bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of Imatinib and Hypusination Inhibitors Represents a Novel Therapeutic Strategy in Bcr-Abl Positive Leukemias.
Authors: Balabanov Stefan , Gontarewicz Artur , Ziegler Patrick , Hartmann Ulrike , Copland Mhairi , Brassat Ute , Hauber Ilona , Kanz Lothar , Bokemeyer Carsten , Nordheim Alfred , Hauber Joachim , Holyoake Tessa L. , Brummendorf Tim H.
Published: April 2020

In order to identify new cellular Bcr-Abl downstream targets, we have recently detected differential regulation of eukaryotic initiation factor 5A (eIF-5A) in Bcr-Abl positive cells. EIF-5A represents the only known eukaryotic protein activated by post-translational hypusination. Hypusination inhibitors (HI) GC7 and ciclopirox alone exerted an anti-proliferative effect on Bcr-Abl positive (K562, BA/F3p210) and negative human leukemia cell lines (HL-60, BA/F3) in vitro. No direct effect of HI treatment on phosphorylation of Bcr-Abl downstream targets (global p-tyrosination, p-CRKL, p-STAT5) was observed. However, the synergistic dose-effect relationship for apoptosis induction, cytotoxicity and inhibition of proliferation found for the combination of Imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell cycle analysis and CFSE-labeling of primary CD34+ CML cells in comparison to CD34+ cells from healthy donors. In parallel to these findings, eIF-5A mRNA is expressed at clearly (if significant then say significant and add p-value, otherwise delete clearly) elevated levels in PBMCs from CML patients at different disease stages when compared with PBMCs from healthy donors. Specificity of this effect could be demonstrated by co-treatment of K562 cells with Imatinib and a stable lentiviral siRNA against eIF-5A. Based on this observation, we observed a strong antiprolifrative effect of HI as single agents on BA/F3p210 cells, including the Imatinib-resistant mutants T315I, M351T or E255K. Furthermore, a combination of Imatinib and HI had a synergistic effect in inducing cytotoxicity, apoptosis and inhibition of proliferation in leukemic cell lines with moderate resistance to imatinib (M351T). In contrast, in BA/F3p210T315I (T315I) cells, which are resistant to Imatinib as a single agent, the co-treatment of Imatinib and HI did not lead to additive or synergistic effects. In conclusion, we have identified inhibition of eIF-5A hypusination as a promising new approach for single agent or combination therapy in Bcr-Abl
P0-Related Protein Accelerates the Migration of Human Mesenchymal Stem Cells and Their Progeny by Modulating VLA-5 Interactions with Fibronectin.
Authors: Watt Suzanne M. , Roubelakis Maria , Forde Sinead , Lu Fengjuan , Tsaknakis Grigorios , Martin-Rendon Enca
Published: April 2020

Human mesenchymal stem cells (hMSCs) are capable of expansion, self renewal and generation of several tissues such as bone, fat and cartilage. They also generate microenviromental niche cells that regulate angiogenesis and hematopoiesis. During development and in the adult, hMSCs and their progeny migrate to specific niches where they provide support for hematopoietic cells or they play an important role in tissue regeneration, inflammatory responses and cancer surveillance. There is particular interest in understanding the molecular basis of hMSCs migration which involves numerous transmembrane receptors, cell adhesion and signaling molecules. Here, we demonstrate, for the first time, that human P0 - related protein, hPZR, has the ability to accelerate VLA-5-mediated migration of cultured hMSCs on the extracellular matrix (ECM) protein, fibronectin. This is mediated via the immunotyrosine-inhibitory motif (ITIM) sequences within the hPZR cytoplasmic domain, and the ECM ligand, since it cannot be recapitulated with the hPZR ITIM-less isoform, hPZRb, nor with other ECM components such as laminin or Type IV collagen. It is mediated by interactions with the docking phosphatase, SHP-2. The specificity of these results was confirmed by knocking-down hPZR and hPZRb using siRNA technology. We further demonstrated that hPZR clusters with VLA-5 at the leading edge of the cell, using high-resolution confocal microscopy together with immunoprecipitation and immunoblotting technologies. We propose that hPZR plays a key role in negatively regulating VLA-5 adhesion to fibronectin, a process critical for cell detachment from fibronectin and for the migration of hMSCs and their progeny.
G-CSF Induced Expansion and Mobilization of Hematopoietic Stem Cells Is Altered by the Matrix Protein Osteopontin.
Authors: Forkert Randolf , Ko Yon , Neuhaus Thomas , Gruenewald Elisabeth , Schoeneborn Silke , Vetter Hans , Stier Sebastian
Published: April 2020

