bluuebunny/crossref_metadata_2025 huggingface dataset:
Metylenetetrahydrofolate Reductase Polymorphism and Venous Thrombosis in Children
Authors: Luciani Matteo , Pansini Valeria , Coletti Valentina , Funaro Daria , Soldati Massimiliano , Pancotti Simona , Falappa Piergiorgio , De Rossi Giulio
Published: April 2020

The venous thromboembolism is a serious and complex pathological condition especially for its potential complications. It is related to both congenital and acquired factors. Among the congenital factors, different mutations have been identified leading to a reduced activity of enzymes (even higher than 50%) involved in the homocysteine-methyonine methabolic pathway, such as the methylenetetrahydrofolate reductase enzyme (MTHFR).

The homozygotic TT genotype is found in approximately 10–12% of the population and is associated with a 25% higher homocisteine level in these patients than those without this mutation. The heterozygotic genotype is found in 40% of the population. The aim of this study is to define in children the correlation between venous thrombosis and MTHFR mutations. Between december 2002 and april 2008 nine children aged between 1–132 months were admitted in the Department of Hematology of Children Hospital Bambino Gesù in Rome. They were affected by deep venous thrombosis and were submitted to thrombophilic screening and vascular imaging. The MTHFR polymorphism C677CT was found in all the patients (four in homozygosis and seven in heterozygosis) and also in their parents. Four heterozygous and three homozygous patients presented increased plasmatic and urinary levels of homocysteine. Since venous thrombosis was only referred in the clinical history, two patients did not received treatments. Nine were treated with low molecular weight heparin (enoxaparin 100 U/kg s.c. × 2 daily), then seven continued the treatment with oral anticoagulant for 6–12 months and all of them performed long term profilaxis with folic acid and B group vitamins. Only one patient presented further thrombotic event and restarded the oral anticoagulant therapy. These data confirm that the measurement of homocysteinemia and MTHFR may be indicated in unexplained idiopathic venous thrombosis or recurrent episodes or venous thrombosis occurred at an early age or at an uncommon site. It is to be discussed whether to perform anticoagulant profilaxis in all patients with MTHFR mutation, in homozygosis or heterozygosis, with or without hyperhomocysteinaemia, with previous thrombotic events, with other thrombotic risk factors (pregnancy, oral contracceptives, sepsis and immobilization). In addition in presence of hyperhomocysteinemia, it might be reccomended to perform therapy with folic acid and B6 vitamin.
The Prevalence of Anemia in Rio Grande Do Sul, Brazil: Is Iron Deficiency the Silent Burden Hidden Behind “Normal” Anemia?
Authors: Silla Lucia , Zelmanowicz Alice , Mito Ingrid , Daudt Liane , Bem Carlos do , Alves Marta , Lehuguer Denise , Fridrisch João , Bittar Christina , Fisher Gustavo , Fogliatto Laura , Paz Alessandra , Bittencourt Rosane , Bortolini Gisele , Brouwers Larissa , Shilling Marco , Michalowski Mariana , Hellwig Tania
Published: April 2020

We ran a study on the prevalence of anemia in the state of Rio Grande do Sul (RS), southern Brazil. RS is in a sub-tropical Latin American area, near Uruguay and Argentina. There is no Malaria, a low prevalence of hemoglobinopaties, and HIV/AIDS.

METHODS: Capillary blood of children less then 7 and women from 14 to 30 years of age were tested. Socioeconomic status was determined utilizing the classification designed by the Brazilian Association of Market Research Agencies for this purpose. A second sample of 1 year old children was submitted to total blood cell count and serum ferritin determination, and in a third group of individuals, total blood count was done along with stool parasitic analysis.

RESULTS: A total of 2198 children and 1999 women with capillary blood measured were analyzed. The median prevalence of anemia in the whole state and the median hemoglobin concentration was 45, 4% and 9.90g/dl for children, and 36.1% and 11 g/dl for women, respectively. Of the aforementioned group, before two years of age, 76% were anemic, and anemia prevalence decreased with age, suggesting a nutrition deficiency. There were very few cases of severe anemia. A significant percentage of children and women belong to the middle class, with access to food and medical care. Of the 356 children tested at one year of age, 67% were anemic and of these, 90% had ferritin < 15μg/L. In the third group of individuals, 31% of 424 children less then 6 years of age were anemic and of these, only 13% tested positive for parasitic infection.

