bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Thrombin-Induced Platelet Aggregation Using Human Single Chain Fv Antibodies Specific for TREM-Like Transcript-1.
Authors: Washington A. Valance , Giomarelli Barbara , Chisholm Maia M. , Quigley Laura , McMahon James B. , Mori Toshiyuki , McVicar Daniel W.
Published: October 2019

Platelets play a duel role in our cardiovascular system. They are important in maintaining our vascular integrity. They also facilitate the progression of cardiovascular diseases (CVD) such as arteriosclerosis. In more severe cases, displaced platelet aggregates can lead to myocardial infarction or stroke. Because of their prominent role, anti-platelet therapies are used to reduce mortality associated with CVD. However, the use of these therapies is sometimes associated with bleeding complications, thrombocytopenia or non-responsiveness and the search for new therapeutic targets continues. Some of the recent treatment strategies include molecules that target platelet membrane receptors. The success shown by this class of therapeutics has prompted us to evaluate the recently characterized murine platelet receptor, TREM like transcript 1 (TLT-1), as a therapeutic target. Murine studies suggest that TLT-1 is a platelet specific protein found in α-granules of platelets and megakaryocytes. Here we asked if human TLT-1 was also linage specific and if platelet aggregation could be modulated through interaction with TLT-1 specific antibodies. We evaluated human TLT-1 (hTLT-1) for pattern of expression in the peripheral blood and we found that hTLT-1, like its mouse counterpart, is platelet specific. To address TLT-1 function we panned a naïve human library of single chain fragments (scFvs) with a TLT-1 extracellular domain/human fc fusion protein to produce monoclonal antibodies specific for hTLT-1. We isolated six scFv clones using ELISA to detect reactivity to the fusion protein and confirmed their specificity by flow cytometry on cells transfected with TLT-1 and purified human platelets. These scFvs were then tested to see if they would disrupt or enhance platelet aggregation. Here we demonstrate, for the first time, inhibition of thrombin induced platelet activation using scFv that are specific for TLT-1. Taken together these results suggest TLT-1 is a potential target for therapeutic intervention of cardiovascular disease.
Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.
Authors: Zorca Suzana M. , Josefsson Emma C. , Rumjantseva Viktoria , Hartwig John H. , Hoffmeister Karin M.
Published: October 2019

We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation.

The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.
Intravenous (IV) Iron Supplementation in Patients with Chemotherapy-Induced Anemia (CIA) Receiving Darbepoetin alfa Every 3 Weeks (Q3W): Iron Parameters in a Randomized Controlled Trial.
Authors: Lerchenmueller Christian , Husseini Faress , Gaede Bernd , Mossman Tony , Suto Tamas , Vanderbroek An
Published: October 2019

Background and Aim: The role of IV iron supplementation during treatment with erythropoiesis-stimulating agents (ESAs) in patients with CIA is of increasing interest as a possible means of improving response. This randomized, open-label, multicenter study was designed to evaluate the safety and efficacy of IV iron vs standard practice in CIA patients receiving darbepoetin alfa. Interim efficacy analyses showed a higher response rate for darbepoetin alfa with IV iron compared to darbepoetin alfa with standard iron practice with no difference in the safety profile between the treatment arms (Vanderbroek et al, EHA 2006). Iron parameters are reported here.

Methods: Eligible patients were diagnosed with a non-myeloid malignancy and had CIA with a baseline hemoglobin (Hb) value < 11g/dL. All patients received darbepoetin alfa 500 mcg administered Q3W with the SureClick™ prefilled autoinjector. Patients were randomized 1:1 to IV iron 200 mg (single dose Q3W at the same time as darbepoetin alfa or in 2 doses of 100 mg within 3 weeks) or standard practice (oral iron or no iron). Randomization was stratified by tumor type and baseline Hb category (< 10 or ≥10 g/dL).

Results: A total of 400 patients were randomized. Mean (SD) age of the study population was 61.4 (11.5) years; range, 20–86. Sixty percent (n=241) of participants were women; 28% (n=114) had lung or gynecological tumors; and 52% (n=208) had a baseline Hb value ≥10 g/dL. In the interim analysis population (n=196), the mean (SD) weekly dose of IV iron was 64.8 (6.6) mg in the IV iron group (n=100). In the standard practice group, 28 of 96 patients (29%) received oral iron and 2 (2%) received IV iron (these patients were analyzed as randomized). Mean (standard error) serum ferritin concentrations and percent transferrin saturation (TSAT) in the 2 groups from baseline (BL) to end of study (EOS) are shown in the figure.

Conclusions: The combination of darbepoetin alfa Q3W and IV iron appeared to be associated with a trend toward increased mean serum ferritin levels compared to the standard practice control arm. In contrast, mean TSAT surprisingly appeared to be similar in the 2 groups for most of the study period, perhaps suggesting that TSAT is influenced by other factors. Iron management appears to be an important factor in the response to ESAs and the findings presented here suggest the need for additional exploration into iron uptake and demand in cancer patients treated with darbepoetin alfa.