Stem cells reside in a physical microenvironment or niche where a balance of signals controls their proliferation, differentiation and death. Components of the specialized microenvironment have generally been defined in terms of cells and signaling pathways affecting stem cell maintenance or expansion. We have defined a role for a matrix glycoprotein that provides a constraining function on hematopoietic stem cells within the bone marrow microenvironment. Osteopontin (OPN) is an abundant glycoprotein in bone that modifies primitive hematopoietic cell number and function in a stem cell non-autonomous manner. Here we analyzed the role of OPN for regulating stem cell mobilization and pool size in times of G-CSF induced marrow stress, a context close to the clinical setting of stem cell mobilization not well understood so far. Bone marrow stromal cells show an enhanced expression of OPN under stimulation with G-CSF, which prompted us to analyze the role of OPN in G-CSF mediated activation of the stem cell niche. First we treated OPN deficient mice and their wild-type littermates with G-CSF for 5 days. We could observe a significant increased stem cell fraction in the peripheral blood and in the bone marrow in the absence of OPN in comparison to the wild-type controls. To evaluate, if this effect is stroma dependent, we first transplanted wild-type bone marrow into wild-type or OPN-deficient recipients. 6 weeks after transplantation we treated these mice with G-CSF for 5 days and analyzed the peripheral blood and the bone marrow for the contents of primitive hematopoietic cells. Here we could detect a significantly increased stem cell fraction in peripheral blood and bone marrow of the OPN−/− recipients in comparison to wild type controls detected by FACS and functional in vitro stem cell assays. We then transplanted the stressed bone marrow in a competitive repopulation assay into wild-type recipients and observed a significant increase of CD45.2 cells from OPN−/− recipient mice up to 12 weeks after transplantation in comparison to wild-type controls, demonstrating an enhanced G-CSF induced expansion of hematopioetic stem cells in the OPN-deficient stem cell niche. Furthermore, we could observe an enhanced expression of Angiopoietin and N-Cadherin in OPN-deficient bone marrow stromal cells after stimulation with G-CSF in comparison to wild-type controls, supporting the stroma dependent expansion of stem cells in the absence of OPN in the G-CSF stimulated stem cell niche. Therefore, OPN is a restricting element of the stem cell niche limiting the size of the stem cell pool and may provide a dynamic mechanism by which excess stem cell expansion is prevented during times of niche stimulation. These findings may provide new insight into expansion and mobilization of hematopoietic stem cells by G-CSF mediated by components of the stem cell niche.
Large Scale Copy Number Variation Upregulates the Expression of MYB in Human T-ALL.
Authors: O’Neil Jennifer , Tchinda Joelle , Gutierrez Alejandro , Moreau Lisa , McKenna Keith , Maser Richard , Wong Kwok-Kin , Wu Wen-shu , DePinho Ronald , Chin Lynda , Lee Charles , Look A. Thomas
Published: April 2020

Using comparative genome hybridization (array CGH), we have discovered a small area of increased copy number on the long arm of chromosome 6 in 8 out of 20 (40%) T-ALL cell lines. The region of increased copy number is very small, containing only one gene, MYB, the cellular homolog of the avian oncogene v-myb. By performing fiber-FISH on these cell lines, we have shown that the increased copy number results from a discrete tandem duplication of the MYB gene on one allele. Although myb is a frequent target of retroviral insertional activation in screens for oncogenes whose overexpression accelerates the onset of murine T-ALL, its overexpression and increased copy number has not previously been implicated in human T-ALL. Using gene expression profiling and Western blotting, we have demonstrated that the duplication in human T-ALL results in increased levels of MYB expression. Furthermore, using quantitative PCR we have confirmed that this tandem duplication occurs in primary human T-ALL samples. In our studies to date, MYB tandem duplication and overexpression appears to occur as part of the major multistep molecular pathway in T-ALL that affects a majority of cases, in which the leukemic cells also have TAL1/SCL and LMO1/2 overexpression, and NOTCH1 gene activating mutations, together with homozygous deletion of the INK4A/ARF locus. We are currently determining the mechanism through which MYB overexpression contributes to the pathogenesis of T-ALL by siRNA knockdown in T-ALL cell lines. Our finding of MYB tandem gene duplication differs from classic forms of oncogene amplification involving double minutes or homogenously staining regions. It is possible that MYB copy number is increased through a novel somatic mechanism of allele-specific, tandem duplication of a small genomic region during the process of malignant transformation. Another possibility is suggested by recent studies documenting that inherited large-scale copy number variation (CNV) accounts for much of the phenotypic diversity within human populations. Further studies will be needed to determine whether MYB tandem duplication is present in the germline DNA of T-ALL patients, which if identified, would provide the first example of an inherited CNV functioning as a mechanism of cancer susceptibility.
Treatment of High-Risk (HR) Philadelphia Chromosome-Negative (Ph-) Adult Acute Lymphoblastic Leukemia (ALL) According to Classical Risk Factors and Minimal Residual Disease (MRD). Interim Results of the PETHEMA ALL-AR-03 Trial.
Authors: Ribera Josep , Oriol Albert , Morgades Mireia , Sarrá Josep , Brunet Salut , Llorente Andreu , Guardia Ramon , Parody Ricardo , Bernal Teresa , Esteve Jordi , Sanz Miguel , Potro Eloy Del , Bergua Juan , Moreno Maria-Jose , Grande Carlos , Bueno Javier , Cladera Antonia , Fernández María-Carmen , García-Boyero Raimundo , Bermúdez Arancha , Feliu Evarist
Published: April 2020

Current therapeutic protocols for adult ALL consider MRD together with the classical risk factors (age, WBC count, immunophenotype, cytogenetics and speed in response to therapy) for treatment decisions. The aim of the prospective study ALL-AR-03 from the Spanish PETHEMA Group was to evaluate the feasibility of sequential MRD detection and the response to a differentiated therapy according to MRD levels (assessed by cytofluorometry at the end of induction and consolidation therapy) in HR Ph- adult ALL pts. HR ALL included one or more of the following: age 30–60 yr, WBC count >25×109/L and 11q23 or MLL rearrangements. Induction therapy included VCR, DNR and PDN for 4 wk. In pts with slow cytologic response (≥ 10% blasts in BM assessed on d14) intensified induction with HD-ARA-C and mitoxantrone was administered. Early consolidation therapy included 3 cycles with rotating cytotoxic drugs including HD-MTX, HD-ARA-C and HD-ASP. Pts. with slow cytologic response on d14 or MRD level >0.05% after consolidation were assigned to allogeneic SCT (related or unrelated) and those with standard cytologic response on d14 and MRD level <0.05% after consolidation received 3 additional cycles of delayed consolidation (identical to those of early consolidation) followed by maintenance therapy up to 2yr in CR.