CONCLUSION: The prevalence of iron deficiency anemia is very high and constitutes a severe public health problem for 1 year old children in RS, and probably for those younger then 7, and for fertile women as well, irrespectively of their socioeconomic level. Micronutrients supplementation has being neglected in RS health system. The definition of anemia plays a central role in this and has to be addressed if we want to overcome the biologic factors involved in childhood psychosocial development in Brazil.
An IgM Lambda Monoclonal Antibody in a Patient with Anti-Pr Cold Agglutinin Causing Erythrocyte Agglutination and Catastrophic Thrombosis
Authors: Kwaan Hau C. , McMahon Brandon , Ruch Joshua , Weiss Ivy
Published: April 2020

Erythrocyte agglutination causing microvascular occlusion is a rare albeit clinically significant event. This is seen in cold agglutinin disease and in cerebral malaria caused by Plasmodium falciparum. We studied a patient with autoimmune hemolytic anemia, characterized by massive hemolysis and the presence of both an anti-Pr antibody and a cold hemagglutinin. In addition, he suffered from catastrophic mesenteric ischemia necessitating surgical resection and extensive and multiple ischemia strokes. His surgical specimens, including colon, ileum and gall bladder, were studied with informed consent. Eluate from his agglutinated erythrocytes showed that he had an IgM lambda monoclonal antibody against the Pr antigen. The patient’s peripheral blood showed extensive erythrocyte agglutination that was worse at cold temperatures. H&E staining of the colon and ileum showed ischemia and necrosis with widespread microvascular occlusion caused by agglutinated erythrocytes. To characterize the intravascular agglutinated erythrocytes, immunoperoxidase staining was done and positive for IgM and lambda light chain and negative for IgG and kappa light chain. We thus concluded that the cold agglutinin on the intravascular agglutinated erythrocytes was an IgM lambda. To investigate why the circulating agglutinated erythrocytes resulted in widespread microvascular occlusion, we studied adhesive molecules including CD 62E (E-selectin), CD62P (P-selectin), CD54 (ICAM-1) and CD 106 (VCAM-1) and found all of them expressed on the agglutinated erythrocytes within the occluded vessels and on the endothelial cells of the involved vessels. The data suggests that the endothelial cells were activated at the site of vascular occlusion, thereby promoting adhesion of the agglutinated erythrocytes to the vascular endothelium.
Shape Recovery of Human Crenated Erythrocyte Ghosts Depends on the Cytoplasmic Endoskeletal Actin Filaments
Authors: Terasawa Kazutaka , Taguchi Takehito , Momota Ryusuke , Naito Ichiro , Ohtsuka Aiji
Published: April 2020

Previously, we showed the human erythrocyte cytoplasmic endoskeleton composed of actin filaments and intermediate filaments [Terasawa K, et al. Arch. Histol. Cytol.2006;69:326–340]. The present study aims to determine whether endoskeletal actin filaments contribute to regulate the shape recovery of human erythrocytes. We prepared two types of ghosts: prefixed ghosts which contain endoskeletal network and non-fixed usual ghosts which lack endoskeletal network. To prepare prefixed ghosts, fresh erythrocytes were mildly fixed with 2 % paraformaldehyde and 0.05 % glutaraldehyde in phosphate buffered saline and haemolysed by mild hypotonic Tris buffer with 2 mM Mg-ATP. These prefixed ghosts contained an endoskeletal network which could change their shape quickly by Mg-ATP. In transmission electron microscopy, all the spherical and crenated prefixed ghosts possessed the endoskeletal network as an electron dense matter. Gold particles immuno-labeled for beta actin dispersed over the whole endoskeleton in both crenated and discoid prefixed ghosts. Moreover, cytochalasin B, a blocking agent of fast growing end of long actin polymer, inhibited crenated to discoid shape transition in prefixed ghosts. In non-fixed ghosts, addition of cytochalasin B did not affect recovery to the original shape. Thus, these results suggest that shape transition induced by Mg-ATP is driven through the action of the endoskeletal actin network.
Serum Prohepcidin Levels in Infants with Anemia of Inflammation and Iron Deficiency Anemia
Authors: Park Eun Sil , Kim In-Suk , Lim Jae-Young
Published: April 2020

Backgroud . The aims of this study were to measure the serum levels of prohepcidin in anemic and non anemic infants and to evaluate the correlation between prohepcidin and iron parameters and inflammatory cytokines.

