrating lymphocytes (MILs) from myeloma patients proliferate upon stimulation with anti-CD3/CD28 beads more efficiently and exhibit greater specificity to autologous tumor than their peripheral blood lymphocyte (PBL) counterparts. In attempting to dissect the mechanisms responsible for greater tumor specificity of MILs, we examined the frequency and function of the putative CD4+/CD25+ regulatory T cells (Tregs) in both compartments. Phenotypic and functional differences in the CD4+/CD25+ populations were examined in MILs and PBLs from 12 multiple myeloma patients and 3 normal donors. CD4+/CD25+ cells were stained for the suppressive marker: FoxP3; or the activation markers: CD40L, CD71 (transferrin receptor). CD4+/CD25+ MILs of myeloma patients are CD25lo, FoxP3lo and predominantly express activation markers. They expand more readily upon anti-CD3/CD28 stimulation, produce high amounts of the Th1 cytokines: IFNγ and IL-2, and low amounts of IL-10. Importantly, they fail to suppress the tumor specific T cell proliferation. These findings are all consistent with an activated T cell phenotype. In contrast, CD4+/CD25+ cells from PBLs of myeloma patients are CD25hi, FoxP3+, primarily produce IL-10 and IL-4 and markedly suppress T cell expansion in a tumor specific assay, suggestive of a regulatory T cell phenotype. Although MILs possess a lower number of FoxP3+ cells as compared to PBLs, the higher MFI may indicate the presence of a small yet highly regulatory T cell population. Interestingly, MILs from normal donors possess a more regulatory phenotype then their myeloma counterparts as determined by high FoxP3 and low CD40L and CD71 expression as well as reduced expansion upon activation. Taken together, these data underscore major differences in the immuno-inhibitory pathways present in blood as compared to marrow. Notably, MILs of myeloma patients demonstrate a paucity of Tregs. These differences may explain the disparities seen in the tumor-specificity of T cells from these two compartments. The ability to expand a T cell population with fewer endogenous Tregs and heightened tumor-specificity may have significant implications for the implementation of adoptive immunotherapy.
%CD25 (MFI) %FoxP3 (MFI) %CD40L %CD71 Fold Expansion Ability to Suppress Myeloma CD4/CD25 MILs 4.7 (29.6) 2.2 (2932.1) 88.8 90.1 12.2 − CD4/CD25 PBLs 19.1 (115.2) 52.3 (163.7) 15.7 19.6 2.5 +++++ Normal CD4/CD25 MILs 14.0 (56.8) 70.1 (82.7) 37.3 6.4 3.3 +++ CD4/CD25 PBLs 24.0 (19.4) 30.9 (261.8) 12.1 4.2 4.0 ++
Reaction of Factor VIIa (VIIa) with Plasma Inhibitors Ex Vivo and In Vivo.
Authors: Petersen Lars C. , Christoffersen Carsten L. , Krogh Thomas N. , Elm Torben 
Published: October 2019

Abstract
Activated proteases are typically short-lived in the circulation where they react with plasma inhibitors and are removed as complexes by scavenger receptors. In contrast VIIa is characterized by a relatively long in vivo clearance half-life of 2–3 hr. Free VIIa exists in a latent state that is fully activated only upon binding to tissue factor (TF). Ex vivo VIIa is described to react predominantly with anti-thrombin (AT) in a slow reaction enhanced by heparin and by TF. Free VIIa is assumed to be rather inert towards plasma inhibitors also in vivo, and this may account for its slow clearance. Still it is not known whether the reaction with AT or other plasma inhibitors is an obligatory step in the in vivo clearance of VIIa. To clarify this question we studied the interaction between VIIa and plasma inhibitors in human and murine serum and plasma, and also studied this interaction during in vivo clearance of VIIa in mice. The VIIa-inhibitor complexes formed after spiking of serum with VIIa for 24 h were purified by affinity chromatography and subjected to SDS-PAGE and characterized by MALDI analysis. In the absence of heparin we identified VIIa complexes with alpha 2-macroglobulin (alpha 2M), AT, alpha 2-plasmin inhibitor and inter-alpha-trypsin inhibitor with the complex with alpha 2M as the most prominent component, whereas the AT complex was pre-dominant in the presence of heparin. The interaction between VIIa and plasma inhibitors might be differently affected by contact with the vasculature in the circulation. We therefore next studied VIIa complex formation in vivo in mice after i.v. injection of 125I-VIIa. Complex formation was followed by SDS PAGE and autoradiography, and clearance was followed by measurements of the VII antigen level. The data suggest that alpha 2M and AT complexes are formed in vivo, however, without affecting VIIa antigen clearance. This was confirmed by studies of in vivo clearance of VIIa or a preformed VIIa-AT complex. Measurements of VIIa clot activity, and of VII- and VIIa-AT antigen concentrations suggestedthat in vivo clearance of VIIa from circulation does not include complex formation with a plasma inhibitor as an obligatory intermediate,that the VIIa-AT complex is cleared with the same rate as VIIa andthat a transient VIIa-AT complex is formed subsequent to VIIa bolus injection which may account for a faster clearance of VIIa when measured by clot activity relative to VII clearance when this is measured by antigen concentration.
Heat Shock Prevents Tissue Factor Induction after Rewarming from Lethal Hypothermia in a c-Jun-Dependent Manner.
Authors: Egorina Elena M. , Sovershaev Mikhail A. , Kondratiev Timofei V. , Olsen Jan O. , Tveita Torkjel , Østerud Bjarne 
Published: October 2019