On May 2006,119 patients were evaluable (mean (SD) age 37(14) yr, 64 males, 75 precursor B-ALL, 44 T-ALL, WBC 67(87) ×109/L). Induction death 7(%), resistance 20 (%), CR 93 (78%) and MRD<0.1% in 64% were observed. Early consolidation was completed in 63 patients. MRD<0.05% was observed in 77% at the end of consolidation. Allo-SCT was performed in 20 pts (TRM 6, relapse 1, CCR 13) and delayed consolidation and maintenance in 38 (toxic death 2, relapse 7, CCR 29). Two-yr DFS for the whole series was 35±13%. No significant differences were observed between pts receiving chemotherapy or SCT. Slow cytologic response was associated with a lower CR. No initial variable was associated with a higher probability of MRD negativity after induction or consolidation. Neither the negativity of MRD nor the pattern of clearance of MRD were associated with a better DFS. These preliminary results suggest that in HR adult ALL pts with adequate response to induction and adequate clearance of MDR the results of late consolidation and maintenance are not hampered by avoiding allo-SCT.
Circulating Levels of Adhesion Molecules and Markers of Endothelial Dysfunction in Acute Inflammation Induced by Prolonged Brisk Exercise: Rapid Endothelial Recovery and Potential Beneficial Effects.
Authors: Papassotiriou Ioannis , Bartzeliotou Anastasia , Margeli Alexandra , Tsironi Maria , Skenderi Katerina , Bacoula Chryssa , Chrousos George
Published: April 2020

The “Spartathlon” ultradistance foot race (246 Km continuous, prolonged, brisk exercise for up to 36 hours) provides a unique model of prolonged duration exercise that reveals dramatic systemic inflammatory changes. In this study we investigated endothelial function of athletes participating in this extreme physical stress paradigm. The circulating levels of adhesion molecules E-, L- and P-selectins, intracellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1), along with those of thrombomodulin (TM), N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and cardiac troponin T, were measured before, at the end of and at 48 h postrace. The E-, L-, P-selectins were quantified in plasma with a biochip array analyzer, the Evidence® (Randox Laboratories, Crumlin, UK). The determination of soluble ICAM-1 and soluble VCAM-1 in plasma was performed on the Luminex-100 system using the multiplex assay kit Linco Human Sepsis/Apoptosis (Linco Research, Inc., Missouri, USA), TM was measured with an immunoassay technique (Euroclone Life Sciences, Pero, Italy), while NT-pro-BNP and cardiac troponin T were quantitatively determined on the Roche Elecsys 2010 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany). The table summarizes the results of the study.

Levels of L- and P-selectins remained the same before and at the end of the “Spartathlon” race, but had a similar decline 48 h postrace. E-selectin, ICAM-1 and TM reached a maximum value at the end of the race and returned to normal 48 h after the race. A similar profile was observed for VCAM-1 and NT-pro-BNP, with a tendency for a decrease 48 h postrace but without reaching complete normalization. Cardiac troponin T remained below the detection limit at all time points examined. The indices of endothelial damage and dysfunction were strongly affected during exercise, with a peak at the end of the “Spartathlon” race and a return to normal 48 h later. In parallel, the indices of endothelial protection were also affected, while no evidence for myocardial damage was observed. These findings suggest that although prolonged brisk exercise activates the endothelium, the latter expresses protective molecules and rapidly recovers, possibly participating in the beneficial effects of exercise on health.

Plasma Biochemical Parameters (mean±SE) Examined in Athletes that Successfully Completed the “Spartathlon” race pre end post ND* non detectable s-VCAM-1 (ng/mL) 328.4±20.7 717.3±102.4 379.5±21.9 s-ICAM-1 (ng/mL) 107.6±12.5 137.7±18.1 107.3±12.6 s-L-selectin (ng/mL) 982.9±35.3 1008.5±51.8 870.9±34.1 s-P-selectin (ng/mL) 175.0±29.5 170.1±11.7 119.8±8.3 s-E-selectin (ng/mL) 10.3±1.0 19.2±1.5 12.1±1.0 s-TM (ng/mL) 4.0±0.3 6.9±0.7 4.5±0.2 NT-pro-BNP (ng/L) 40.1±9.6 964.6±153.9 82.1±12.1 Troponin T (μg/L) ND* ND* ND*
Tissue Factor Firmly Immobilized on Surfaces Promotes Platelet Adhesion and Fibrin Formation in Studies with Circulating Human Blood: Importance of Shear Rate Conditions and FVIIa.
Authors: Tonda Raul , Lopez-Vilchez Irene , Galan Ana M. , Navalon Fulgencio , Pino Marcos , Hernandez Rosa , Ordinas Antonio , Escolar Gines
Published: April 2020

While procoagulant activity of tissue factor (TF) has been widely investigated, its possible proadhesive properties towards platelets have not been studied in detail.