Serum Ferritin Concentration Serum Ferritin Concentration Transferrin Saturation (%) Transferrin Saturation (%)
Characterisation of the Laminin 10/11 Binding Site on the Lutheran Glycoprotein Suggests a Novel Type of Protein-Protein Interaction.
Authors: Mankelow Tosti J. , Burton Nicholas , Stefansdottir Fanney O. , Spring Frances A. , Parsons Stephen F. , Gilstring C. Fredrik , Brady R. Leo , Narla Mohandas , Chasis Joel Anne , Anstee David J.
Published: October 2019

The Lutheran glycoprotein is a five domain member of the immunoglobulin superfamily (IgSF) with a wide tissue distribution. It is a ligand for Laminin isoforms containing the alpha5 chain (Laminins 10 and 11). Lutheran glycoprotein on erythrocytes is thought to play a role in vasocclusive events that are a serious cause of morbidity in sickle cell anaemia. We have investigated the molecular basis of the Lutheran:Laminin 10/11 interaction. Lutheran binding to Laminin 10/11 is pH and salt dependent suggesting the interaction is influenced by surface charge. Since Laminins are known to contain areas of positive charge that are of importance in binding to other ligands (heparin, alpha-dystroglycan), a molecular model of Lutheran glycoprotein was constructed to identify surface exposed areas of negatively charged aspartic and glutamic acid residues. Selected residues were mutated to alanine and the mutant proteins examined for binding to Laminin 10/11 using ELISA and Surface Plasmon Resonance. Mutations E309A and D310A greatly reduced binding to Laminin 10/11 while D312A completely abolished binding. The Lutheran model predicts a rod-like structure with a flexible hinge region of 6–8 residues between the 2nd and 3rd IgSF domains. Residues E309, D310 and D312 are located on domain 3 proximal to the hinge region. Mutations (H235P, and delta 233–235) within the hinge region also abolished Laminin binding showing the hinge region to be essential for ligand interaction. Electron tomography on recombinant Lutheran-Fc chimeric protein bound to Laminin 10/11 suggested Lutheran glycoprotein bends at the hinge region to expose the critical negatively charged residues on domain 3 and thereby allow Laminin binding. These data suggest Lutheran-Laminin 10/11 interaction is a novel type of protein:protein interaction and provide a foundation for further investigation of its biological significance.
Comprehensive Analysis of Murine and Human RBC Ghost Proteomes.
Authors: Wooden Jason M. , Finney Greg , MacCoss Michael , Gilligan Diana M.
Published: October 2019

Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. Mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton. Genetic knock-out of various components of this apparatus has led to the creation of mouse models which have contributed significantly to our understanding of these disorders in humans. However, the mouse and human RBC protein complements have not been comprehensively compared.

Using newly developed proteomic methodology, we conducted a peptide level ‘bottom-up’ analysis of the normal mouse and human RBC ghost (i.e., RBC membrane skeleton and associated proteins). RBCs were purified using cellulose acetate chromatography from whole blood taken from three genetically identical mice and two hematologically normal yet genetically diverse humans. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin and analyzed by shotgun proteomics using microcapillary liquid chromatography coupled with tandem mass spectrometry. In total, 400 and 491 unique proteins were identified in human samples A and B respectively while 469 proteins were found in common across the three mouse samples. All previously identified membrane skeleton proteins were found in the human and mouse samples. Likewise, well-known RBC membrane proteins were represented. Of interest, a surprising number of proteins were found associated with the RBC ghost involved in processes such as protein repair (15–20), protein degradation (30–43), oxidative stress response (4–6), Ras oncogene biology (28–30), and glycolysis (3–6). Collectively, the two human samples represented 544 unique proteins. These results affirm the usefulness of inherited anemia mouse models given the observed conservation of membrane skeleton components and the inherent challenges with doing normal versus diseased RBC analysis in humans due to genetic variation.
Genomic Composition of Howell-Jolly Bodies.
Authors: Gantt Nicole M. , Walker Bob , Lee Y. Terry , Jiang Yuan , Staker Pamela , Oneal Patricia A. , Bhanu Natarajan V. , Meltzer Paul S. , Miller Jeffery L.
Published: October 2019

