bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bone Marrow Biopsy and Aspirate Evaluation in 90 Patients with Essential Thrombocythemia Treated with PEG Interferon alpha-2b. Preliminary Results.
Authors: Gugliotta L. , Bulgarelli S. , Tieghi A. , Asioli S. , Gardini G. , Vianelli N. , Candoni A. , Latagliata R. , Rupoli S. , Martinelli V. , Zaccaria A. , De Biasi E. , Sacchi S. , Lauria F. , Miglino M. , Passamonti F. , Tabilio A. , Cacciola E. , Grossi A. , Pisapia G. , Favini P. , Baccarani M. 
Published: October 2019

Abstract
Ninety patients with Essential Thrombocythemia (ET) where object of a phase II prospective multicentre study designed to evaluate efficacy, safety and tolerability of a two years treatment with PEG Interferon α-2b (PEG Intron, Schering Plough). The patients, 30 M and 60 F, 18–72 years old (median 45), observed in 16 Hematological Institutions of the Gruppo Italiano Malattie Mieloproliferative Croniche (GIMMC), received the ET diagnosis according to the PVSG criteria. At PEG Intron treatment start the patients showed: previous cytoreduction 97% (IFN α 31%), platelet count >1000 x 109/L 81%, splenomegaly 22%. At the end of the first year The PEG Intron starting dose of 25 μg/week resulted increased to a mean value of 55 μg/week and the Hematological Response (HR = Plts <500x109/L) was registered in 79% of the patients still on treatment. At the end of second year 65 patient still receiving PEG Intron (mean dose 31 μg/week) showed a maintenance of the HR (66%), a Partial Response (17%) and a Minor Response (17%). By utilizing the data included in the study CRFs we preliminarily evaluated the bone marrow biopsy and aspirate both performed at baseline, after 1 and 2 years in 89 and 86, 79 and 67, 57 and 50 patients, respectively. Data concerning the bone marrow biopsies after 1 year of treatment are reported:
BONE MARROW BIOPSY BASELINE % 1 YR % 2 YRS % Cellularity increased 56 51 48 Granulopoiesis increased 51 54 39 Erytropoiesis increased 29 24 23 MK number increased 99 90 84 MK size increased 78 69 62 MK ploidy 54 51 42 MK dystrophy 52 56 59 Fibrosis mild 40 37 46 Fibrosis moderate 7 25 26
The increase of bone marrow fibrosis registered after one year (representative also of second year data) resulted not related to patient gender, age >45 years, platelet count >1000 x109/L, Hb <12 g/dL, splenomegaly, previous IFN treatment, PEG Intron dose >50 μg/week.
In conclusion, the present study shows that in ET patients a two years PEG Intron treatment, able to induce and to maintain the Hematological Response in the majority of cases, is associated to a decrease of bone marrow cellularity, granulopoiesis, erytropoiesis, MK number, size and ploidy and, moreover, with an increase of MK dystrophy and of bone marrow fibrosis. These preliminary data on bone biopsy and aspirate will be object of a planned centralized reevaluation by a Panel of Pathologists and Clinicians.
Possible Role of Rap1B in the Cross-Talk between Integrins α2β1 and αIIbβ3.
Authors: Guidetti Gianni , Bernardi Bruno , Balduini Cesare , Torti Mauro 
Published: October 2019