We explored the interaction of platelets with human TF (hTF) firmly attached to a surface using anticoagulated blood with low molecular weight heparin (20 U/ml) at different shear rates. For studies at 250 s−1 and 600 s−1, TF adsorbed on a synthetic surface was exposed to circulating blood in flat perfusion devices. Deposition of platelets and fibrin formation were evaluated by morphometric, immunocytochemical and ultrastructural methods. For experiments at 5000 s−1, we used the PFA-100™ with experimental cartridges with collagen or collagen-hTF. Effect of rFVIIa was assessed in all experimental settings. Prothrombin fragment F1+2 levels were also measured.

At 250 and 600 s−1 platelet interaction was 19.84±1.33% and 26.12±3.42% of the total surface respectively. Our inmunocytochemical results suggest that von Willebrand factor could mediate these interactions. Fibrin formation was significantly higher at 250 s−1 than at 600 s−1 (p<0.05). FVIIa tended to increase platelet deposition without reaching statistical significance, and raised fibrin formation and thrombin generation (p<0.05). Our At 5000 s−1, closure times in the PFA-100 were significantly shortened in the presence of hTF (154.09 ±14.69 s vs 191.45± 16.09 s with collagen alone; p<0.05). Addition of rFVIIa did not result in a further reduction of closure time.

Our studies demonstrate that hTF is reactive for platelets. von Willebrand factor could mediate these interactions. Recombinant FVIIa enhances the procoagulant action of hTF at low and intermediate shear rates, but has no impact on the hemostatic performance at very elevated shear rates.
The Leech Product Saratin Is a Potent Inhibitor of Both VWF and Integrin α2β1 Binding to Collagen.
Authors: White Tara C. , Robinson David K. , Yin Hang , DeGrado William F. , Hanson Stephen R. , McCarty Owen J.T.
Published: April 2020

Background: Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor (VWF), which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors GPVI and α2β1 to collagen is critical for platelet activation and stable adhesion. Hematophages, such as leeches, have evolved a number of inhibitors directed toward platelet-colagen interactions so as to prevent haemostasis in the host during haematophagy.

Objective: To elucidate the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen.

Results: In the presence of inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), both saratin and 6D1, a blocking α2β1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated α2β1-dependent platelet adhesion to soluble collagen in the presence of a Src kinase inhibitor. Moreover, saratin prevented platelet-rich-plasma adhesion to fibrillar collagen, a process dependent upon both α2β1 and VWF binding to collagen. Furthermore, saratin specifically inhibited the binding of the α2I-domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of an anti-GPIb and anti-α2β1 mAb.

Conclusions: The present study demonstrates that saratin interferes with integrin α2β1 binding to collagen in addition to inhibiting VWF-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.
Endothelial Microparticles and Their Binding to Leukocytes in Patients with End-Stage Renal Disease.
Authors: Zuckerman David , Hofmeyer Mark , Chirinos Julio A. , Jy Wenche , Jimenez Joaquin J. , Horstman Lawrence L. , Corrales-Medina Vicente F. , Pennel Philip , Ahn Yeon-Soong
Published: April 2020

Background: End-stage renal disease (ESRD) has been associated with endothelial dysfunction, a proinflammatory state and markedly increased cardiovascular risk. Endothelial microparticles (EMP) are released by activated or apoptotic endothelial cells. In vitro studies have shown that EMP can bind and activate neutrophils, suggesting that they may represent not only markers of endothelial status but also mediators of inflammation. The objectives of this study were to:

Assess the levels of free EMP and leukocyte-bound EMP in ESRD; Determine whether EMP binding to neutrophils predicts the degree of neutrophil activation in patients with ESRD.

Methods: We measured the levels of endothelial microparticles (EMP) and EMP-leukocyte conjugates in 80 patients with ESRD and 35 normal controls. We performed flow cytometry in peripheral venous blood before hemodialysis to measure (1) EMP positive for CD31 (EMP31), E-selectin (EMP62E), CD54 (EMP54); (2) EMP54- and EMP62E-leukocyte conjugates; (3) Neutrophil activation, assessed by expression of activation marker CD11b.

Results: Assessment of free EMP revealed that patients with ESRD had slightly elevated levels of free EMP54 but not of free EMP62E or EMP31. However, levels of EMP54-Lymphocyte, EMP54-Monocyte, EMP54-Neutrophil, EMP62E-Lymphocyte and EMP62E-Monocyte conjugates were clearly elevated in patients with ESRD. CD11b expression in leukocytes strongly correlated with levels of EMP54-Neutrophil conjugates (R=0.67; p<0.0001) and EMP62E-Neutrophil conjugates (R=0.66; p<0.0001). Furthermore, both types of conjugates independently predicted CD11b expression (adjusted p<0.05).

Conclusions: ESRD is associated with increased binding of EMP to leukocytes, resulting in normal levels of some species of free EMP. Therefore, measurement of both free EMP and EMP-leukocyte conjugates is necessary for adequately assessing EMP release in ESRD. Levels of EMP bound to neutrophils strongly predict neutrophil activation, suggesting that EMP induce neutrophil activation in vivo, consistent with in vitro studies. EMP may represent important mediators of the proinflammatory state in ESRD.

