bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Leukemia-Inducing Genes Using the Retroviral cDNA Library
Authors: Jang Suhwa , Lee Young , Kim Byoung Kook , Chung Hee Yong
Published: April 2020

Acute myeloid leukemia is a disease caused by oncogenic change(s) of hematopoietic stem cells or progenitor cells. The purpose of this research is to characterize the gene involved in the leukemia induction processes by screening the retroviral cDNA libraries of cellular oncogenes, homeobox genes. The cDNA libraries were constructed by cloning the individual genes to a MSCV retroviral vector backbone. For convenience of detecting the transduced cells and their protein products, the MSCV retroviral vector was modified to include HA tag and GFP marker and ninety cellular oncogenes and 30 homeobox genes were individually cloned and the structural integrity was verified. To screen for the leukemia-inducing genes, 5-FU treated mouse bone marrow cells were transduced with retroviral mixtures of oncogenes, and the cells were transplanted into ten lethally irradiated mice. All ten mice developed acute leukemia between the eight and ten weeks post-transplantation. The oncogenes that were responsible for the leukemia induction were characterized by genomic DNA PCR of the leukemic cells of each mouse. Surprisingly, all ten mice had c-myc genes in their leukemic cells. However, except for three mice, all the mice have additional oncogenes within the leukemic cells. The list of the additional genes include; RAB3D, RAB7B, PDGF-beta, CRK, and PIM-2 and Ras. Theses results show that the c-myc is the major leukemia-inducing oncogenes in our system. In addition, since the initial transduction rate of 5-FU-treated bone marrow just prior to in vivo transplantation was 10.56%, it is highly unlikely that all these additional oncogenes were present in the transplantable leukemic cells just by chance. Therefore, the retroviral cDNA library-mediated leukemia induction system we developed may be an useful system in systematically screening the cooperating oncogenes in leukemia induction. We are currently verifying the cooperating potential of the genes co-transduced with c-myc in inducing leukemia in the same animal model.
Risk Determinants of Arterial Thrombosis Increase the Risk of Intrauterine Growth Restriction (IGR)
Authors: Gerhardt Andrea , Scharf Rudiger E. , Zotz Rainer B.
Published: April 2020

Background: The HPA1-b allele of the beta3-subunit of the essential platelet integrin AlphaIIb-beta3 is a risk factor for increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess hereditary risk factor for arterial thrombosis in addition to the known risk determinants for venous thrombosis as risk determinants for fetal IGR.

Materials and Methods: 116 women with severe fetal IGR (defined by birth weight below the 5th-percentile for gestational age and sex) and 300 normal women were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia, neonatale immunthrombocytopenia) were excluded. The fetuses were born alive after the 24th week of gestation.

Results: A significant risk association could be shown for the HPA-1b/1b genotype OR 3.1 (95%CI 1.2–8.8), increased levels of lipoprotein (a) (>0.2g/l) OR 1.7 (95%CI 1.0–2.9), increased levels of fibrinogen (>median 284mg/dl) OR 2.8 (95%CI 1.8–4.4) increased levels of FVIII:C (>median 147%) OR 1.7 (95%CI 1.1–2.7), and increased levels of von-Willebrand-factor antigen (vWF-Ag) (>median) OR 2.7 (95%CI 1.1–2.7). The combination of risk determinants led to a further increase in risk (e.g. HPA-1b allele, increased lipoprotein (a) and vWF-Ag OR 14.0) There was no significant risk association for factor V Leiden G1691A OR 1.3 (95% CI 0.7–2.81), and prothrombin G20210A mutation. OR 2.0 (95% CI 0.6–6.5)

Conclusions: Increased platelet thrombogenicity via interaction with an inflammatory vascular process and/or via plasma levels of components of hemostasis/receptor ligands appear to be more important in women with IGR than the established risk determinants for venous thrombosis.
Irinotecan and Increased Triglyceride Level in Paraneoplastic Deep Venous Thrombosis (DVT): A Strange Link in An Old Problem
Authors: Giordano Giulio , Farina Giuliana , Tambaro Rosa , Piano Silvia , Papini Stefano , Zappacosta Bruno , Storti Sergio
Published: April 2020