Erythrocyte inclusion bodies that are now commonly referred to as Howell-Jolly Bodies (HJB) were originally discovered over 100 years ago by microscopy. These nuclear fragments are observed in the erythrocytes of patients lacking splenic function, but their precise genomic origins are vague. Here we report studies of the genetic content of HJB using a molecular cytogenetic method known as Comparative Genomic Hybridization (CGH). Peripheral blood samples from six splenectomized human donors were studied. All donors had undergone splenectomies prior to this study for diagnostic and therapeutic indications related to underlying neoplastic and autoimmune disease. Ultrasound testing was used to rule out the presence of accessory splenic tissue, and peripheral blood smears confirmed the presence of HJB. Nucleated cells were removed by density gradients and leukocyte filtration for use as patient-matched controls. DNA was isolated from the leukocytes and HJB-containing erythrocytes. The presence of high molecular weight HJB DNA was confirmed in each sample using gel electrophoresis. As expected, it was not possible to obtain sufficient quantities of DNA from the control erythrocytes of healthy individuals with normal splenic function. CGH was performed using equivalent amounts of reference genomic DNA co-hybridized with HJB DNA or matched donor leukocyte DNA. Agilent oligonucleotide CGH arrays were hybridized and scanned according to the manufacturer’s instructions. The control hybridizations (genomic DNA isolated from donor leukocytes) demonstrated standard patterns consistent with the donor’s karyotype. In contrast, the HJB hybridization signals demonstrated an unusual pattern of centromeric-to-telomeric enrichment on each chromatid of each chromosome. The HJB hybridization signals also increased as a function of distance from the centromere according to the chromatid size. The enhanced signal relative to centromeric distance varied considerably between chromosomes, but was remarkably consistent between donors. These data suggest Howell-Jolly Bodies contain a non-random distribution of centromere-depleted chromatin generated after DNA double-strand breakage during erythroid differentiation.
Age-Related Homeostasis: Molecular Mechanism, Disease and Predictions from Global Analyses of Mouse Liver Proteins.
Authors: Kurachi Sumiko , Tanaka Taku , Kanai Muneyoshi , Suenaga Emi , Solovieva Elena , Kurachi Kotoku
Published: October 2019

We previously reported the first molecular mechanism underlying the age-related homeostasis, ASE/AIE-mediated genetic mechanism for age-related regulation of gene expression, which turned out to be a mechanism of puberty-onset gene switching, specifically controlling a group of genes for their expression from puberty into old age (Kurachi & Kurachi J Thromb Haemost 2005; Zahng et al J Biol Chem277:4532, 2002; Kurachi et al Science285:739, 1999). This led us to successful construction of a transgenic mouse model of hemophilia B Leyden, a unique subset of hemophilia B with its mechanism being remained mysterious, robustly mimicking its unusual pattern of puberty-onset spontaneous amelioration. With this background, we hypothesized that besides the ASE/AIE-mediated mechanism, there exist more unidentified fundamental regulatory mechanisms for age-related homeostasis, which individually or in various combinations generate age-related complex and dynamic regulatory patterns of liver proteins. We launched a series of global and quantitative analysis of age-related changes in expression of liver nuclear proteins of mice (C57BL/6xSJL, [male], 1 through-24 month of age) by taking a procedure composed of two-dimensional gel electrophoresis (2DE) for separation and quantification of liver nuclear protein spots and of MALDI-TOF/MS PMF analyses to identify proteins in the spots. Out of over 6000 spots recognized and quantified in 2DE, 4547 protein spots were subjected to MALDI-TOF/MS analysis for protein identification. Finally, 2765 protein spots including many isomers were found unique. Systematic analyses of their age-related expression identified several major phases in protein expression throughout the lifespan. These findings supported our hypothesis that there exist multiple novel molecular mechanisms responsible for maintaining age-related homeostasis. The comprehensive liver nuclear protein data set was then used to construct a comprehensive database, which allows rapid and reliable searches for expression of specific proteins, their age-related dynamic profiles, isomers, protein identification from 2DE image, and other related information. This will serve as a valuable platform resource for studying epigenetic challenges, evaluation of drugs as well as gaining further insights into the molecular mechanisms underlying age-related homeostasis.
Characterization of an Ideal Amphipathic Peptide as a Potential Hemostatic Agent.
Authors: Ganopolsky Jorge G. , Charbonneau Sophie , Peng Henry , Blostein Mark
Published: October 2019

We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.
The Specific Aurora Kinase Inhibitor AZD1152 Significantly Affects the Growth of Human Leukaemic Cells in an In Vivo AML Model.
Authors: Pearce Daniel , Odedra Rajesh , Wilkinson Robert , Bonnet Dominique
Published: October 2019

AZD1152 is a specific Aurora kinase inhibitor with selectivity for Aurora B kinase, designed to target cell division in proliferating tumour cells. It has been shown in model systems that inhibition of Aurora B kinase with AZD1152 reduces histone H3 phosphorylation (phH3) and inhibits cytokinesis, thus inducing cellular multi-nucleation and polyploidy, leading to apoptosis and cell death.