EMP and EMP-Leukocyte Conjugates in ESRD patients compared to normal controls ESRD (n=80) Controls (n=35) P value IQR=Interquartile range EMP31 (IQR) 842 (484–1546) 807 (566–1240) 0.97 EMP62E (IQR) 69 (39–128) 60 (43–113) 0.96 EMP54 (IQR) 75 (33–144) 34 (18–141) 0.04 EMP54-Lymphocyte Conjugates (±SD) 1.45±0.2 1.21±0.1 <0.0001 EMP54-Monocyte Conjugates (±SD) 1.77±0.6 1.27±0.3 <0.0001 EMP54-Neutrophil Conjugates (±SD) 2.12±0.6 1.85±0.6 0.02 EMP62E-Lymphocyte Conjugates (±SD) 1.30±0.2 1.13±0.1 <0.0001 EMP62E-Monocyte Conjugates (±SD) 1.61±0.5 1.40±0.3 0.01 EMP62E-Neutrophil Conjugates (±SD) 2.04±0.6 2.05±0.5 0.95
Blocking the SDF1/CXCR4 Axis Inhibits Marrow Derived Vasculogenesis in Cancer.
Authors: Russell Kathryn J. , Otero Jacklyn , Fu DongTao , Jorgenesen Marda L. , Scott Edward W. , Cogle Christopher R.
Published: April 2020

Cancer growth and metastasis depend upon a rich supply of oxygen and nutrients from blood vessels. Our previous work found that the hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in settings of physiologic repair (i.e., retinal ischemia) as well as tumor vasculogenesis. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. The SDF1/CXCR4 axis is pivotal for marrow cell homing and migration, thus was targeted in these set of experiments. To test this hypothesis, we transplanted green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice and then inoculated these mice with lung cancer (LLC). An experimental cohort of mice (n=4) received intra-tumoral anti-SDF1. A control cohort included mice receiving intra-tumoral PBS (n=4). Over the ensuing 14 days, tumors in the anti-SDF1 treated group grew at a much slower rate and to a much smaller size, if at all. After 14 days of injections and tumor growth, microvessel density was markedly decreased in the anti-SDF1 cohort compared to the control cohort. Moreover, marrow-derived tumor vasculogenesis was decreased in the anti-SDF1 treated tumors compared to controls (18% of vessels with marrow-derived endothelial cells vs. 26%). As a complementary approach, we used the same transplant and tumor model, but administered anti-CXCR4 in the experimental group (n=4), compared to controls receiving PBS injections (n=4). The tumors in the anti-CXCR4 group had the same growth kinetics as the control; however, had lower tumor microvessel density and markedly decreased marrow-derived tumor vasculogenesis compared to controls (12% of vessels with marrow-derived endothelial cells vs. 26%). In conclusion, perturbing the SDF1/CXCR4 axis inhibits marrow-derived tumor vasculogenesis and may represent an excellent target for future anti-neoplastic therapy.
In Vitro Characterization of Magnetic Resonance Imaging Contrast Agents for Molecular Imaging.
Authors: Yu Zhi , Grafe Michael , Meyborg Heike , Fleck Eckart , Li Yangqiu
Published: April 2020

The aim of this work was to evaluate the biological properties of one citrate-coated and two different dextran-coated paramagnetic particles with comparable size (iron core 4–10 nm).

Endothelial cells from humans and mice as well as human macrophages were incubated for different time intervals with different particle suspensions. The cellular uptake was semi-quantitatively measured using the Prussian blue staining and, in addition, by cellular iron content. Furthermore the effect of known inhibitors of endocytosis was evaluated. In addition, it was evaluated whether linking of monoclonal antibodies to dextran-coated particles can make them bind specifically to certain cell surface structures.

The results showed that the bEnd.3 cell line, human umbilical vein endothelial cells (HUVECs) and THP-1/macrophage cell lines internalize paramagnetic particles. The ranking of cellular uptake was: VSOP > CMD-coated particles >> CLIO. The carboxydextran-coated SPIO uptake by endothelial cells is reduced by colchicine (50%). Conversely, cytochalasin B down-regulates the endocytosis of citrate-coated particle. Our data imply that the major mechanism of uptake would be pinocytosis for the VSOP and phagocytosis for the carboxydextran-coated particle CMD. The different surface coating can influence not only the quantity of the internalization, but also the pathway of internalization. CLIO linked to CD40 antibodies or to CD62E antibodies bound significantly better than IgG-linked CLIO. This was true especially for the anti-CD40-CLIO constructs where fluorescence increased two fold. Comparable results were observed with anti-CD62E-CLIO constructs; however, increase in fluorescence was higher than with CD40 binding; it increased on 3.9-fold (median) and 4.5-fold (mean).

In conclusions, the binding of antibody-conjugated CLIO to the antigen-expressing cells was specific, with an affinity similar to that of the free antibody. Thus, it seems feasible to use antibody linked SPIOs for molecular imaging.
Prevalence of Anti-HLA and Anti-GPIIB/IIIA Allo-Immunization in Patients with Glanzmann Thromboasthenia: Experience of a Single Center.
Authors: Santoro Cristina , Rago Angela , Antenucci Angela , Chistolini Antonio , Conti Laura , De Vellis Annalisa , Di Renzo Lucia , Laurenti Luca , Perrone Mariapaola , Foa Robert , Mazzucconi Mariagabriella
Published: April 2020

Background. Platelet transfusions, the main therapy of Glanzmann Thromboasthenia (GT), can induce an allo-immunization against HLA antigens and GPIIb/IIIa complexes, with a possible reduction of efficacy of subsequent treatments.

Aims. To investigate the development of anti-HLA antigen and anti-GPIIb/IIIa complexe allo-immunization in GT transfused patients, and to evaluate the efficacy of replacement therapy.