Procedure. One hundred and sixty nine patients aged from 6 months to 24 months were enrolled and divided into 4 groups according to hemoglobin and serum ferritin (SF). Routine hematologic labs, soluble transferrin receptor (sTfR), serum prohepcidin, C reactive protein (CRP), and interleukin-6 (IL-6) were assessed. The subgroup of anemia of inflammation (AI) was defined as Hb <11 g/dL and SF >50 μg/L, the subgroup of iron deficiency anemia (IDA) as Hb <11 g/dL and SF <12 μg/L, the normal group as Hb ≥11 g/dL and SF ≥12 μg/L, and the unclassified anemia group (UCA) as <11 g/dL and SF 12–50 μg/L.

Results. The prohepcidin levels ranged from 38.86 to 874.25 ng/mL throughout all the infants (258.60±130.03 ng/mL) but were significantly lower in the AI group than in the normal group (201.93±71.74 vs 299.97±120.68 ng/mL, P=0.007). However there was no significant difference between anemic subgroups. Prohepcidin levels were positively correlated with CRP (r=0.400, P=0.048) in the AI group and were negatively correlated with sTfR in the UCA group (r=−0.376, P=0.022). However in both groups there were no independent predictors in multiple regression analysis.

Conclusions. This study examines the prohepcidin levels in infants with anemia and without anemia. The prohepcidin levels of anemic infants were significantly lower than those of infants without anemia. However there was no correlation between prohepcidin and inflammatory cytokines or iron parameters in the groups of AI and IDA, thus we cannot differentiate between them using prohepcidin levels.
Matrix Metalloproteinase 9 Levels Are Increased in Pegfilgrastim and Filgrastim Induced Mobilisation but Do Not Predict Efficiency of Mobilisation
Authors: Willis Fenella M , Theti Davinder , Gordon-Smith Edward C. , Pettengell Ruth
Published: April 2020

Release of matrix metalloproteinase 9 (MMP-9), neutrophil elastase and cathespin-G from bone marrow stromal cells and granulocytic precursors in response to cytokines and chemotherapy results in degradation of important retention signals such as SDF-1 and V-CAM and promotes egress of progenitor cells from the bone marrow into the peripheral blood. In this study we investigated whether plasma MMP-9 levels could predict efficiency of haemopoietic progenitor cell mobilization

Methods: Plasma MMP-9 levels were assessed at 0, 24 and 48 hours in chemotherapy naïve patients with solid tumours. Patients were randomised to receive a single administration of 6, 12 or 18mg of pegfilgrastim on day 1 or daily administration of filgrastim 10 μg/kg/day from day 1 until day 7. Eight subjects were studied in each group. Each group was balanced in terms of baseline characteristics. Blood samples for peripheral CD34+ cell count and colony assays were collected on the first 7 days and day 9 and 12 post therapy.

Results: The most common tumour types were non-small cell lung cancer (n = 15; 47%) and ovarian cancer (n = 9; 28%). In the filgrastim and pegfilgrastim 6mg, 12mg and 18mg groups a significant rise in MMP-9 levels were seen between 0 and 24 hours (380 vs. 2020 ng/ml, p=0.002; 220 vs 1920 ng/ml, p=0.015; 610 vs. 2510 ng/ml, p=0.0002 and 250 vs. 2050 ng/ml, p=0.003, respectively). In all groups, there was no significant increase in plasma MMP-9 levels between 24 and 48 hours. No significant differences in MMP-9 levels were observed between the 4 cytokine groups over 0 to 48 hours. Across all the groups no significant correlation was seen between plasma MMP-9 levels and peak colony forming units (CFU) per millilitre of blood and peak CD34 positive cells per millilitre of blood (r2=0.103, p=0.08; r2=0.09, p=0.1, respectively).