Abstract
Decrease in body core temperature is associated with life threatening coagulation disturbances in humans. Although rewarming from hypothermia is a prerequisite for survival, it can lead to mobilization of large amounts of tissue factor (TF) from damaged tissues, which in turn can induce disseminated intravascular coagulation. Using immunoblotting, In-Cell Western assay, fluorescence activated cell sorting (FACS) and TF activity assay, we studied post-rewarming TF levels, activity and surface redistribution, along with the regulation of TF gene transcription in mononuclear cells (MNCs) obtained from an in vivo rat model. Twenty-four male Wistar rats were randomly assigned into four groups: normothermic control group (n=6); hypothermic followed by rewarming group (n=6); heat shock pretreated hypothermic followed by rewarming group (n=6); and heat shock pretreated normothermic group (n=6). Rewarming after a 5-hour episode of 15oC hypothermia caused a 5-fold rise of TF protein levels and 2-fold increase in surface TF signal intensity and serface redistribution of TF antigen, judged by 2-fold increase in surface-to-total TF ratio. TF activity was enhanced from 5.6±0.7 mU 106/cells in normothermic group to 13.9±3.6 mU 106/cells after hypothermia/rewarming. This was accompanied by a dramatic elevation of c-Jun and c-Jun-N-terminal kinase (JNK) phosphorylation, and the absence of early growth response-1 and NF-kB activation. Reflecting certain limitations of the method when dealing with detection of low levels of antigens, FACS analysis of TF-positive population of MNCs failed to detect any differences between the groups. To search for a stimulus to counteract c-Jun-mediated induction of TF activity in MNCs from rewarmed rats, we applied heat shock pretreatment one day before the hypothermia/rewarming experiment. Heat shock pretreatment restored post-rewarming TF activity, protein levels and surface-to-total TF ratio in rat MNCs to normothermic levels. Furthermore, in heat shock-pretreated animals, rewarming from hypothermia failed to increase phosphorylated c-Jun and JNK levels. We attribute this to the profound overexpression of heat shock protein 70 and inhibition of JNK. Taken together, in our present study, we report an induction of TF activity, protein levels and redistribution of TF antigen to the surface of MNCs, isolated from rats rewarmed from hypothermia. This increase was counteracted by a full body heat shock 24 hours prior to hypothermia via suppression of hypothermia-induced activation of c-Jun.
O-Linked Glycosylation with Sialylated T-Antigen: A Novel Carbohydrate Determinant of von Willebrand Factor Antigen Levels.
Authors: Lenting Peter J. , van Schooten Carina J.M. , Lisman Ton , van den Berg Marijke , Eikenboom Jeroen C.J. , Leebeek Frank W.G. , Goudemand Jenny , Fressinaud Edith , de Groot Philip G. , Denis Cécile V. 
Published: October 2019

Abstract
The glycosylation-profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of the sialylated T-antigen (NeuAc( α 2–3)Gal( β 1–3)[NeuAc( α 2–6)]GalNAc). It is unknown yet if O-linked carbohydrates affect VWF levels. We therefore developed an immunosorbent-based assay using the lectin Peanut Agglutinin (PNA) to determine O-linked T-antigen on VWF. Control experiments showed that PNA-binding was: (i) specific to T-antigen that was de-sialylated via neuramidase-treatment; (ii) proportional to the number of O-linked glycosylation sites and (iii) independent of the extent of multimerisation. Using this assay, we found a strong correlation between PNA binding and VWF-antigen levels in a series of randomly selected plasma samples (252±125% for VWF<0.5 U/ml (n=15); 131±36% for VWF between 0.5 and 1.5 U/ml (n=32); 92±40% for VWF>1.5 U/ml (n=19); p<0.003). Reduced or increased PNA binding was also observed for patients characterized by increased (liver cirrhosis; n=58) or reduced (von Willebrand disease (VWD)-type 1; n=32) VWF-antigen levels, respectively. VWD-type 1 patients further displayed increased ratios of propeptide over mature VWF-antigen levels (0.38±0.18 versus 0.17±0.03 for patients (n=32) and controls (n=20), respectively; p<0.0001), which is indicative for reduced VWF survival in these patients. Interestingly, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank= 0.50; p=0.0039), suggesting a potential association between T-antigen glycosylation and VWF survival. Finally, we observed a marked decrease in PNA binding in post-DDAVP samples from various patients (131±45% versus 85±33% in pre- and post DDAVP, respectively; n=18; p=0.0013). Since these patients had distinct underlying pathologies (primary platelet function disorder, hemophilia A or VWD type 1, 2A or 2N), this seems to represent a general phenomenon that is not restricted to VWD. Moreover, it indicates that the O-linked glycosylation profile of VWF stored in endothelial storage-organelles may differ from that of constitutively secreted VWF.