Patients and Methods. From 1975 onwards, we have followed 17 GT patients; 13 type I, 3 type III, 1 not classified; 8 men, 9 women; median age at diagnosis 9.8 years (range 1–44.5); the median age at the time of this study was 35.5 years (range 23.6–68.5). Our patients had shown at least once in their life the following symptoms: 10/17 epistaxis; 5/17 gastrointestinal hemorrhage; 5/17 oropharingeal hemorrhage; 4/17 muscle hematoma; 2/17 bleeding for traumatic injury; 2/17 hemarthrosis; 2/17 hematuria; 1/17 intracranial hemorrhage; 1/17 hematothorax; 1/17 otorrhagia. Five out of 9 women had experienced meno-metrorrhagia. Ten major and 22 minor surgical procedures had been performed. Two spontaneous deliveries and 3 cesarian sections with 5 live births had been reported; moreover, 2 abortions had occurred, 1 spontaneous and 1 voluntary. Globally, 9/17 patients had been transfused with platelets and red blood cells (RBC); 5/17 only with platelets; 2/17 only with RBC. One patient has never been transfused. Platelet transfusions have always been hemostatically effective. All transfused patients have been investigated for anti-HLA and anti-GPIIb/IIIa allo-antibodies,.

Results. Positivity for allo-antibodies could be demonstrated in 4/15 patients (27%): isolated for anti-HLA in 2; isolated for anti-GPIIb/IIIa in 1; combined in 1.

Conclusions. The prevalence of allo-immunization (27%) is inferior to recent literature data (50%). While positivity for anti-HLA (3/15, 20%) agrees with the recently reported data (22%), positivity for anti-GPIIb/IIIa (13%) is inferior (35%). Presence of allo-immunization did not compromise the efficacy of platelet transfusions.
Assessment of Menorrhagia in Females Followed for Bleeding Disorders at the Hemophilia Center of Western New York-Correlation between Clinical Presentation and Laboratory Parameters.
Authors: Bush Eric E. , Kepner James , Smiley Shannon , Belling Linda , Bernstein Zale P.
Published: April 2020

Menorrhagia is a common problem among women. The cause of which, in some individuals, has been attributed to von Willebrand’s disease. The assessment of this condition has been hampered by the lack of easily available and consistent laboratory assessment of levels of this factor that correlate with the patient’s clinical presentation. Therefore we initiated a study to correlate the patient’s self assessment of their menorrhagia with their levels of von Willebrand’s factor (VWF) and platelet function. A peripheral blood assessment chart (PBAC) was utilized for patient self-assessment of blood loss during their menstrual cycle. The PBAC is a validated, well-established method which utilized a point system that quantifies the number and extent of soiling of tampons and pads used during a patient’s menstrual cycle. The PFA-100 has been used as an adjunct for lab assessment of platelet function. This is a high shear-inducing device which simulates primary hemostasis after injury to small vessel. This apparatus consists of a reservoir for whole blood and a small capillary surmounted by a collagen-coated membrane with a central aperture. Platelet agonist which is either epinephrine or ADP is present on the membrane. Closure time is reported as a variable which inversely corresponds with von Willebrand factor levels (or platelet function). Clinical correlation with its results are needed. In addition, blood samples were analyzed for Von Willebrand’s factor antigen (VWFA) and Ristocetin co-factor activity (RCOA) using a CLIA approved laboratory. We explored the relationship between PBAC, PFA-100 and VWFA and RCOA levels. Twenty-six patients were enrolled after obtaining an institutional board approved informed consent. Pearson association estimates and P values to assess the association between one month PBAC scores and PFA-100 showed 0.463 (with a p-value of 0.017). A similar analysis between one month PBAC scores and VWFA and RCOA showed 0.099 (p=0.632) and the association between PFA-100 and VWFA and RCOA showed an inverse relationship of −0.481 (p=0.013). These results confirm a correlation between VWFA and RCOA and assessment of platelet function utilizing a PFA-100. Furthermore, there was a correlation between PBAC scores and the results of the PFA-100. The association between PBAC scores and VWAF screening levels did not show significance. These results suggest further data are necessary to understand the relationship between these variables and further suggests that the PFA-100 may offer a more sensitive assessment of platelet function that correlates with clinical presentation than levels of VWFA or RCOA.
Stability and Sterility of Sucrose-Formulated Recombinant Factor VIII (Kogenate® FS/Bayer) for Use during Continuous Infusion.
Authors: Regan Lisa , Chew Jordan , Vo Khang , Leveque Joyce , Khabbaz Naji , Gorina Eduard
Published: April 2020