Abstract
Platelet adhesion to subendothelial collagen plays a key role in cell activation and thrombus formation. Two main receptors promote platelet binding to collagen, GPVI and integrin α2β1. It is now clear that both receptors are involved in signal transduction and platelet activation. Rap1B is a small GTPase highly expressed in platelets, and its activation is mediated by a number of agonists through multiple pathways involving PKC, Ca2+, as well as PI-3K. Although a role for Rap1B in integrin αIIbβ3 regulation has been documented, its involvement in integrin-mediated outside-in signaling and cross-talk between integrins has been poorly investigated. In this work we investigated the possible relationship between Rap1B and integrin αIIbβ3 activation mediated by platelet adhesion via integrin α2β1. Integrin α2β1-mediated adhesion has been investigated under static conditions. Monomeric collagen, decorin, and two different peptides obtained by digestion of collagen type II with CNBr (CB8 and CB11) have been used as integrin ligands. Rap1B activation in adherent cells has been evaluated by pull-down experiments using GST-RalGDS-RBD. Integrin αIIbβ3 activation in collagen adherent platelets has been monitored by measuring binding of biotinylated fibrinogen in a colorimetric assay. Integrin α2β1-mediated adhesion to monomeric collagen, decorin and collagen-derived peptides induced a rapid and sustained activation of Rap1B independently of secretion of ADP, production of thromboxane A2, or integrin αIIbβ3-dependent platelet aggregation. We further analysed the effect of several pharmacological inhibitors on Rap1B activation supported by integrin α2β1. Integrin α2β1-mediated platelet adhesion resulted in the activation of PLC, as revealed by the strong and sustained phosphorylation of pleckstrin. Inhibition of PLC by U73122 completely prevented Rap1B activation. We found that both PKC activation and intracellular calcium increase contributed to Rap1B activation in collagen adherent platelets. Platelet adhesion through integrin α2β1 also caused tyrosine phosphorylation of Src, Syk and PLCγ2. Inhibition of Src kinase by PP2 prevented tyrosine phosphorylation of PLCγ2, but had minimal effect on integrin α2β1-promoted pleckstrin phosphorylation. Moreover, inhibition of Src kinase by PP2 did not affect integrin α2β1-mediated Rap1B activation. Analysis of fibrinogen binding revealed that platelet adhesion via integrin α2β1 induces activation of integrin αIIbβ3 indicating a cross-talk between platelet integrin receptors. Binding of fibrinogen to integrin α2β1-adherent platelets was blocked by the same pharmacological inhibitors able to prevent Rap1B activation. By contrast, Src kinases did not appear to be involved in integrin α2β1-mediated integrin αIIbβ3 activation. These results demonstrated that Rap1B is activated downstream integrin α2β1-mediated platelet adhesion and suggest that it may be involved in the cross-talk between the collagen and the fibrinogen receptors.
Identification of Apolipoprotein E Receptor 2′ as the Low-Density Lipoprotein Receptor on Platelets.
Authors: Akkerman Jan-Willem N. , Korporaal Suzanne J.A. , Relou Ingrid A.M. , Van Eck Miranda , Bezemer Martineke , Gorter Gertie , Van Berkel Theo J.C. , Nimf Johannes , Lenting Peter J. 
Published: October 2019

Abstract
The interaction of platelets with low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis and thrombosis. Previously, we have shown that native LDL (nLDL) is a mild activator of platelets increasing their sensitivity to aggregation-inducing agents. Binding of nLDL to platelets was saturable, reversible and initiated signal transduction to p38MAPK suggesting the involvement of a receptor. A peptide mimic of the B-site in apoB100 resembled nLDL in its platelet-activating properties, suggesting that the receptor is a member of the LDL-receptor family. Platelets from familial hypercholesterolemia patients, who lack or have a defective apoB/E receptor, responded normally to nLDL and an antibody against this receptor left nLDL-induced activation of normal platelets undisturbed, excluding the involvement of the classical LDL (apoB/E)-receptor. In this study, we provide evidence that nLDL initiates platelet signaling to p38MAPK via a splice variant of the LDL-receptor family member Apolipoprotein E Receptor 2 (ApoER2′). This conclusion is based on (i) blockade of nLDL-induced p38MAPK activation by receptor-associated protein (RAP), an inhibitor of ligand binding to members of the LDL-receptor family, (ii) confocal microscopy showing a high degree of co-localization of nLDL and ApoER2′ at the platelet surface, (iii) binding of both nLDL and the B-site peptide to soluble ApoER2′, (iv) activation of p38MAPK by an anti-ApoER2 antibody with similar kinetics as nLDL, and (v) tyrosine phosphorylation of ApoER2′ upon binding of nLDL. The nLDL-induced phosphorylation of ApoER2′ could be abolished by PP1, an inhibitor of Src-like tyrosine kinases. In the absence of PP1, ApoER2′ phosphorylation was accompanied by co-association with the Src-family member Fgr. We conclude that binding of nLDL to platelets involves ApoER2′. Upon nLDL binding, the receptor is phosphorylated which induces the recruitment of Fgr, a kinase known to activate p38MAPK. The ApoER2′-Fgr complex subsequently activates p38MAPK, an upstream element in the formation of thromboxane A2 that primes the platelets to further stimulation by aggregation-inducing agents.
A Soluble Truncated Form of the Mer Tyrosine Kinase Inhibits Gas6 Activity in Platelets and Monocytes.
Authors: Graham Douglas K. , Sather Susan 
Published: October 2019