DVT is present in about 3–15% of cancer patients. Paraneoplastic thrombosis pathogenesis is multifactorial. Chemotherapy is frequently involved in DVT genesis as pro-inflammatory/pro-thrombotic factor. There are few data about incidence of DVT in patients treated with Irinotecan. Aim of our study is to define if thrombotic risk in patients treated with Irinotecan is increased and then which mechanism is probably involved. For this purpose we considered in our patients the complement fractions C3 and C4 and the immune circulating complex (ICC), because they activate macrophages and platelets and increase tissue factor level. Moreover we evaluated also total cholesterol and triglyceride level, cause linked with factor VII activation. We considered C3, C4, ICC, cholesterol and triglyceride level, dose density and chemotherapeutic agents used in 116 patients with solid neoplasms (62colon, 26lung, 18gastric,) and not receiving anticoagulant prophylaxis. Median age was 68.5 years (R 57–83). M/F ratio was 66/40. The threshold value of third quartile was chosen as risk cut-off (C3: 130mg/dl; C4: 32mg/dl; ICC: 2.9 mcg/ml; total cholesterol: 205mg/dl; triglycerides 120mg/dl). The statistical analysis was conducted with Yates corrected chi square test, Fisher’s exact test, Odds Ratio (OR), relative risk (RR). Twenty-four patients (20%) developed DVT. The patients treated with Irinotecan were 42. DVT occurred in 14 of these patients (32%) and in 10 of the 74 patients treated without Irinotecan (14%), with a Yates corrected chi square test of 4.6 (p=0.03), a Fisher’s exact test with p=0.03, an OR of 2.98 (95%CI 1.2–7.3) and a RR of 2.35 (95%CI 1.1– 4.8). C-Reactive Protein and Erythocyte Sedimentation Rate at Pearson’s test were not related with C3, C4, ICC, total cholesterol and triglyceride levels. All patients treated with Irinotecan had advanced stage colon cancer. High levels of triglycerides (>120mg/dl) were present in 17 out of 42 patients treated with Irinotecan (40%) and in 17 out of 74 patients treated without Irinotecan (23%) with a Yates corrected chi square test of 3.1 (p=0.07), a Fisher’s exact test with p=0.05, an OR of 2.2 (95%CI 1–5) and a RR of 1.7 (95%CI 1–3). Although most patients developing DVT have an advanced stage colon cancer and so receiving Irinotecan, the influence of chemotherapy in DVT pathogenesis in our study cannot be excluded. Probably Irinotecan could contribute to DVT increasing triglyceride levels that are linked with in vivo factor VII activation. This relief suggests also that risk factors, other than classical, should be considered in the thrombosis onset; then other drugs (i.e. hypolipemizing drugs) could be useful in paraneoplastic DVT prevention. Nevertheless these data should be confirmed in a larger cohort of patients.
Platelet Reactivity Is Associated with VAMP8 Expression and a VAMP8 3′UTR Polymorphism
Authors: Kondkar Altaf A , Dajiang Liu , Leal Suzanne M , Nagalla Srikanth , Jin Ying , Khan Farhan , Bray Paul F
Published: April 2020

Variations in platelet reactivity can promote either a hemorrhagic or thrombotic phenotype, and platelet granule secretion is essential for platelet activation and normal hemostasis. We have earlier rigorously phenotyped platelet reactivity in 337 healthy subjects and found excellent reproducibility for secretion-dependent platelet functions. Vesicle associated membrane protein (VAMP)-8, a v-SNARE, is the primary regulator of platelet secretion. Our goals were to determine whether the expression of 9 key molecules involved in the platelet secretory pathway – SNAP23, SNAP25, VAMP3, VAMP8, STX2, STX4, STX7, STXBP3, and RAB27B are associated with the well-known variability in platelet reactivity and investigate potential mechanisms. Leukocyte-depleted platelets were prepared from 10 subjects with hyperreactive platelets and 6 subjects with hyporeactive platelets. No significant variations were observed for SNAP25, STX2, STX4 and RAB27B mRNA levels. Although small variations were observed for other genes examined VAMP8 mRNA levels showed the highest variation. VAMP8 mRNA levels were 4.8-fold higher in hyperreactive platelets than hyporeactive platelets (P=0.0023). Immunoblotting showed that VAMP8 proteins were also significantly elevated by 2.5-fold in the hyperresponsive subjects (P = 0.04). An A-to-G transition in the 3′UTR region of the VAMP8 gene (rs1010) has been consistently shown to be associated with a high risk of myocardial infarction (MI) in European Americans [EA]. We genotyped all 337 subjects (182 EA and 155 African Americans [AA]) in our cohort for this rs1010 SNP. The ‘G’ allele frequency was 0.44 and 0.62 in EA and AA, respectively. The ‘G’ allele was associated with enhanced aggregation to epinephrine (P=0.001) and ADP (P=0.001) in an age dependent manner. The 3′ UTR of VAMP8 has at least 10 predicted microRNAs (miRNAs), 8 of which we have identified in platelets and 5 of which encompass the rs1010 SNP. In summary, variable VAMP8 expression levels appear to be a major regulator of platelet reactivity, and a common VAMP8 SNP known to be associated with MI is associated with the platelet aggregation response to epinephrine and ADP. Studies are currently on-going to assess whether this SNP modifies miRNA binding and whether this miRNA regulates VAMP8 expression.
Caffeic Acid Phenyl Ester, a Component of the Chinese Herb Propolis, Inhibits Platelet Aggregation Via Competition with Fibrinogen for Binding to aIIbb3
Authors: Wu Ya Ping , Huisman Albert , Weeterings Cees , de Groot Philip G. , Zhang Yun Xiang , PAN Shao Dong , Wei Ran , Wang Jia Fu
Published: April 2020

Background: Propolis is a resinous substance collected by honeybees from various plant sources. Honeybees collect resin from cracks in the bark of trees and leaf buds, masticate it and the partially digested material is mixed with beeswax. The precise composition of propolis varies with the source. There is a long history of the use of propolis, at least to 300BC and its use continues till today. Propolis has a reputation to have a large number of healthy properties among others anti-thrombotic properties. Over 180 different compounds have been identified in propolis, among others caffeic acid phenyl esters (CAPE). We have investigated the effects of CAPE on platelet aggregation.

Results: Platelet aggregation was induced with ADP, collagen or the thrombin analogue TRAP. Different flavoid components present in propolis (CAPE, galangin, acacetin, pinostrobin) were able to inhibit platelet aggregation, CAPE was the most potent one, especially for the collagen induced platelet aggregation. We examined the effect of 100 mM CAPE on the expression of a number of platelet membrane activation markers after addition of collagen and we observed that CAPE inhibited fibrinogen binding and the binding of LIBS antibody against aIIbb3 for 93% and 82%, respectively, while the expression of P-selectin was only inhibited for 37%. To explain this discrepancy, we studied the binding of CAPE to purified aIIbb3 with a Biacore. CAPE binds to aIIbb3 with an affinity constant of about 400 μM. In an ELISA set-up CAPE was able to inhibit the binding of fibrinogen to purified aIIbb3 (figure).