Here, we present results of preliminary studies on the in vivo action of AZD1152 on the growth and development of both a human acute myeloid leukaemia (AML) cell line (HL-60) and primary AML samples in the NOD/SCID xeno-transplantation model. Two weeks of AZD1152 at 25 mg/kg/day is well tolerated in NOD/SCID mice. HL-60 cells were injected iv into sub-lethally irradiated NOD/SCID mice (n=24) and allowed to engraft in the bone marrow. Three weeks later, when malignant cell growth was well established in the bone marrow, mice were split into treatment groups. Either vehicle (n=12) or AZD1152 (25 mg/kg/day via minipump, n=12) was administered for 7 days. One AZD1152-treated group was sacrificed immediately post-treatment (n=9) and another was left for a further 2 weeks before bone marrow analysis for HL-60 cell content (defined by human CD45+/CD33+/CD19− expression). The mean percentage HL-60 cell content in the marrow of untreated mice was 64.5 ± 19.0 (range 14.0–86.6, n=11), whereas in mice that had received AZD1152 and were sacrificed at the end of dosing period, the mean percentage was 0.29 ± 0.74 (range 0–2.26, n=9; p<0.001). In AZD1152-treated mice that were left for 2 weeks post-treatment, the mean percentage of HL-60 cell content in the bone marrow was 0.008 ± 0.74 (n=3).

Similar experiments were performed with two primary AML samples. AML-1 is from a patient with French-American-British (FAB)-type M1 AML and AML-2 is from a FAB-type M2 AML patient. Ten million cells from both AML samples were injected into NOD/SCID mice (n=12 for AML-1; n=6 for AML-2). Ten weeks later, mice were split into treatment groups and dosed with either control vehicle or 25 mg/kg/day of AZD1152 via minipumps for 7 days. All mice were sacrificed after 1 week of treatment in these initial experiments. The mean percentage engraftment in untreated mice was 59.7 ± 36.0 (range 6.2–84.0, n=4) for AML-1 and 21.8 ± 22.5 (range 5.9–37.7, n=2) for AML-2. Whereas, in mice that had received 25 mg/kg/day of AZD1152 for 7 days, the percentage of engraftment was 1.8 ± 1.7 (range 0.38–4.53, n=5; p<0.01) and 0.07 ± 0.07 (range 0.12–0.014, n=2) for AML-1 and AML-2, respectively.

These data indicate that AZD1152 has a profound effect on the engraftment of human AML cell lines and primary AML samples in an in vivo model. Furthermore, initial results may indicate a long-term effect of AZD1152 treatment in vivo. AZD1152 is currently undergoing Phase I clinical trials in AML patients.
Implications of Heparinoid-Induced Inactivation of Coagulation Cascade Factors and In Vivo Proenzyme Concentration.
Authors: Patel Sanjay , Berry Leslie R. , Hatton Mark W.C. , Chan Anthony
Published: October 2019

Heparin is a commonly used anticoagulant in the treatment of thrombosis. We have compared heparinoid-stimulated inhibition rates with the in vivo plasma concentration of antithrombin (AT)-inhibitable coagulation factors. Second order rate constants (k2) for inhibition of activated factors by either AT + unfractionated heparin (AT+UFH) mixtures or a novel covalent complex of AT and heparin (ATH) (Chan et al, J Biol Chem, 272:22111, 1997) were determined by discontinuous assay. A plot of k2 values (mean ± SEM; n ≥5) versus the respective human plasma concentration of coagulation factors revealed a linear correlation (with R2 values of 0.93 for AT+UFH and 0.90 for ATH, excluding factor XII), in which neutralization efficiency was proportional to in vivo factor level (see Figure). Anticoagulant actions of AT+UFH and ATH were more oriented towards treatment than prophylaxis since inhibition of cascade end point enzymes (thrombin and factor Xa) was more rapid than factors involved in coagulation initiation (factors VIIa and XIa). However, ATH exhibited more enhanced inhibition rates against factors VIIa, IXa and XIa than against factor Xa and thrombin, suggestive of an improved prophylactic profile compared to AT+UFH. Intriguingly, factor XII did not follow this trend, further challenging its role in the coagulation cascade. The impact of these assertions requires confirmation by in vivo inhibition studies.

Figure Figure
Interaction of Plasminogen and Angiostatin with Heat Shock Proteins.
Authors: Dudani Anil K. , Mehic Jelica , Martyres Anthony
Published: October 2019

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60 and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was dose-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, thirty-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, thirty-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. FACS analyses indicated the presence of hsps 27, 60 and 70 on the surface of MCF-7 breast cancer cells but not on human umbilical vein ECs. Polyclonal antibodies to hsp 27 significantly inhibited the interaction of plasminogen and angiostatin with MCF-7 surface-associated hsp27 in a dose-dependent manner. Collectively, these data indicate that

while plasminogen interacts specifically with hsp 27, 60 and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; plasminogen only partially displaces angiostatins binding to hsp 27; actin only partially displaces plasminogen/angiostatin binding to hsps and surface-associated hsp 27 can mediate the binding of both plasminogen and angiostatin to MCF-7 cells.
Critical Role of Hem1 in Hematopoietic Cell Chemotaxis and Innate Immunity to S. Pneumoniae
Authors: Lorsbach Robert B. , Kim Yoon-Sang , Moore Jennifer , Smith-Sielicki Hope , Jones Tiffany M. , Sun Weili , Tuomanen Elaine , Downing James R.
Published: October 2019