Abstract
Several receptor tyrosine kinases generate soluble ligand binding domains either by proteolytic cleavage of the extracellular domain or differential splicing yielding in a truncated RNA transcript. Mer is a member of a receptor tyrosine kinase family which includes Axl and Tyro-3. All three members of this family have an extracellular domain which includes two immunoglobulin and two fibronectin type III motifs. Mer, Axl, and Tyro-3 are transforming in vitro in numerous cell types and are overexpressed in several different human cancers. In addition, Mer, Axl, and Tyro-3 and the common ligand Gas6 are all expressed in platelets and directly affect platelet activation and clot stability. We have found that the 205 kD Mer protein in platelets and monocytes is post-translationally processed by cleavage of the extracellular domain via a TACE-like metalloprotease. The cleavage results in a soluble extracellular domain protein (approximately 150kD) and a membrane-bound kinase domain. The proteolytic cleavage of Mer can be enhanced by LPS and PMA, and specifically inhibited by a TAPI, a TACE inhibitor. Significant amounts of the soluble Mer protein are present in human and mouse serum. The soluble Mer protein directly binds the ligand Gas6, and inhibits Gas6 stimulation of full-length Mer. Gas6, related to anticoagulant protein S, is also a ligand for Axl and Tyro-3. The cleavage of Mer may represent a mechanism of directly regulating the numerous functions of Gas6 including promoting platelet adhesion and clot stability, stimulating cell proliferation, inducing cell adhesion and chemotaxis, and preventing apoptosis. Furthermore, the ability of soluble Mer to inhibit Gas6 activity in platelets may provide a potential novel therapeutic intervention in the treatment of clotting disorders.
Ammonium Transport by the Erythrocyte Rh-Associated Protein.
Authors: Westhoff Connie M. , Dang Binh , Daniel Mak , Kevin Foskett J. 
Published: October 2019

Abstract
The erythrocyte Rh and Rh-associated (RhAG) proteins have distant sequence identity to a family of ammonium transporters found in yeast and bacteria. We previously showed that RhAG mediates movement of ammonium when expressed in yeast and in Xenopus oocytes. Importantly, these are the first mammalian proteins found to transport ammonium as a principal substrate. Elucidation of the mechanism and actual substrate(s) transported (protonated NH4+ or unprotonated NH3, or both) is important to understand their role in elimination of ammonium, proton recycling, and their impact on cellular pH and acid/base regulation. Functional characterization revealed that uptake was independent of the membrane potential and the Na+ gradient, but was dramatically stimulated by raising extracellular pH or lowering intracellular pH. This suggested that uptake was coupled to an outwardly directed H+ gradient and led us to hypothesize that RhAG might function by an H+-coupled, counter-transport mechanism. To further define the mechanism and actual substrate transported, RhAG-expressing oocytes were exposed to varying concentrations of NH4+ with constant NH3, and vice versa, by manipulation of the NH4Cl concentration and the pH of the buffer. A voltage-ramping protocol was used to evaluate changes in membrane conductance and reverse potential to measure membrane depolarization. Radioactive flux uptake of 14C-methylammonium, an analogue of ammonium, was used to measure transport. In the presence of substrate in the physiologic range (20 uM-500 uM), RhAG-mediated transport responded to the concentration of protonated NH4+ rather than the amount of unprotonated NH3 present. Currents in RhAG-expressing oocytes did not differ from water-injected controls. No significant changes in membrane conductance or membrane depolarization and reverse potential were observed. Taken together these data support a role for RhAG in the electronuetral transport of NH4+ by exchange with H+, and for erythrocytes in the maintenance of total blood ammonia levels. Sequestration of ammonium by erythrocytes would keep blood plasma levels low, preventing exposure of cells to toxic levels. Erythrocytes are ideally postioned to then transport ammonium to be exchanged in the liver and kidney, where other Rh-related proteins (RhBG and RhCG) are expressed.
L-arginine Administration Reverses Anemia of Renal Disease.
Authors: Imagawa Shigehiko , Tarumoto Takahisa , Kobayashi Makoto , Ozawa Keiya 
Published: October 2019