Figure Figure

Conclusion: Propolis contains components such as CAPE that inhibit platelet aggregation via competition with fibrinogen for the binding to aIIbb3.
Interaction Between Genotypes for HFE and TFR2 Genes Mutations and Iron Status in Brazilian Blood Donors
Authors: Cancado Rodolfo D , Santos Paulo CJL , Rostelato Samuel , Terada Cristiane T , Gonzales Iris , Hirata Rosário DC , Hirata Mario H , Chiattone Carlos , Guerra-Shinohara Elvira M
Published: April 2020

Hereditary hemochromatosis (HH) is a disorder characterized by increased intestinal iron absorption, which leads to a progressive accumulation of iron in the body. This iron overload has been associated with mutations in HFE gene (C282Y, H63D and S65C) and other genes. The objectives of this study were to assess the frequencies of functional mutations in HFE and TFR2 genes and to investigate their relationship with the iron status in a sample of blood donors. Blood donors (n=542) were recruited at the Hemocenter of the Santa Casa Hospital, Sao Paulo, Brazil. The genotypes for HFE (C282Y, H63D and S65C) TFR2 (Y250X and Q690P) gene mutations were evaluated by PCR-RFLP. The concentrations of serum iron and total iron-binding capacity (TIBC) were measured by automation system Advia®(Bayer Diagnostics) and serum ferritin by Axsym System®(Abbott Laboratories). The frequencies of HFE 282Y, HFE 63D and HFE 65C alleles were 2.1, 13.6 and 0.6%, respectively. The frequency C282Y allele (2.1%) in Brazilian blood donors is lower than that observed in blood donors from Northern Europe (5.1 to 8.2%, P<0.05). The TFR2 250X and TFR2 690P alleles were not found in these subjects. The iron status was similar between HFE genotypes in women. However, men carrying HFE 282CY genotype had higher serum ferritin and lower TIBC concentrations when compared to the HFE 282CC genotype carriers. HFE 282CY genotype was also associated with higher transferrin saturation in men who donated blood at the first time. Moreover, male donors with HFE 63DD plus 63HD genotypes had higher serum iron and transferrin saturation when compared to those with HFE 63HH genotype. A relationship between HFE CY/HH/SS haplotype and lower TIBC concentrations was also found in men. The HFE 282Y and HFE 65C alleles were rare while the HFE 63D was frequent in blood donors. The mutations in TFR2 gene were not found in this study. The HFE 282Y and HFE 63D alleles were associated with alterations on iron status only in male blood donors.
Intravascular Hemolytic Anemia with Acanthocytosis Following Alternative Treatment with Ozone, UV Light, and Perflurocarbon
Authors: Hymes Kenneth B. , Huynh Chan
Published: April 2020

Alternative medicine has become more common as patients seek approaches to diseases where traditional medicine has failed. Treatment with ozone has been purported to have benefits for a variety of infectious, inflammatory and neoplastic conditions. Treatment can be administered by the intra arterial, intravenous, intra rectal and subcutaneous routes as well as ozonated autohaemotherapy. We describe a 44 year old woman who received treatment at an alternative medical center for recurrent breast cancer including laetrile, perflurocarbon emulsion, high dose ascorbic acid, vitamin K and extracorporeal treatment of her blood with ozone and ultraviolet light. After receiving her second treatment, she presented to our hospital with a syncopal episode and was found to be anemic (Hb 4 gm/dl). The LDH was 8X> ULN, the serum haptoglobin was undetectable, and the reticulocyte count was increased. The peripheral blood smear showed aniscocytosis, poikilocytosis and polychromasia. There was also severe acanthocytosis. Heinz bodies were not detected. The patient refused blood products and after 48h of hospitalization, her Hb rose to 7gm and she was discharged. She did not return for follow-up evaluation.

Acanthocytosis has been associated with impairment of cholesterol membrane fluidity seen with acquired hepatic disease as well as some congenital diseases. Oxidative stress can lead to peroxidation of membrane phospholipids, and if the intrinsic repair mechanism of the RBC is overwhelmed, the RBCs transform into acanthocytes. To date, there has been no literature describing hemolysis associated with any of the individual treatments which this patient received; however the combination of treatments exposed the RBC’s to multiple oxidative stresses which might lead to lipid peroxidation and formation of acanthocytes. This case illustrates the potential problems that face clinicians when encountering patients who seek alternative therapy.
High Resolution Tertiary and Quaternary Structural Changes in Deoxy and Liganded Hemoglobin E
Authors: Hirsch Rhoda Elison , Malashkevich Vladimir , Balazs Tatiana C. , Almo Steven C.
Published: April 2020