Hem1 is a hematopoietic specific member of the HEM family proteins, which have been identified as components of the WAVE regulatory complex. To further characterize the role of Hem1 in hematopoietic cell function, we have generated mice deficient in Hem1 using gene targeting methodology. Hem1-deficient mice manifest several phenotypic abnormalities, including peripheral blood lymphopenia and neutrophilia, splenomegaly, premature mortality, and with variable penetrance thymic hypoplasia and alopecia. Flow cytometric analysis of peripheral blood and spleen demonstrated that Hem1−/ − mice have a marked reduction in peripheral B cells, changes that were evident in both adult and neonatal mice; the splenomegaly in Hem1−/ − mice was attributable to increased extramedullary hematopoiesis. The frequency of bone marrow B cell progenitors was also markedly reduced in Hem1−/ − mice. To assess the role of Hem1 in hematopoietic stem cell (HSC) function, competitive bone marrow transplantation assays were performed. In contrast to wild-type HSCs, Hem1-deficient HSCs had poor competitive repopulating activity in irradiated recipient mice. KSL cell analysis demonstrated no significant difference in the frequency of lin-c-kit+Sca1+ HSCs between wild-type and Hem1−/ − bone marrow, suggesting that the defective competitive repopulating activity of Hem1−/ − HSCs is attributable to defective bone marrow homing or stem cell niche interaction. Given the biochemical evidence implicating the HEM proteins as a component of the WAVE regulatory complex, we also assessed the ability of Hem1-deficient leukocytes to undergo cytoskeletal remodeling in vitro. Using a transwell assay, Hem1−/ − bone marrow storage pool neutrophils demonstrated markedly blunted chemotactic responses to formylated peptide which was attributable to defective f-actin formation. Hem1−/ − peripheral CD4+ T cells similarly manifested chemotactic defects in response to SDF-1, and showed blunted proliferation when stimulated with antibodies against CD3 and CD28. Finally, a model of Streptococcus pneumoniae infection was employed to test the role of Hem1 in the in vivo function of neutrophils. Hem1−/ − mice were dramatically more sensitive to S. pneumoniae than wild-type littermates, as manifested by the inability to eliminate S. pneumoniae organisms in vivo and higher mortality. In summary, Hem1 deficiency results in deficiencies and functional defects in multiple hematopoietic lineages due to defective signaling to the actin cytoskeleton, and importantly, Hem1 plays a critical role in innate immunity to S. pneumoniae in vivo.
Do Lipocalins Play a Role in Mast Cell Physiology?.
Authors: Logdberg Lennart E. , Akerstrom Bo , Hair Gregory A. , Allhorn Maria , Vikulina Tatyana , Kirshenbaum Arnold S. , Sundstrom J. Bruce
Published: October 2019

Introduction. Aiming to identify molecular properties of allergens responsible for their “intrinsic allergenicity”, we focus on a subset of xenogeneic lipocalins (LCs) that comprise the major mammalian respiratory allergens. These structurally and functionally homologous molecules likely possess conserved molecular motifs promoting IgE-dependent allergy. We hypothesize that such LC “allergenicity” depends on non-IgE interactions of LCs with components of the innate immune system. Herein we describe a possible basis for such interactions between LCs and mast cells.

Materials and Methods. Two sources of human mast cells were used; primary cultures derived from peripheral blood CD34+ progenitor cells; or the LAD-2 cell line. Cells were cultured in serum-free medium with recombinant human stem cell factor (SCF; 100 ng/ml). Monoclonal antibodies to human gp330/megalin (MAb E11) were a kind gift of Prof. Lars Rask (Uppsala University, Sweden). Cell surface protein expression was assessed by flow cytometry and gene transcription was measured by real-time PCR.

Results. Monoclonal antibodies to an endocytic cell surface receptor (megalin, also known as low-density lipoprotein receptor-related protein (LRP)-2) known to bind multiple LCs stained the mast cell lines. This putative expression of megalin by the mast cells corresponded to their transcription of megalin mRNA as shown as by PCR. Moreover, mast cell megalin gene transcription could be induced (≥ 1000-fold) by overnight culture with monomeric IgE myeloma protein (100 ng/ml), and such induction of the megalin message correlated with both an increase in cell surface expression of the molecule and the specific binding of a purified human LC (a-1 microglobulin).