Continuous infusion of factor VIII (FVIII) is an alternative treatment approach to bolus dosing for providing hemostatic control in patients with hemophilia A undergoing surgery. Continuous infusion maintains consistent circulating FVIII, thus avoiding wastefully high or dangerously low levels. Potential advantages of continuous infusion include improved protection from excessive bleeding, reduced factor consumption, and easier monitoring. Continuous infusion requires FVIII usage parameters that are typically outside of the tested and approved ranges for the product, including extended time in an aqueous state at room temperature and exposure to a large surface area of tubing. Here we present in vitro data on the stability and sterility of a sucrose-formulated recombinant FVIII product (Kogenate® FS/Bayer; rFVIII-FS) under conditions comparable to those that would be experienced during the continuous infusion of patients. Three lots for each of 3 vial sizes (250, 500, and 1000 IU) were tested. Twenty vials of each size were reconstituted in Water for Injection, pooled into a 50 mL volume (100, 200, or 400 IU/mL for the 250, 500, and 1000 IU vial sizes, respectively), and aseptically transferred to pump reservoirs. Two mini-pumps were evaluated (WalkMed 350 Ambulatory Infusion Pump and CADD-Legacy PLUS Ambulatory Infusion Pump Model 6500) using a flow rate of 0.6 mL/hr. Reservoir bags containing reconstituted rFVIII-FS, pumps, and tubing were incubated at 30°C and samples were withdrawn to test coagulation activity via one-stage and chromogenic assays after 0.5, 1, 2, 3, 6, 12, 24, and 48 hours. Sample remaining in the reservoir after 48 hours was tested for sterility. The reservoirs and tubing used in this study were made from polyvinyl chloride (PVC), which has a high FVIII adsorption potential. Results for all 1000 IU lots based on the recoveries obtained at the first time point demonstrated >80% activity at the end of the study using both the one-stage and chromogenic assays on both pumps. Results for all 500 IU lots showed similar recoveries (≥80%) for both pumps at study end using the chromogenic assay. However, the one-stage assay gave higher recovery values for the WalkMed pump (>90%) compared to the CADD pump (>75%). Results for all 250 IU lots demonstrated ≥79% activity at the end of the study using both pumps and both assays. All lots passed the sterility test. These findings indicate that rFVIII-FS is stable in both the WalkMed and CADD pumps and that sterility of rFVIII-FS is maintained for at least 48 hours. This suggests that rFVIII-FS is suitable for use in continuous infusion.
The Effect of the Order of Draw of Evacuated Blood Collection Tubes on Coagulation Parameters in Normal Volunteers.
Authors: Wakeman Lisa J. , Munro Roger C. , Al-Ismail Saad , Benton Ann , Beddall Andrew , Dorward Nick , Morris Keith
Published: April 2020

Introduction: The recommended order of draw for multiple tube collections (NCCLS [CLSI] H3-A5) clearly indicate that citrate tubes for coagulation tests should be taken before any other tubes (except blood cultures) and that a discard tube should be used if specialised coagulation tests are to be performed. This is to avoid the possibility of tissue activation and the theoretical risk of additive carryover on the parameters being measured. Because of the paucity of published evidence, this study was performed to determine the effect of the order of draw and whether the use of a discard tube is really necessary.

Methods: Three consecutive early morning venous samples were collected into siliconised glass B–D Vacutainers containing tri-sodium citrate (Ref: 367691) from 116 healthy laboratory personnel (F= 74; M = 42) aged 20–63 yrs. Age groups were equally represented. Samples were processed on a Sysmex CA1500 analyser within 1 hour of collection. Appropriate CLSI guidelines were followed throughout. All parameters were measured using Dade-Behring reagents: Activated partial thromboplastin time (APTT) (Actin FSL), prothrombin time (PT) and derived fibrinogen (DF) (Innovin), thrombin clotting time (TCT) (Thromboclotin) and Clauss fibrinogen (CF) (Bovine thrombin and Owren’s veronal buffer). For each parameter, the data from each of the three samples were analysed for significant differences by one way analysis of variance (ANOVA).

Results: Data obtained on measurements of basic coagulation parameters are shown in the table below. SDs are shown in parenthesis. (ns = not significant).

Coagulation Parameter Results and Statistical Analysis Parameter First Sample Second Sample Third Sample ANOVA (p) ns=not significant APTT (secs) 28.3 (1.73) 28.3 (1.73) 27.9 (1.64) 0.230 (ns) PT (secs) 10.9 (0.47) 10.9 (0.47) 10.8 (0.45) 0.368 (ns) TCT (secs) 15.8 (1.03) 15.8 (1.02) 15.7 (1.02) 0.740 (ns) DF (g/L −1) 2.44 (0.54) 2.47 (0.55) 2.48 (0.55) 0.866 (ns) CF (g/L −1) 3.03(0.67) 3.04 (0.67) 3.10 (0.67) 0.825 (ns)

No statistically significant differences were found between the first, second or third samples for any of the measured parameters.

Conclusions: The CLSI recommends an order of draw for evacuated blood collection tubes in order to reduce the possibility of tissue activation in coagulation samples and the theoretical risk of additive carryover on the parameters being measured. Until now, this was based largely on theoretical probability. This comprehensive study demonstrates that the use of a discard tube is probably unnecessary since there is no statistical difference in any of the parameters measured between the first, second or third samples. Although this potentially obviates the expensive use of a discard tube in normal subjects, further work is required to determine whether it is necessary when measuring abnormally prolonged parameters in various pathological states.
Unexpected Harmful Side Effects Triggered by Immune Modulation.
Authors: Sternbach Marion , Dragomir Alina , Crowther Mark A. , Holbrook Ann
Published: April 2020

A 66 year old man was referred urgently to Hematology for severe, spontaneous bruising, bleeding and acute onset anemia. His Past History revealed long standing, poorly controlled rheumatoid arthritis, ischemic cardiac disease with a prior MI, hypertension and elevated Homocystein. Medications included: Leflunomide, Hydroxychloroquine, Ramipril, Bisoprolol, vitamins B12 and B6 and the recently initiated Adalimumab (Humira), an anti-Tumor Necrosis Factor (TNF) immune modulator. On examination he was covered by extensive ecchymoses, large hematomas and had a knee effusion, feeling tired and light headed. He denied any injury. Hemostatic screening showed a normal PT and a markedly prolonged PTT, first 71, later over 100 seconds. A mix of patient and control plasma did not correct the PTT. An inhibitor was detected. Factors XI and IX were normal, Factor VIII was less than 1%. Factor VIII inhibitor was 60 Units Bethesda. White cell and Platelet counts were normal. Hemoglobin was 91 g/l from a previous 120.