Hemoglobin (Hb) E (β26 Glu→Lys), known to be more unstable than HbA in vitro, remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms. EE individuals exhibit a mild, chronic anemia while HbE-β thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac disease. To determine pathophysiological consequences originating from HbE, we created a transgenic mouse expressing exclusively human HbE with equivalent α- and β-chains [Chen et al., Blood 106(11):892a, 2005]. This mice model exhibited hematological characteristics similar to human EE individuals: microcytic RBC with low MCV, low MCH but normal MCHC; target RBC; mild anemia with low Hb, low HCT, elevated reticulocytes; and decreased osmotic fragility. These RBC parameters correlated with indications of mild RBC oxidative stress. Importantly, the transgenic mouse model allows us to obtain purified HbE for crystal growth and dynamic studies, without the presence of human HbA2 (co-expressed in human HbE RBC and difficult to separate). We now present the low [PDB 1YVQ] and high salt [PDB 1YVT] liganded COHbE structures (1.8Å resolution) at or near physiological pH. The known in vitro instability of oxy HbE and its propensity for oxidation may be explained by the substitution of the β26 side-chain with Lys resulting in a positive charge repulsion with consequential changes to β117, and other key stabilizing residues at the α1β1 interface that impart normal association and oxidative stability to HbA. The specific alterations observed in this microenvironment may be the key to HbE instability, given the findings by Adachi et al. (Biochemistry, 42:10252, 2003) that β116 in normal HbA plays a critical role in stabilizing α1β1 interactions and inhibiting oxidation. Additionally, the difference in water structure observed in each of our COHbE structures from crystals generated under different conditions clearly suggests that resolved water structure is dependent simply upon crystallization conditions, and caution must be taken in interpretation of thermal instability. The 1.6 Å structure of deoxy HbE [PDB 3DUT] (crystallization conditions: pH 6.8, 1.8M ammonium phosphate/ammonium sulfate/citrate) exhibits definitive quaternary change compared to the deoxy HbA structure obtained under similar conditions. These structural findings, explain in part, our recent spectroscopic findings of alterations in oxy and deoxy HbE-nitrite interactions.
Pharmacokinetics and Pharamcodynamics of Recombinant Human Thrombopoietin Following Single Injection in Rats
Authors: Krzyzanski Wojciech , Rekic Dinko , Gebreegziabher Asfiha G.
Published: April 2020

Background: Thrombopoietin (TPO) is the primary regulator of platelet production. Recombinant human thrombopoietin (rHuTPO) and its derivative have been reported as potent drugs in treatment of chemotherapy induced thrombocytopenia and idiopathic thrombocytopenic purpura. Due to severe immunogenic adverse affects clinical applications of rHuTPO have been limited. However, new TPO receptor agonists that lack this adverse effect are developed. RHuTPO exhibits nonlinear kinetics and complex dynamics both mediated by binding to TPO receptor. Studies of rHuTPO pharmacokinetics (PK) and pharmacodynamics (PD) will facilitate future use of new TPO receptor agonists.

Purpose: To analyze PK and PD of rHuTPO following a single IV bolus administration in rats.

Methods: Adult male Wistar rats of body weight 300–600 g received a single dose of rHuTPO (ProSpec Ltd) (2.4, 4.8 and 9.6 μg/kg) and a placebo via an intravenous injection in tail. Blood samples (100–200μL) were collected at various hours during 3 days for PK analysis and every day for consecutive 14 days for PD assessment. RHuTPO plasma concentrations were measured using ELISA (R&D Systems Inc). Platelet count was determined using hematology analyzer (BC-2800 VET, Mindray). Noncompartmental parameters such as area under concentration curve (AUC), clearance (CL), volume at steady-state (Vss), and terminal half-life (t1/2) were calculated by WinNonlin 5.2 (Pharsight). Percent increase over the baseline for platelet maximum (%MPC) and area under the platelet response curve (%AUCPLT) were applied as PD markers. Student t-test was used to compare the means between control and treatment groups (SAS 9.1, SAS Institute).

Results: CL decreased with increasing dose (0.185, 0.131, and 0.047 L/h/kg), similarly to Vss (3.28, 1.10, and 0.51 L). The t1/2 values were also dose dependent (13.2, 6.1, and 8.2 h). The %MPC values were 43, 77, and 45% for corresponding doses and the peaks occurred on days 5, 6, and 6 following rHuTPO administration. The %AUCPLT values were relatively insensitive to increasing doses (20, 23, and 13%). Both mean peak platelet counts and mean AUCPLT were significantly different (p < 0.05) from the control values with an exception of AUCPLT for dose 2.4 μg/kg.

Conclusions: RHuTPO exhibits nonlinear pharmacokinetics in rat. CL and Vss dependence on dose suggests receptor mediated drug disposition as a major reason of nonlinear PK. IV bolus doses 2.4, 4.8 and 9.6 μg/kg of rHuTPO moderately increase platelet count in rats that peaks on day 5–6 after drug administration. The observed lack of the PD marker increase with increased doses implicates that the applied doses are in a flat region of the dose-response curve. Compared to similar data reported for humans, rHuTPO is less efficacious in rats than in humans.
Traditional Chinese Whole-Body Acupuncture Mobilizes CD133(+)CD34(−) Cells
Authors: Moldenhauer Sonja , Burgauner Miriam , Hellweg Rainer , Lun Andreas , Kiesewetter Holger , Salama Abdulgabar , Moldenhauer Anja
Published: April 2020

The objective of this study was to analyze the effects of traditional Chinese acupuncture for spinal cord lesions on the mobilization of stem cells. Therefore, 14 healthy study participants were acupunctured and gave blood samples before, immediately after as well as 24 and 48 hours after acupuncture. At these time points, the frequency of CD133, CD34, CD4, CD14, CD19 and CD45 positive cells were determined by flow cytometry. Furthermore, serum concentrations of matrix metalloproteinases (MMP) 8 and 9, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), stroma-derived factor (SDF) 1 and granulocyte-colony stimulating factor (G-CSF) were measured by enzyme-linked immunosorbent assays. CD133(+)34(−) cells were significantly increased 48 hours after acupuncture (29.1±5.8% versus 11.4±4.8%, p= 0.015), which was paralleled by significant decreases of BDNF (7.5±1 ng/ml versus 10.8±1.6 ng/ml, p= 0.013) and MMP-9 serum levels (46.4±2.6ng/ml versus 42.5±2.4 ng/ml, p= 0.009). Values in non-treated controls were not affected. No changes in SDF-1, NGF, interleukin-6 and G-CSF concentrations occurred. In conclusion, acupuncture does mobilize non-hematopoietic CD133(+) cells.
Soluble Endoglin and Its Interaction with Adhesion Molecules in Patients with Thalassemia Intermedia
Authors: Kattamis Antonios , Lazaropoulou Christina , Siopi Maria , Kanavaki Ino , Goussetis Evgenios , Ladis Vassilios , Papassotiriou Ioannis
Published: April 2020