Conclusion: In all cytokine groups, a significant increase in plasma MMP-9 levels occurred between day 1 and 2. No association between MMP-9, levels numbers of peripheral blood CD34 positive cells and CFU levels could be established. Plasma MMP-9 levels were not affected by pegfilgrastim dose. MMP-9 does not appear to be a predictor of mobilization efficiency for filgrastim or pegfilgrastim at doses up to 18mg.
Iron Chelation, Blood Transfusion and Splenectomy Decreases Angiogenesis in Patients with Thalassemia Major
Authors: Canatan Duran , Olgar Seref , Kara Aslihan , Hicyilmaz Hicran , Balta Nihal
Published: April 2020

Introduction: Thalassemia major (TM) is an important cause of severe anemia that necessitates regular blood transfusion to prevent the profound weakness and cardiac decompensation caused by the anemia. However, iron overloading is an inevitable consequence of prolonged transfusion therapy. In addition, extramedullary hematopoiesis and hemosiderosis cause spleen, liver and marrow enlargement. In recent years the role of angiogenesis has been investigated in physiologic and pathologic conditions. However, it is known that angiogenetic factors, especially vascular endothelial growth factor (VEGF), cause differentiation of the hemangioblast. The role of angiogenesis has been investigated in different kinds of anemia, such as malignancy related anemia and sickle cell anemia. However, the role of angiogenesis has not been investigated in thalassemia major (TM) patients. In this study the angiogenesis was researched in thalassemic patients by serum VEGF measurement.

Material and method: Forty-four consecutive patients with TM were included in this prospective study. TM patients’ findings were compared with those of a healthy control group (n=12). Blood samples were analyzed using commercially available ELISA kits for VEGF

Results: VEGF levels were not affected by hemoglobin levels, ferritin levels, or chelation type (p>0.05). However, VEGF was positively affected by chelation starting age and negatively affected by yearly blood transfusion rate (p<0.05). In addition, VEGF of patients who underwent splenectomy was higher than those who didn’t undergo splenectomy (p<0.05).

In conclusion, VEGF causes differentiation of hemangioblasts, however, early starting transfusion age and regular blood transfusion decrease angiogenesis in thalassemic patients. The besides regular blood transfusion and effective chelation therapy, splenectomy decreases angiogenesis in this group of patients.
Comparing a Novel PET Agent, Yttrium-86 Oxine, with Biopsy and FACS Analysis for Tracking in Vivo Migration Patterns of Different Preparations of DC from a Single Donor
Authors: Harrison Si mon J. , Thompson Mick , Wall Dominic , Loudovaris Maureen , Mackensen Andreas , Bartholeyns Jacques , Ferrantini Maria , Santodonato Laura , Felzmann Thomas , Hicks Rod , Prince H. Miles , Ritchie David
Published: April 2020

Aim: The objective of this study was compare the in vivo distribution of different preparations of dendritic cells (DC) from a single donor after intradermal (ID) administration and correlate FACS analysis of excised tissue and imaging findings.