He was treated with Prednisone and Cyclophosphamide as well as Hexacapron. Activated Factor VIIa was on standby and was used on one occasion for a huge lower lip hematoma oozing intractably, that required also topical Aprotinin.

Investigations for an underlying lympho-proliferative disorder or solid tumor neoplasia were negative. His complete response to chemotherapy took several months.

Factor VIII inhibitors in non-hemophiliacs are the commonest autoantibodies against clotting factors. Their incidence is 0.2 to 1.0 case per million per year and mostly associated with hematological neoplasias, solid tumors and post-partum. This appears to be first occurrence of Anti-Factor VIII triggered by this type of immune modulation.

Since Monocyte-Macrophages are a major source of TNF, it is possible, that by the interaction of Adalimumab, as Anti-TNF and the TNF receptor, the antigen presenting cell becomes altered, permitting in a patient with underlying autoimmune disease, a monoclonal B-Lymphocyte expansion with anti-Factor VIII properties.
Factor VII Deficiency - A Restrospective Case Review.
Authors: Katz Randi J. , Steinberg Amir , Klafter Robert
Published: April 2020

Factor VII deficiency is a rare autosomal recessive disorder. Its incidence is thought to be 1 in 500,000. Symptoms vary from mild to severe. Factor VII deficient patients usually do not experience bleeding if their level of factor VII is less than 10%. Manifestations are seen with levels less than 5%, and severe bleeding can occur with levels less than 1%. Surgical hemostasis is obtained with levels greater than 25%. Factor VII deficiency is notable for causing a prolonged PT with a normal aPTT, making the diagnosis easy to determine.

This is a descriptive study based on a population of patients from our community based teaching hospital with severe Factor VII deficiency. We reviewed lab data and found any patients with less than a 5% factor deficiency from March 2004- June 2006. In our experience we have noted varying manifestations in moderate factor deficiency patients. We describe four patients with varying symptoms, all with factor levels less than 3%. Their charts were reviewed, and three of the patients were available for a telephone interview.

Patient A is a 75 yo African American female found to have factor deficiency after an increased Prothrombin time (PT) of 31.2 seconds, INR 5.9, and Partial thromboplastin time (PTT) of 34.9 seconds on routine blood work. This patieny’s factor level was found to be 1.86%. Patient A denied any history of bleeding complications, including an uncomplicated tooth extraction. Patient B is a 46 yo African American female with a long history of bleeding complications. Laboratory data revealed a PT of 26 seconds, INR 4.2, PTT 22 seconds and a Factor level of 1%. She experienced years of heavy menstrual periods and nose bleeds. A hysterectomy was performed secondary to bleeding fibroids. She required multiple doses of novoseven as well as FFP. Patient C was diagnosed at the age of 11. Laboratory results revealed a factor level of 1.87 %, and a PT of 25.1 seconds and an INR of 4.1. She has had minor bleeding events, such as gum bleeding. She did receive FFP prior to a foot surgery, and tooth extraction. Patient D is a 46 year old Hispanic man found to have and INR of 4.3 and, factor VII 1.45. Pt had an uncomplicated cholocystectomy.

This is a descriptive study of four different patients with factor VII levels of less than 2%. These four cases demonstrate the wide range of clinical manifestations that factor VII patients may experience. One patient did require novoseven prior to a hysterectomy. Though, as our cases illustrate, some patients do not experience any bleeding manifestations, and therefore, prophylactic plasma may not be required. Furthermore, treatment of factor VII deficient patients needs to be individualized, and guided by personal history.

Coagulation Studies of our Patients Patient Prothrombin Time INR Factor VII A 31.2 5.9 1.86% B 26 4.2 1% C 25.1 4.1 1.87% D 46 4.3 1.45%
Vascularization of the Synovial Membrane in Hemophilic Synovitis.
Authors: McClain Ramona , Hakobyan Narine , Valentino Leonard A.
Published: April 2020

Hemophilia is due to the deficiency of the activity of coagulation factor VIII or IX and results in bleeding. Hemarthrosis frequently leads to synovitis and arthropathy in adolescents and adults with severe disease. The molecular basis for this disorder is only recently being interrogated in detail (Wen, Jabbar et al. 2002; Hakobyan, Kazarian et al. 2004; Valentino, Hakobyan et al. 2004; Hakobyan, Kazarian et al. 2005). In addition to proliferation of synovial fibroblasts, vessels are prominent and likely play a fundamental role. We hypothesized that synovial hyperplasia was limited by the degree of tissue vascularization and a direct relationship between the expansions of these two tissues existed. To test this hypothesis, mouse knee joints were injured by blunt or surgical trauma to induce hemarthrosis or blood was directly injected into the joint capsule to induce synovitis then the knees isolated and the tissues examined by seven reviewers blinded to the experimental conditions for the extent of synovial and vessel hyperplasia (Valentino and Hakobyan 2006, in press) and their individual scores averaged. The distribution of the scores for the 271 sections is given in the table. A direct relationship (r2=0.90, p<0.001) between synovial and vessel hyperplasia was present (figure). It is likely that the synovial membrane can expand up to a point (grade 1) but beyond this, vessel hyperplasia is necessary and that progressive hypertrophy of the synovial membrane is dependent upon the development of vessels to provide oxygen and nutrients. The production of vascular trophic factors by synovial fibroblast cells is under investigation.

Types and numbers of hyperplasia Hyperplasia 0 1 2 3 Synovial 149 67 18 37 Vascular 180 58 19 14 Figure Figure