Conclusion. Megalin, an LC-binding cell surface receptor, appears to be constitutively expressed by both progenitor and mature mast cells, and its expression seems to be strongly upregulated by culture with monomeric IgE. This is consistent with a role for direct mast cell-LC interactions in the development of IgE-dependent allergy. In addition, and also of potential clinical relevance, endogenous LCs may play a functional role in normal mast cell physiology.
Hematopoietic Stem Cells Contribute to Lymphatic Endothelium.
Authors: Jiang Shuguang , Bailey Alexis S. , Swain John R. , Hewett Erin M. , Wong Melissa H. , Streeter Philip R. , Fleming William H.
Published: October 2019

The lymphatic system plays an important physiological role in vascular and immune homeostasis. Lymphatic vessel function is implicated in a number of pathological conditions including tumor metastasis and impaired wound healing. The identity and origin of lymphatic endothelial precursors is poorly understood. Previously we have shown that adult bone marrow-derived, hematopoietic stem cells (HSCs, c-kit+, Sca-1+, lineage−) can differentiate into functional blood vascular endothelial cells. Given the close relationship between the blood and lymphatic vascular systems, we have investigated whether HSCs also give rise to lymphatic endothelial cells (LEC). GFP+ HSCs were transplanted into lethally irradiated (1200 cGy) recipient mice. Donor-derived LEC expressing lymphatic endothelial markers including LYVE-1 and VEGFR3 were clearly distinguished from hematopoietic cells by the absence of CD45 and F4/80 expression. Deconvolution microscopy confirmed the co-localization of donor and LEC marker expression in individual cells. Transplanted HSCs gave rise to LEC in the liver, gut, gastric and kidney. Donor-derived LEC were detected in 2.4% of liver lymphatic vessels at 4 weeks and persisted for at least 12 months (mean of 3.4%). The self-renewal capacity of HSC-derived lymphatic progenitor cells was demonstrated by serial transplantation. The contribution of these progenitors to tumor lymphatics was evaluated. Transplantation of HSCs into young Min−/− mice resulted in the incorporation of donor-derived LEC into the lymphatics of intestinal adenomas that spontaneously develop in these mice. In addition, CD45+F4/80+ leukocytes were detected in the vessel lumens indicating that these are functional tumor lymphatic vessels. Finally, to determine if LEC progenitors contribute to lymphatic vessels in the absence of radiation injury or tumorigenesis, a parabiosis model was evaluated. Donor-derived LEC were detected in parabiotic mice at a frequency similar to that observed for donor-derived blood vascular endothelial cells. This finding suggests that circulating progenitor cells contribute to lymphangiogenesis during steady-state conditions. Our results indicate that hematopoietic stem cells have the potential to contribute to lymphatic endothelium and therefore HSC-derived progenitors may be potential therapeutic targets for hematopoietic and lymphatic disease.
Characterization of Post Natal Human Lymphoid Progenitors.
Authors: Six Emmanuelle , Bonhomme Delphine , Beldjord Kheira , Jurkowska Monika , Cortivo Liliane Dal , Monteiro Marta , Garrigue Alexandrine , Fischer Alain , Cavazzana-Calvo Marina , Andre-Schmutz Isabelle
Published: October 2019

In humans, little is known about post-natal lymphoid progenitors, especially those able to circulate, colonize the thymus and generate T lymphocytes. On the basis of a previous work published by Anne Galy in 1995, we have detected in the human post-natal bone marrow up to 60 yrs of age a population of progenitors characterized by their CD34+Lin-CD10+ phenotype. Their differentiation potential analysed by culture in methylcellulose medium indicated that in contrast with their CD10− counterparts, CD10+ progenitors have lost erythroid and myeloid potential. On the other hand, CD10+ progenitors cultured on MS5 or OP9/hDL1 stroma demonstrated an enriched capacity to generate B, T and NK lymphocytes as compared to CD10− precursors. In limiting dilution assays, the high lymhoid potential of CD10+ population was confirmed, since 1 out 15 of them gave rise to T cells, 1 out 23 to B cells and 1 out 90 to NK cells. Gene expression profile shows that CD10+ cells express both B and T restricted factors, such as RAG, Gata3, Pax 5 and TdT. In addition, recombination at the IgH locus is already going on, with multiple DJ, but also VDJ recombination products detected. More importantly, CD10+ precursors circulate in the peripheral blood and are detected in the thymus where they are part of the most immature thymocytes CD34+CD1a-CD38-. Altogether, our results demonstrate for the first time the existence of a post-natal lymphoid progenitor population with a broad lymphoid potential and the ability to reach the thymus and generate efficiently T cells. On the long term, their full characterization will pave the way for their enrichment and usage in therapy of primary lymphoid immunodeficiencies.
Clonal Analysis Reveals a Common Progenitor for Endothelial, Myeloid, and Lymphoid Precursors in Cord Blood.
Authors: Akbarloo Nasrin , Perlingeiro Rita R.
Published: October 2019