Endoglin (CD105) is an accessory protein of the transforming growth factor-β receptor system expressed on vascular endothelial cells. Mutations on the endoglin gene are associated with hereditary hemorrhagic telangiectasias (Osler-Weber-Rendu syndrome) and, thus, endoglin has been extensively studied in the context of this disease. Endoglin is highly expressed on endothelial cells in healing wounds, developing embryos, inflammatory tissues and solid tumors. It is a marker of activated endothelium, while its vascular expression is limited to proliferating cells. Previous studies have shown that the endothelial function is impaired in patients with Thalassemia Intermedia (TI). Oxidative damage resulting from hemolysis and iron load, leads to increased expression of the intercellular and vascular adhesion molecules-1 (ICAM-1 and VCAM-1) and impaired nitric oxide (NO) bioavailability. As endoglin plays a critical role in angiogenesis and dysregulation of its expression and/or activity has been implicated in multiple vascular diseases, we aimed to investigate the expression of endoglin and its correlation with factors of endothelial dysfunction in patients with TI. Thirty adult patients with TI were included in the study, while 20 healthy individuals served as controls. Soluble endoglin, soluble forms of adhesion molecules ICAM-1, VCAM-1, E and P-Selectins, thrombomodulin, von Willebrand factor, as well as NO and vascular endothelial growth factor (VEGF), were measured in patients and controls, by immunoenzymatic methods. The main results of the study are:

levels of endoglin, E-selectin, thrombomodulin, VCAM-1, ICAM-1 and VEGF in patients with TI (5.5±0.4 ng/ml, 91.4±20.0 ng/ml, 48.8±16.5 ng/ml, 1413.7±176.1 ng/ml, 658.8±34.6 ng/ml and 619.1±227.8 pg/ml, respectively) were higher compared to controls (4.9±0.3 ng/ml, 10.3±2.2 ng/ml, 4.0±0.6 ng/ml, 328.3±43.8 ng/ml, 107.6±26.5 ng/ml, 81.3±58.1 pg/ml), (p<0.01), endoglin levels in patients with TI correlated positively with concentrations of ICAM-1 (r=0.760, p<0.003), VCAM-1 (r=0.520, p<0.05), E-Selectin (r=0.790, p<0.0020, P-Selectin (r=0.530, p<0.04), while these correlations were absent in normal individuals.

Angiogenesis is a highly coordinated process in which VEGF, endoglin and hypoxia inducible factor 1 (HIF-1) play a pivotal role by coordinating interaction between endothelial cells, extracellular matrix and the surrounding cells. Taking into assumption that endoglin is a protective-repair tissue protein, our findings support the hypothesis that patients with TI exhibit increased degree of angiogenesis and endothelial regeneration, which are probably compensatory mechanisms in response to tissue hypoxia and damage.
CD44 Signaling Via PI3K/AKT and MAPK/ERK Pathways Protects CLL Cells from Spontaneous and Drug Induced Apoptosis
Authors: Herishanu Yair , Gibellini Federica , Njuguna Ndegwa , Keyvanfar Keyvan , Wiestner Adrian
Published: April 2020

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-cells. CLL cell proliferation and survival depends on host factors that are present in the tissue microenvironment. In–vitro these cells rapidly undergo apoptosis but can be supported by culture on stroma cells. The extracellular matrix is an important component of the microenvironment. Interactions between tumor cells and the extracellular matrix are, in part, mediated by CD44, whose principle ligand is hyaluronic acid. To explore a possible role of this adhesion receptor in CLL, we evaluated the effect of CD44 engagement on the survival of CLL cells and the induction of drug resistance. Dimerization of CD44 activated the PI3K/AKT and MAPK/ERK pathways and resulted in increased levels of MCL-1. There was no increase in MCL mRNA in CD44 stimulated cells, consistent with an effect on protein stabilization. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous apoptosis: CD44 stimulated CLL cells had a 46% increase (range 7% – 181%) in viability over the corresponding unstimulated control cells (n=20, p<0.0001). Furthermore, CD44 activation also protected from fludarabine-induced apoptosis: cell viability for isotype treated cells 76 ±6%, for isotype and fludarabine treated cells 47 ±18% and for CD44 stimulated and fludarabine treated cells 69 ±16% (p=0.005). CLL cells of the IgVH unmutated subtype expressed CD44 more highly than IgVH mutated CLL cells (MFI ratio 224 ±43 to 122 ±44, respectively, p<0.0001) and derived a more pronounced survival advantage from CD44 activation: % live cells (CD44 stimulated - control) 21 ±9% vs. 13 ±6%, respectively (p=0.04), which could contribute to the more progressive nature of this subtype. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44 activation. Furthermore, obatoclax was able to sensitize CLL cells to fludarabine. In conclusion, CD44 engagement protects CLL cells from spontaneous and fludarabine induced apoptosis. This prosurvival effect is mediated through PI3K/Akt and MAPK/ERK pathways and increased MCL-1 levels. Targeting either signaling pathway or MCL-1 with specific inhibitors could overcome the protective effect of the microenvironment in CLL and provides a rationale for combination therapy.
Relevance of α-Globin Genotyping in Patients Expressing HbH in Cord Blood: Prevalence of α-Globin Gene Deletions and α2-Globin Termination Codon Mutations in a Neonatal Hemoglobinopathy Screening Program
Authors: Lavallée Vincent-Philippe , Soulieres Denis , Weng Xiaoduan , Robin Louise , Girard Steven , Cornut Gilbert , Audette Marie-Lise
Published: April 2020

Introduction: α-Thalassemia results from an heterogeneous group of disorders involving the α-Globin genes. Common deletions are responsible for 95% of cases of α-thalassemia, whereas Constant Spring mutation (Hb CS), one of the known mutations involving the termination codon of α2-globin gene, is the most common non-deletional cause of α-thalassemia worldwide. Its prevalence has never been studied in occidental countries.