Method: DC were generated from a single healthy donor apheresis product. The DC types were GM-CSF + IL-13 (IL-13 imDC), then further matured with FMKp+IFNg (IL-13mDC), GM-CSF + IL-4 (IL-4 imDC) and matured (IL-4 mDC) and GM-CSF+IFN-α (IFN-α DC). Antigen naive DC preparations were labelled with Yttrium-86 oxine (prepared on site) by incubation at room temperature for 30 minutes followed by centrifugation and resuspension. 5 × 105 radio-labelled cells in 50_L was injected ID into the footpad and the volar aspect of the foreleg of Balb/c nude mice (n=5 per group). A cohort (n=5) of control mice were similarly injected with Yttrium-86 oxine only. Images were acquired on a small animal PET (SAPET) (Mosaic, Philips Medical Systems) at 2, 24 and 45 hours post administration. Mice were then euthanized and popliteal, axillary and inguinal lymph nodes (LN) harvested. LN were processed into single cell suspension, stained with anti-human HLA-DR and CD11c and analysed by flow cytometry. SAPET images were graded: 0 – no signal, 1 – marginal, 2 – definite nodal signal, 3 – strong signal. DC detection by FACS was graded: 0 – no detection, 1 – <0.5% DC, 2 – 0.5.⋄1.5% DC, 3 – >1.5% DC of all viable cellular events. Results: Pre-injection analysis of DC showed expression of HLA-DR, HLA-ABC, CD11c, CD80 and CD86, low levels of CD83, CCR5 and CCR7 along with down regulation of CD14. Matured DC populations showed increased levels of CD83 and CD86. Similar phenotypic expression on the DC populations were observed prior to cryopreservation and upon thawing. Mean DC radiolabelling efficiency of 72% (54% – 88%). There was a minor decrease in cell numbers and viability after labelling most likely due to oxine related toxicity. There were no substantial differences in tracking between cell types. In 28 of 43 nodes assessed, FACS and SAPET results were congruent (27 FACS and SAPET positive and 1 FACS and SAPET negative). Of the 15 discrepant samples, 13 were SAPET positive and FACS negative, while 2 were FACS positive and PET negative. Average FACS and SAPET grades at 45 hours were higher for popliteal nodes than axillary nodes. No false positive nodal accumulation was visualised at 45 hours in control mice. Conclusion: This novel PET technique provides a robust, reproducible method for quantification of in vivo functional DC tracking to LN. Detection of DC migration using this novel PET agent is a more reliable technique than biopsy and FACS analysis, which has an inherent sampling error.
Comparative Analysis of CD8+ t Cell Priming Efficiencies against a Panel of Leukemia-Associated HLA-A2 Restricted Epitopes Identifies PRAME as a Possible Vaccination Target in AML
Authors: van den Ancker Willemijn , Westers Theresia M , Bontkes Hetty J , Hooijberg Erik , Stam Anita GM , Santegoets Saskia JAM , Ossenkoppele Gert J , de Gruijl Tanja D , van de Loosdrecht Arjan A
Published: April 2020

Background Therapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from Leukemia-Associated Antigens (LAA) are an attractive source of antigen. WT1, PRAME, hTERT, and survivin are all LAA that are expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative Cytotoxic T Lymphocyte (CTL) priming efficiencies in response to a panel of LAA-derived HLA-A2 binding epitopes.

Methods CD8+ cells were isolated from peripheral blood of healthy donors (HD) and stimulated with peptide-loaded DCs. DCs were derived from the human AML cell line MUTZ-3 (MUTZ3-DCs).1 The peptides used were WT1 (126–134), PRAME (100–108), hTERT (540–548), and survivin (96–104). Irradiated CD8− cells from the same donor were also added. CD8+ cells were plated at one million per well in 8 to 30 wells. Weekly CD8+ cells were restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for WT1, PRAME, hTERT or survivin epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant LAA peptides or with an irrelevant control peptide (BCR-ABL).

Results Percentages of the induced peptide-specific CTLs were low as shown in the table below. Priming efficiencies were highest for PRAME. Furthermore, PRAME and WT1 specific CTLs produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells.

Number of HD that were Tm+ after co-culture Number of wells that were Tm+ after co-culture Median % Tm+ cells per positive well (range) Number of restimulations at maximum of Tm+ PRAME 3/4 28/60 0.10% (0.02–1.07) 1–5 WT1 2/5 4/104 0.07% (0.03–0.09) 1–4 hTERT 1/2 1/24 0.25% 5 Survivin 1/1 2/18 0.17% (0.12–0.21) 8

Conclusions On the whole, the detected LAA-specific CD8+ T cell frequencies were low. Most consistently successful priming results were obtained for PRAME, indicating that this peptide may be an attractive candidate for inclusion in AML-specific DC-based vaccine regimens.
Active Von Willebrand Factor in Plasma of Sickle Cell Patients Is An Indicator for Severity of Sickle Cell Anemia
Authors: Chen Junmei , Hobbs William , Le Jennie , Lenting Peter J , Lopez Jose A
Published: April 2020