Abstract
Erythropoietin (EPO) gene expression is positively regulated by hypoxia-inducible factor-1, which binds to the 3′ enhancer of the EPO gene. EPO expression is negatively regulated by GATA, which binds to the GATA site in the 5′ promoter. Anemia of renal disease is believed to be caused by damage to the EPO-producing cells in the kidney as a result of renal failure. However, even mild renal failure can reduce the serum concentrations of EPO, suggesting that, in addition to the damage to EPO-producing kidney cells, uremic toxin(s) may play an important role in the pathogenesis of the anemia of renal disease. NG-monomethyl-L-arginine (L-NMMA), which competes with L-arginine as a substrate for nitric oxide synthase (NOS), is undetectable in nonuremic subjects, but is markedly elevated in patients with chronic renal failure. We have previously shown in the Hep3B cell line that L-NMMA suppresses EPO expression by upregulating GATA through the suppression of NOS activity, and that inhibition of EPO production with NG -nitro-L-arginine methyl ester (L-NAME) is reversed by pretreatment with L-arginine in mice. In this study, eight patients with anemia of renal disease, who were in the predialysis state of chronic renal failure, were enrolled in a nonrandomized study. All patients were anemic, and had hemoglobin (Hb) levels ranging between 7.9 and 10.8 g/dL. Patients with iron deficiency anemia, hemolytic anemia, and hematological malignancies were excluded. Blood urea nitorgen (BUN) levels were 29.7–90.1 mg/dL, and creatinine (Cre) levels were 1.1–3.8 mg/dL. Eight patients with anemia of renal disease were treated orally with L-arginine at 1.3 g/day for 8 to 22 wks. During this period, patients did not receive blood transfusions, or treatment with iron, vitamins or recombinant human EPO. Six patients responded to the treatment with increased levels of Hb and EPO, and three of these showed improved renal function. The EPO concentrations decreased in two nonresponders. There were no significant adverse effects. These limited data suggest that oral administration of 1.3 g/day of L-arginine can significantly improve EPO production and reverse anemia without adverse effects in patients with anemia of renal disease who are in the predialysis state of chronic renal failure. More studies including younger patients with severe renal dysfunction are in progress.
Incompatibilities of HA-1 and CD62L Polymorphic Adhesion Molecule Induce Graft-versus-Leukemia Effect Rather Than GVHD Resulted in Long-Term Survival in HLA Identical Myeloablative Stem Cell Transplantation.
Authors: Katagiri Takamasa , Shiobara Shintaro , Furukawa Tatsuo , Tsukada Jyunichi , Nakao Shinji , Wakano Miki , Muranaka Eriko , Harada Mine 
Published: October 2019

Abstract
Mismatches of minor histocompatibility antigen ( mHag) between HLA identical stem cell donor and host are known as a major risk factor for graft-versus-host disease (GVHD). We determined alleles of 5 polymorphic molecules including HA-1 and 4 adhesion molecules for 107 patients stem cell transplanted from HLA identical donors and investigated the association of their mismatches with relapse rate and GVHD. All of 107 recipients received stem cell transplantation ( SCT) after myeloablative conditioning regimen from 1978 to 2002, 89 received marrow, 15 received PBSC and 3 received both. Stage of the disease at SCT was 63 in standard risk and 43 in high risk. After SCT, 37 developed acute GVHD( ≥2), 50 developed chronic GVHD and 31 relapsed during at least 2 years follow up period.
We observed 16.1% of relapse rate in patients with at least one or more incompatibilities and 39.3% of relapse rate without incompatibilities (P= 0.018). Relapse rate of patients with CD62L, HA-1, CD31 at codon 563, CD31 at codon 125 and 49b incompatibilities are as followed 6.1%, 14.3%, 11.7%, 20% and 40% respectively. No difference of acute GVHD was observed in patients with and without incompatibilities. Among standard risk patients, 11 patients with HA-1 incompatibility showed 0% of relapse rate which was better than 45% (P= 0.003) in 47 patients without HA-1 incompatibility. In addition, 10 year survival rate was 100% in the former patients which was better than 52% of survival rate (p=0.03) in the latter patients. These data suggests that polymorphic molecules of HA-1 and CD62L contribute to GVL effect rather than the developing of GVHD which resulted in long term survivor after HLA identical stem cell transplantation. Mhag analysis of donor and host in addition to HLA antigen will help a patient to find a suitable donor in HLA identical family and voluntary donor.
Figure Figure
Genetic Modifiers of Cerebrovascular Large Vessel Stenosis in Sickle Cell Anemia.
Authors: Driscoll M. Catherine , Devaney Joseph , Gordish Heather , Minniti Caterina , Hoffman Eric P. 
Published: October 2019