Patients and methods: as part of our neonatal hemoglobinopathy screening program, fetal cord blood samples are systematically tested by HPLC. When an abnormal Hb or HbH is found, α-Globin deletion is carried by multiplex-PCR. A two part observational study was done at our center based on the available data from our hemoglobinopathy screening program available since 2002. Firstly, we aimed to determine the level of Hb H present and to correlate it to a specific type of α-Globin gene deletion by m-PCR sequencing (table 1). 306 samples were analyzed and demonstrated a statistically significant difference between categories of α-globin gene deletions: 2 del cis vs 1 del, 2 del trans vs 1 del, 2 del cis vs 2 del trans (difference of means of 8.07, 4.33 and 3.75 respectively, P<0.001). With HbH ≤3.15% a CI of 95% excludes a condition other that 1 α -globin gene deletion. With Hb H ≤7.1%, a CI of 99% excludes 2 α -globin gene deletions in cis.

Table 1

Deletion/allelic distribution Hb H level (range) Hb H level (means) Std Dev 1 deletion 1.0 – 12.4 2.03 1.12 2 deletions cis - 10.1 2.16 2 deletions trans - 6.35 1.5

Our second goal was to establish the prevalence of α-Globin termination codon mutation in the fetal blood sampling containing ≥1% of Hb H in absence of α-Globin gene deletion by m-PCR. 395 fetal blood samples were analysed. Of these, 59 lacked α-Globin gene deletions. 17 were excluded from our analysis for lack of personal data or DNA. The 42 remaining samples were studied by amplification of the termination codon followed by an enzymatic digestion for the recognition site TTAA. 2 samples (5%) were found to be heterozygotes for α2-globin mutation, presumably CS mutation. The small number of positive cases does not permit the conduct of a powerful statistical analysis, however the two mutated samples had a high level of Hb H (2,9%) whereas only 21% of the 42 studied samples had an Hb H ≥2%.

Conclusion: These results represent a step forward in diagnosis of α-thalassemia and in identification of patients requiring particular genetic counselling based on HbH level at birth in cord blood. These results also indicate that 5% of Hb H positive/α-Globin deletion negative samples were caused by termination codon mutation in an occidental urban based population, and that it could be of interest to search for these mutations, especially when the Hb H level is high.
Fluctuation of Cystatin C Concentration during Deferasirox Treatment in Thalassemic Patients May Reflect Hemodynamic Changes
Authors: Papassotiriou Ioannis , Kattamis Antonios , Hantzi Eugenia , Delaporta Polyxeni , Sergounioti Athanassia , Margeli Alexandra , Ladis Vassilios
Published: April 2020

Deferasirox (Exjade®, ICL670) is an once-daily, oral iron chelator for the treatment of transfusional iron overload and its efficacy and tolerability have been established in adults and children with a range of transfusion-dependent anemias, such as thalassemia. We observed alterations in cystatin C plasma concentration during longterm treatment of thalassemia patients with deferasirox. Cystatin-C is a non-glycosylated protein that belongs to the cystatin superfamily of cysteine protease inhibitors. Plasma cystatin-C has been suggested to be an ideal endogenous marker of glomerular filtration rate (GFR). We aimed to investigate if these changes in cystatin C concentration were solely due to deferasirox treatment, or whether other factors contribute to these changes. 150 b-thalassemia patients treated with deferasirox at doses between 20–40 mg/Kg/day were followed longitudinally. Cystatin C concentrations were measured by an immunonephelometric technique (Dade Behring, Siemens Healthcare Diagnostics) at regular monthly intervals (5–12 monthly serial measurements per patient), while GFR was calculated according to the recently proposed cystatin C–based prediction equation using only its concentration in mg/L: GFR [mL/min/1.73m2] = 76.7 x Cystatin C−1.18. To further evaluate the effect of deferasirox on renal function we measured: a) plasma levels of Neutrophil-Gelatinase-Associated-Lipocalin (NGAL) (R&D Systems) a protein expressed on tubular cells and which production is markedly increased in response to harmful stimuli, such as ischemia or toxicity, b) N-terminal pro-B-type natriuretic peptide (NT-proBNP) (Roche Diagnostics), in order to correlate cystatin C fluctuation with left ventricular ejection fraction (LVEF) and c) ferritin level, as a marker of iron overload/mobilization. The main results of the study showed that cystatin C concentration fluctuated during deferasirox treatment (ANOVA repeat measures p>0.850). Baseline mean values were 0.97±0.27 mg/L and reached a maximum of 1.01±0.29 mg/L at 4 months of treatment. According to the guidelines and as no significant increase of creatinine levels was observed, the drug dose was not reduced. Analysis of data showed that: a) there is no correlation between cystatin C fluctuation and NGAL levels, p > 0.6745, evidence that the tubuloglomerular feedback is not activated b) cystatin C and NT-proBNP levels correlated positively with a binomial equation (p<0.004) and c) cystatin C and ferritin levels correlated positively with also a binomial equation (p<0.001). These findings suggest that cystatin C fluctuation during deferasirox treatment do not reflect renal injury. Hemodynamic signals, LVEF alterations and iron mobilization seem to play central role on changes of cystatin C.
Induction of Adenovirus Specific T-Cells in Response to 15-Mer Peptides in Cord Blood
Authors: Haveman Lianne Margreet , Bulatovic Maja , Prakken Berent , Bierings Marc
Published: April 2020