Several recent studies demonstrate that hematopoietic tissues, including bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) are a source of endothelial progenitor cells (EPCs), which contribute to newly formed blood vessels during tissue repair in adults. However, it is not clear which cell type in hematopoietic tissues gives rise to EPCs, and therefore might be preferable for vascular regeneration. It has been suggested that EPCs might derive from an adult hemangioblast, the primitive bipotential precursor for hematopoietic and endothelial cells, but to date, this hypothesis has not been proven. To test the possibility that a hemangioblast may exist in UCB, we compared the ability of UCB-derived CD34+CD133+ and CD34+CD133− to form blast colonies using the clonogenic BL-CFC assay, previously described for embryonic hemangioblasts (Perlingeiro et al, 2003). All the blast colonies (BL-CFCs) were present in the CD133+ fraction. Bipotentiality of these BL-CFCs was confirmed by replating in hematopoietic and endothelial conditions. To illuminate the lineage position of the hemangioblast within the hematopoietic system, we added CD38 to the antibody combination and adapted the colony assay to liquid culture in 96-well dishes. Clones derived from single cells deposited by FACS were expanded for a week, and each divided into 4 conditions for T and B lymphoid, myeloid, and endothelial cell growth. Whereas myeloid and lymphoid progeny were evident from the CD133+CD38− fraction, these clones were not able to differentiate into endothelial cells. On the other hand, 86% of clones derived from the triple positive (CD34+CD133+CD38+) population were able to produce all 4 lineages. Endothelial cells were characterized by the expression of VE-cadherin, KDR, vWF (and lack of CD45 and c-fms), and the ability to form vessels in vivo in a matrigel plug transplantation assay. Myeloid differentiation was assayed in methylcellulose media, where several BFU-E, CFU-GM, and CFU-GEMM were enumerated. Lymphoid differentiation was assayed by co-culture on OP9-DL1 and MS-5 stroma, for T and B lymphoid differentiation, respectively. Double positive CD4+CD8+ T lymphocytes were observed after 40 days in culture. The presence of B lymphocytes was demonstrated by expression of CD19 and IgM. Final maturation was detected by DJH and VDJH recombination. That the adult hemangioblast should be CD38+ is highly unexpected, as it positions the hemangioblast below the LTR-HSC. An alternate explanation would place the CD38+ hemangioblast upstream, with CD38 being switched off in the LTR-HSC, and back on the STR-HSC. We are currently investigating these possibilities.
Bone Marrow-Derived Cells Regenerate Kit+ Cardiac Stem Cells (CSCs) in Damaged Heart.
Authors: Cerisoli Francesco , Barile Lucio , Gaetani Roberto , Cassinelli Letizia , Frati Giacomo , Giacomello Alessandro , Messina Elisa , Ottolenghi Sergio , Magli Maria Cristina
Published: October 2019

A growing amount of data indicates that the heart harbours stem cells (CSCs) with regenerative potential, however the origin(s) of adult CSCs is still unknown. The expression of Kit a marker of several stem cell types, including hematopoietic and cardiac stem cells, suggests that Kit positive-CSCs may derive, at least in part, from extracardiac sources. In addition, it has been suggested that bone marrow (BM) cells may be mobilized, home into the heart and trans-differentiate into cardiomyocytes, following myocardial infarction.

To investigate whether BM cells can contribute to repopulate the cardiac Kit+ stem cell pool, we transplanted BM cells from a mouse line expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements, into wild type irradiated recipients. After hematological reconstution (4–5 months) and following cardiac infarction, cardiac cells were grown in vitro into typical “cardiospheres” (Messina et al., Circ. Res. 95,911;2004). The cardiospheres obtained, although not numerous, were all GFP fluorescent; this result was confirmed by PCR analysis of genomic DNA of individual CSs. At confocal microscopy, cells at the periphery of CSs showed coexistence of low GFP with cardiac markers, such as Troponin I and the transcription factor NKx2.5, consistent with the expected kit downregulation during cardiac differentiation.

Our results show that cells of bone marrow origin can give rise, after homing into the heart, to cells with properties of Kit+ CSC. In contrast, CSCs isolated from kit/GFP transgenic mice are not able, upon transplantation, to repopulate the bone marrow of wild-type irradiated recipients.

Thus, at least in pathological conditions, part of the Kit-positive CSCs population may be generated by BM-derived cells, capable of adopting in the heart the same function and features of cardiac stem cells.
Improved Endothelial Function with Combined Chelation Therapy in Thalassaemia Major.
Authors: Tanner Mark A. , Galanello Renzo , Dessi Carlo , Agus Annalisa , Smith Gillian C. , Westwood Mark A. , Assomull Ravi , Nair Sunil V. , Walker J. Malcolm , Pennell Dudley J.
Published: October 2019

Background: In β-thalassemia major (TM) myocardial iron toxicity is the dominant cause of ventricular dysfunction, with heart failure responsible for the majority of deaths. Abnormal endothelial function has also been described in these patients and could further contribute to cardiovascular complications. Endothelial function can be determined by measurement of flow mediated dilatation of the brachial artery (FMD). This can be assessed reproducibly by cardiovascular magnetic resonance (CMR).

Aims: To report the changes in endothelial function, LV ejection fraction and ferritin from a randomized placebo controlled trial comparing combined chelation therapy (deferiprone and deferoxamine) with deferoxamine monotherapy.

Methods: 65 patients (male 27, female 38, age 28.7+/−4.8years) with mild-moderate myocardial iron loading (heart T2* 8–20ms) were randomized to receive either deferoxamine with placebo (placebo group), or deferoxamine with deferiprone (combined group). FMD was assessed at baseline and after 12 months.