Abstract
Cerebrovascular disease is a common complication in sickle cell anemia (SCA) with overt stroke occuring in 10% of patients and silent stroke in 24% of patients by age 20 years. Etiologies of overt stroke in SCA are heterogenous and include stenosis of large arteries, hemorrhage, hypertensive and hypoxic encephalopathy. Candidate genes identified as possible modifiers of overt stroke in SCA include adenylate cyclase 9 (ADCY9, rs731471 intron C/T), endothelin-converting enzyme (ECE-1, rs212527, intron A/G), Klotho (KL, rs480780, intron C/A), plasminogen activator inhibitor-1 (PAI-1, rs1799768, − 675 4/5,), transforming growth factor beta receptor 3 (TGFBR3, rs284157, intron G/A), tumor necrosis factor alpha (TNF, rs1800629,-308 G/A), and interlukin 4 receptor (IL4R, rs1805015, 503S/P). We examined the association of these candidate genes in a cohort of patients with SCA and stenosis of large cerebral vessels who are followed at a single institution. Patients with stenosis (n = 42, male = 22) were identified as having overt stroke (n = 29) or positive transcranial Doppler (TCD) (n = 13). The mean age at time of stroke or (+) TCD was 7.8 years. All had large vessel stenosis on magnetic resonance angiogram imaging (MRA). A control group of patients with SCA and normal TCD were identified (n = 71, males = 46, mean age = 14.8 years).This control group has passed through the peak years of stroke risk, ages 2–10 years. Single nucleotide polymorphisms (SNPs) were genotypic using novel Taqman assays. Genotypic associations were determined using logistic regression analysis with odds ratios (OR), 95% confidence intervals, and p-values reported. Among the candidate genes only ADYC9 (OR = 2.75, CI 1.2–6.3, p = 0.017) and TGFBR3 (OR = 2.33, CI 1.0–5.3, p = 0.043) had significant association with stenosis of large vessels in SCA. ADYC9 is a membrane-associated enzyme that catalyzes cAMP formation, which is critical for neuronal signaling and survival. TGFBR3 has a role in cardiac endothelial cell transformation to mesenchyme. Genotypic studies of a complex trait such as stroke in SCA will eventually lead to early diagnosis and new or early interventions. However, such genetic studies require clear definitions of phenotypic subtypes based on neuroimaging studies, including MRA.
Plasma ‘Free’ HB Is Related to Red Cell Derived Vesicle Numbers in Sickle Cell Anemia and Thalassemia Intermedia: Implications for Nitric Oxide (NO) Scavenging and Pulmonary Hypertension.
Authors: Westerman Maxwell , Pizzey Arnold , Hirschman Jocelyn , Cerino Mario , Eze Ada , Ramoton Priya , Weil-Weiner Yonit , Porter John 
Published: October 2019

Abstract
Plasma hemoglobin (Hb) is a scavenger of nitric oxide (NO), which is a likely contributor to the pathogenesis and treatment of clinical abnormalities such as pulmonary hypertension and vasocclusive episodes in sickle cell anemia (SCA) and possibly in thalassemia syndromes, where pulmonary hypertension has been described most frequently in splenectomised patients with thalassemia intermedia (TI). Since plasma Hb consists of both free Hb and RBC-derived microvesicle Hb (Greenwalt. Vox Sang1991;61:14), the relationship between plasma Hb and circulating vesicles may be significant. We have measured plasma Hb and plasma vesicle levels in adults with SCA and thalassemia intermedia (TI). Patients with SCA were all untransfused and TI patients had no transfusions during the previous 3 months. Plasma Hb values in healthy adult controls (1.77±0.2, n=7) were significantly lower than in SCA (11.21±2.08mg/dl, n=15, p=0.003) or in splenectomised TI patients (48.46 ±3.66mg/dl, n=5, p=0.0038). Plasma Hb levels were significantly greater in TI as compared to levels in SCA (p=0.001). Vesicle numbers in SCA were 12.59±3.65 x103/ul (n=21, p< 0.001, SCA v control) and in TI were 24.19 ±12.23 (n=7, p< 0.001, TI v control). Thus both plasma Hb and circulating vesicle levels in SCA and TI were significantly greater than healthy controls. Plasma Hb was significantly greater in TI than in SCA and circulating vesicles markedly greater in TI than in SCA. Furthermore there was a significant correlation between plasma Hb and vesicle numbers in SCA, TI and normal controls (n=26, R2=0.59, p=0.0015). An analysis of splenectomised versus non-splenectomised TI patients revealed a further trend; both plasma Hb and vesicle numbers were significantly higher in splenectomised (n=5, n=7 respectively) than in non-splenectomised patients (n=4, n=4 respectively). Plasma Hb and vesicle numbers were respectively 48.5±3.6 and 24.19±12.2 in splenectomised patients compared to 17.18±5.58 (p=0.014) and 4.36±0.81 (p=0.008) in non-splenectomized TI patients. These findings show that raised plasma Hb levels are related to increments in vesicle numbers in TI and SCA. Splenectomy in TI, which is associated with increased risk of pulmonary hypertension and thrombosis, is associated with increased vesicle numbers and plasma Hb. We suggest that the scavenger characteristics of Hb containing vesicles for NO may differ from the scavenger characteristics of free Hb and thus require detailed study..