Human adenovirus (HAdV) infections are a serious life-threat in haematopoietic stem cell transplantation (HSCT) patients, particularly in children. In recent years, umbilical cord blood transplants (UCBT) emerged as an alternative stem cell source. The advantages are the rapid availability of donor cells, the possibility of using HLA-mismatched transplants and a lower risk of graft versus host disease. However, the immune response after UCBT is often delayed, leading to a higher risk of viral infections. Because no effective antiviral medication exists for severe HAdV infection adoptively transfer of HAdV specific T-cells from cord blood may be a promising treatment. The aim of this study was to see if it was possible to induce HAdV specific T-cells in response to five recently detected 15-mer peptides ( Haveman LM, Int. Imm. 2006), a pool of these five peptides or complete inactivated HAdV (MOI 10). Mitogen Concovalin A ( ConA) was used as control. Cord blood mononuclear cells (CBMC) derived from 34 uncomplicated full term deliveries were isolated and cultured with the different antigens. Proliferation expressed as Stimulation Index (SI) was determined. A SI>1.7 was seen as positive. In 14 neonates a response to in total 36 of the 5 different HAdV peptides could be seen and in 7 of them also a response to the pool of the peptides. Only in 3 neonates a response to complete HAdV could be detected (Figure 1). To induce HAdV specific T-cells CBMCs of 20 neonates were cultured with the different HAdV antigens in addition of interleukin 2 (40IU/ml) for 7 days. By using FACS analysis the upregulation of the activation markers CD25 and CD69 could be detected in response to the HAdV peptides (n=41) or to the complete HAdV (n=26). Induced HAdV specific T-cells expressed the cytotoxins IFN□, TNFα, perforin and granzyme B, produced by CD8+ T-cells, but in higher extent by CD4+ T-cells. This indicates that induced HAdV specific T-cells play an essential role in killing HAdV infections. No clear correlation could be seen between proliferation and the production of the cytotoxins. The induction of a specific immune response to HAdV peptides in cord blood is an important step towards adoptive therapy.

Figure 1. Proliferation of CBMC in response to HAdV peptides, complete HAdV en ConA Figure 1. Proliferation of CBMC in response to HAdV peptides, complete HAdV en ConA
Leukemia, Solid Tumors and Methylarginines: Is There Any Relation?
Authors: Doroszko Adrian , Niedzielska Ewa , Wojcik Dorota , Szuba Andrzej , Andrzejak Ryszard
Published: April 2020

Background: Despite improvements achieving complete remission and progress in supportive care of children with acute leukemias or solid tumors during the last decade, there are no optimal prognostic markers in stratification of risk of adverse events leading to poor outcome. The endogenous nitric oxide synthesis inhibitor asymmetrical dimethyl-L-arginine (ADMA) is associated with atherosclerosis and represents an independent marker for cardiovascular morbidity and mortality.

Aim: To investigate the clinical significance of methylarginines in leukemia and solid tumors in children.

Material and method: Concentration of metabolites of nitric-oxide pathway in children during therapy of acute leukemia (ALL) – N 12 and solid tumors N 15 (osteosarcoma, RMS, PNET, Wilms’ tumor) was determined. Correlations of biochemical markers, blood count parameters and plasma level of methylarginines were investigated. In addition, the influence of cytostatics on NO-pathway metabolites was analyzed.

Results: Table 1: Differences in mean plasma concentration of arginine, ADMA and SDMA in pts with leukemia vs pts with solid tumor

Leukemia N=12 Mean ±SD Median±SEM Solid tumor N=15 Mean ±SD Median±SEM p Arg 31,28±8,63 30,82±2,49 49,59±10,81 49,18±3,00 0,0003 ADMA 1,17±1,10 0,53±0,32 0,98±1,10 0,46±0,31 NS SDMA 1,18±1,10 0,80±0,32 1,56±1,02 1,09±0,28 NS Arg/ADMA 67,40±54,31 69,48±15,68 228,82±451,44 117,35±125,21 NS

Table.2. Differences in mean plasma concentrations of arginine, ADMA and SDMA depending on gender, treatment, etc.

N=27 Arginine ADMA SDMA Arg/ADMA Sex 1. Mail; 2. Femail 1. 0.NS NS NS NS HSCT (1-yes; 0-no) 1. 0.NS NS NS NS Remission (1-yes; 0-no) 1. 0.NS NS NS NS Currently infection (1-yes; 0-no) 1. 0.NS NS NS NS Glucocoricoids (1-yes; 0-no) 1. 31,9±9,30. 51,0±11,2 p=0,0011 NS NS NS Cytostatic – any (1-yes; 0-no) 1. 0. NS NS NS NS Doxorubicine (1-yes; 0-no) 1. 0. NS NS NS NS Vincristine (1-yes; 0-no) 1. 0. NS NS NS NS Cytarabine (1-yes; 0-no) 1. 32,0±10,50. 45,6±10,8 p=0,022 NS NS NS Methotrexate (1-yes; 0-no) 1.0 NS NS NS NS Thioguanine/6-merkaptopurine(1-yes; 0-no) 1. 0. NS NS NS NS Cyclophosphamide/Iphosphamide(1-yes; 0-no) 1. 0. NS NS NS NS

Conclusions: Plasma level of L-Arg was significantly higher in patients with solid tumor The L-Arg/ADMA ratio was not significantly higher in patients with solid tumors and lower plasma level of ADMA was found. Treatment with glucocorticoids and cytarabine was associated with significantly lower level of L-Arg. No significant treatment dependent difference was found in level of nitric oxide metabolic pathway.
Factor VIII and Platelets Cooperatively Accelerate Proteolytic Cleavage of Von Willebrand Factor by ADAMTS13 Under Shear Stress
Authors: Skipwith Christopher G. , Cao Wenjing , Zheng X. Long
Published: April 2020

ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 bond of the central A2 domain. Inability to cleave vWF results in thrombotic thrombocytopenic purpura (TTP), which is characterized by profound thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ involvement. Previous studies have demonstrated that coagulation factor VIII (Cao et al, PNAS, 105:7416–21, 2008) or platelets (Shim et al, Blood, 111:651–7, 2008) can accelerate proteolytic cleavage of multimeric vWF in solution by ADAMTS13 under mechanic induced shear stresses. However, the concentrations of factor VIII or platelets required to achieve a detectable increase in the proteolytic cleavage product (350K) were beyond the physiological ranges. Therefore, the physiological significance of these findings remained to be determined. In this study, we assessed whether factor VIII and platelets, both of which bind vWF with high affinity, cooperatively affected the proteolytic cleavage of vWF by ADAMTS13 under high shear stress. All experiments were performed with a fixed concentration of vWF (150 nM) and ADAMTS13 (25 nM), and various concentrations of factor VIII and platelets in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM CaCl2. The reaction mixtures (total volume 20 μl in a 0.2 ml PCR tube) were subjected to vortexing at 2,500 rpm in MixMate (manufactured by Eppendorf), which generates laminar shear stress of approximately 30 dynes/cm2. We showed that in the absence of factor VIII, lyophilized platelets at the concentration of 1,000×109 per liter did not increase the proteolytic cleavage of plasma-derived vWF by ADAMTS13. An addition of factor VIII (1, 2, and 5 nM) to the reaction mixture markedly accelerated the rate enhancing effect of lyophilized platelets in a concentration-dependent manner. In the presence of physiological concentration of factor VIII (1–2 nM), the proteolytic cleavage product (350K) detected by Western blot under non-reducing conditions reached the maximal intensity at the platelet concentration of 100×109 per liter. B-domain deleted factor VIII (FVIII-SQ) showed the similar cooperativity with platelets accelerating proteolytic cleavage by ADAMTS13. However, a construct with the acidic region (a3) that binds vWF with high affinity removed (FVIII-2RKR) did not show any cooperativity with platelets in vWF proteolysis by ADAMTS13. We conclude that binding of factor VIII and platelets to vWF cooperatively accelerates the proteolytic cleavage of vWF by ADAMTS13 under high (arterial) shear stress. These findings provide novel insight into the regulation of ADAMTS13-mediated vWF proteolysis by a coagulation factor and platelets under physiological conditions. The data also suggest a potential role of factor VIII and platelets in the therapeutic regimen for TTP.
Array-CGH Identifies Both Common and Subtype-Specific Genomic Aberrations in Marginal Zone Lymphomas
Authors: Rinaldi Andrea , Rancoita Paola MV , Bhagat Govind , Novak Urban , Nandula Subhadra , Murty Vundavalli V. , Ponzoni Maurilio , Govi Silvia , Ferreri Andres JM , Baldini Luca , Gascoyne Randy , Soulier Jean , Thieblemont Catherine , Marasca Roberto , Franceschetti Silvia , Gaidano Gianluca , Canzonieri Vincenzo , Gattei Valter , Facchetti Fabio , Tucci Alessandra , Mollejo Manuela , Piris Miguel A. , Uccella Silvia , Tibiletti Maria Grazia , Dirnhofer Stephan , Forconi Francesco , Chigrinova Ekaterina , Doglioni Claudio , Dalla-Favera Riccardo , Zucca Emanuele , Kwee Ivo , Bertoni Francesco
Published: April 2020

Background and Aim. In the current WHO classification, marginal zone B-cell lymphomas (MZL) have been divided into three distinct subtypes (extranodal, EMZL; nodal, NMZL; splenic, SMZL) according to clinical, pathological and cytogenetic features, however the relationship between any of the different subtypes is still unclear. We thus performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question.

Materials and Methods. DNA extracted from frozen samples of MZL was analyzed with Affymetrix Human Mapping 250K SNP arrays.

Results. Samples from 193 patients with MZL (23 NMZL, 63 EMZL, 84 SMZL and 23 not better specified MZL) from 15 Institutions were analyzed. Among EMZL, main primary sites were orbital adnexa in 28%, GI-tract in 16%, lung and salivary glands 5% each. Overall, MZL displayed more recurrent deletions than gains. Gains of 3q and 18 gains were common to all three MZL subtypes as were losses at 1p32.3-pter, 7p22.1-pter, 7q11.21–q11.23, 9q33.3-qter, 16p, 17p and 19. EMZL had significantly more gains at 3p14.2-pter, 6p and 7q31.3-qter and losses at 6q15, 6q23.3 and 14q11.2 than SMZL. Conversely, the latter had deletions of 7q31.32-qter and 14q23.3–q32.13 more frequently than EMZL. NMZL profiles showed the EMZL-specific 6q losses and the SMZL-related 7q losses at very low frequency while presented recurrent gains of chromosome 11, which was unique of NZML among the three subtypes. The 6q23.3 region contains the gene coding for TNFAIP3/A20, a negative regulator of the NFkB pathway. In SMZL a minimal deleted region was observed in 7q32.1. Regarding chromosome 3, gains targeted the whole chromosome in EMZL while, in SMZL and NMZL, the long arm was more commonly affected.

Conclusions. EMZL, NMLZ and SMLZL share genetic alterations, which differ from other B-cell lymphomas. However subtype-specific genetic lesions are observed. In particular, gains of 6p and losses of 6q23.3 (TNFAIP3) are specific for EMZL, and 7q31-qter for SMZL.