Results: There were significant improvements in endothelial function in the combined treatment group compared with the placebo group (+8.8% vs 3.1% p=0.013). This was in accord with improvements in the combined group in left ventricular ejection fraction (+2.4% vs +0.6%, p=0.02), and serum ferritin (−870 vs −194 μg/L; p<0.001). These findings were in accord with improved myocardial T2* in the combined group (+43% vs +23%, p=0.017),

Conclusion: In patients with mild-moderate cardiac iron loading, the combined therapy of deferiprone and deferoxamine is superior to deferoxamine alone in improving endothelial function, cardiac function and ferritin, as well as reducing myocardial iron.
The Role of Platelet Adhesion Receptor GPIb α Far Exceeds That of Its Main Ligand von Willebrand Factor in Arterial Thrombosis.
Authors: Bergmeier Wolfgang , Piffath Crystal L. , Goerge Tobias , Cifuni Stephen M. , Ruggeri Zaverio M. , Ware Jerry , Wagner Denisa D.
Published: October 2019

GPIbα binding to von Willebrand factor (VWF) exposed at a site of vascular injury is thought to be the first step in the formation of a hemostatic plug. However, our previous studies in VWF-deficient mice demonstrated delayed but not absent arterial thrombus formation suggesting that, under these conditions, GPIbα may bind other ligands or that a receptor other than GPIbα can mediate platelet adhesion. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracellular domain was replaced by that of the human interleukin-4 receptor (IL4Rα/GPIbα-tg mice). Platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4Rα/GPIbα-tg mice was virtually absent in contrast to avid adhesion in wild-type (WT) mice. As a consequence, arterial thrombus formation was completely inhibited in the mutant mice. Our studies further show that, when infused into WT recipient mice, IL4Rα/GPIbα-tg platelets or WT platelets lacking the 45 kD N-terminal domain of GPIbα failed to incorporate into growing arterial thrombi, even if the platelets were activated prior to infusion. Surprisingly, platelets lacking β3 integrins, which are unable to form thrombi on their own, incorporated efficiently into WT thrombi. Our studies provide in vivo evidence that GPIbα is absolutely required for recruitment of platelets to both exposed subendothelium and thrombi under arterial flow conditions. Thus, GPIbα contributes to arterial thrombosis by important adhesion mechanisms independent of the binding to VWF.
Association of Endothelial Microparticles (EMP) and Endothelial Progenitors with Serum Cholesterol Levels.
Authors: Jimenez Joaquin J. , Ferreira Alexander , Dodson Hannah J. , Lens Katherine M. , Mauro Lucia M. , Jy Wenche , Peter Arley , De Marchena Eduardo , Horstman Lawrence L. , Ahn Yeon-Soong
Published: October 2019

INTRODUCTION: High cholesterol (HC) is known to adversely affect endothelial cells (EC) and has been shown to correlate with decreased levels of circulating endothelial progenitor cells (CEPC). We assayed endothelial microparticles (EMP), a sensitive indicator of EC perturbation, to investigate relations among HC, CEPC, and injury of coronary artery endothelial cells (CAEC), both in vivo and in vitro.

METHODS: Twelve subjects with normal cholesterol (150 ±30 mg/dL, control) and 12 with HC (250 ±25) were studied. EMP were assayed by flow cytometry using fluorescent antibodies and CAEC were cultured as previously described [Jimenez et al, Thromb Res 109:175, 2003]. CEPC were isolated, cultured, and assayed for endothelial colony formation (CFU) as described [Hill et al, NEJM 348:593, 2003].

RESULTS: Comparing the two groups, EMP measured by CD31+/CD42b− were nearly 2.5-fold elevated in HC as compared to controls (1.7 ±0.5 ×106/mL vs.0.35 ±0.02 ×106/mL; p<0.01). Cholesterol levels correlated well with this measure of EMP (R=0.60, p=0.002). However, no significant correlation was found between CD62E+ EMP and cholesterol levels. Assay of CEPC revealed a nearly 2.5-fold decrease in CFU in HC vs. controls (10 ±2 vs. 25 ±4; p<0.01). In studies in vitro, CEPC from controls were cultured in presence of 20% 0.1μm filtered plasma from members of both groups. The HC group plasma inhibited CEPC colony formation by almost 50% (23 ±3.5 CFU for control plasma vs. 13 ±4 colonies for HC plasma). We next assessed the longer-term effect of HC plasma on CAEC cultures. Six-day culture of CAEC in the presence of 20% plasma resulted in a significant increase of CD31+/CD42b− EMP from CAEC treated with HC plasma vs. normal plasma (6.5 ±0.7 ×106/mL vs. 0.23 ±0.03 ×106/mL; p=0.02).

CONCLUSION: These results suggest that EMP are useful markers to monitor cholesterol mediated-EC changes. High EMP levels inversely reflect the vascular endothelial cell regeneration potential due to decreased circulating endothelial progenitor cells.