Vaso-occlusion, hemolysis and oxidative stress are hallmarks of sickle cell anemia. We and others have shown that oxidants generated in sickle cell anemia can stimulate endothelial cells to release ultra-large von Willebrand factor (ULVWF), which is hyperactive compared to the normal plasma VWF in binding platelets and red blood cells, and is capable of binding sickle erythrocytes especially well. For this reason, we surveyed several parameters of VWF quantity and function in plasma from patients with sickle cell anemia (9 individuals, some with multiple determinations). These parameters included VWF antigen levels, VWF multimer composition, ADAMTS13 (the VWF-cleaving metalloprotease) activity, VWF activation factor, and total active VWF. VWF activation factor was measured using a llama antibody (nanobody) that detects an active conformation in the VWF A1 domain that correlates with an enhanced ability to bind platelets. The activation factor is defined as the ratio of nanobody binding of patient plasma to that of pooled normal plasma at a given VWF antigen level. Thus, the activation factor of pooled normal plasma is 1. Elevated VWF activation factor (≥ 1.5, Curr. Opin. Hematol. 2007 14:284–289) has been demonstrated in the plasma of patients with type 2B von Willebrand disease, thrombotic thrombocytopenic purpura, HELLP syndrome, and other prothrombotic disorders. In this study, plasma was collected from patients with sickle cell anemia at baseline during routine outpatient visits. In all of the patients, the VWF antigen level was higher than in the pooled normal plasma (1.5–3.6 fold) or an ethnically matched control. Activation factors were in the range of 0.2 to 6.8. The total quantity of active VWF in plasma was then determined by multiplying VWF antigen level and activation factor. The total active VWF in plasma correlated with the severity of the patient’s disease, as determined by the number of hospital admissions over the previous year and elevated plasma lactate dehydrogenase (LDH). Elevated LDH is a risk factor for hemolysis associated pulmonary hypertension, leg ulcers, priapism and death in sickle cell anemia. In addition, we found that a sub-set of patents had reduced ADAMTS13 activity, which was correlated with higher VWF multimer composition on agarose gels. This indicates that accumulation of active VWF in patients with sickle cell anemia arises not only from increased endothelial secretion but also from decreased ADAMTS13 activity. These findings suggest that high levels of active VWF predispose patients with sickle cell anemia to vasoocclusive complications and correlate with increased hemolysis.
Glycoprotein IIb/Iiia Antagonist Could Reduce the Inflammatory Factors IL-6, IL-1β, TNF-α of Atherosclerosis Rabbits in Vivo
Authors: Yin Songmei , Xie Shuangfeng , Li Yiqing , Nie Danian , Ma Liping , Wang Xiuju , Xiao Jie
Published: April 2020

Background: Glycoprotein (GP) IIb/IIIa antagonists have been widely used in clinical practice. It had been demonstrated that although GPIIb/IIIa antagonists could reduce 30%~ 50% ischemic events around coronary artery intervention therapy, it had only limited effects on the long term survival. We want to explore the effects of GPIIb/IIIa antagonists besides inhibiting platelet aggregation. So we observed the effects of tirofiban (a GP IIb/IIIa antagonist) in vivo.

Design: the atherosclerosis model rabbits were divided into 4 groups. Each group got a 1.7ml/h nature saline (control group), 3.125mg/L, 12.5 mg/L or 50mg/L tirofiban continuous intravenous injection for 48 hours respectively. Platelet aggregation maximum (PAG(M)), plasma interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α were measured at 0 hour, 12th hour, 24th hour and 48th hour time point.

Results: In vivo, compare to the value at 0 hour, the PAG(M) didn’t change significantly at the 12th hour, 24th hour and 48th hour time point in the control group and 3.125mg/L tirofiban group. While the PAG(M) was reduced at the 12th hour, 24th hour and 48th hour time point in the 12.5mg/L and 3.125mg/L tirofiban group. About the inflammatory factors, in control group, compare to the index at 0 hour, plasma IL-1β, IL-6 and TNF-α concentration were all increased significantly at the 12th hour time point. In the 3.125mg/L tirofiban group, the plasma IL-1β and IL-6 concentration elevated significantly at the 24th hour time point. In the 12.5mg/L tirofiban group, the plasma IL-6 and TNF-α concentration at 24th hour time point, the plasma IL-1β, IL-6 and TNF-α concentration at 48th hour time point were all decreased significantly. In 50mg/L tirofiban group, the plasma IL-6 concentration at 12th hour time point, the plasma IL-1β, IL-6 and TNF-α concentration at 24th hour and 48th hour time point were all decreased. At 0 hour, plasma IL-1β, IL-6 and TNF-α concentration had no difference among the 4 groups (IL-1β PANOVA=0.954, IL-6 PANOVA=0.954, TNF-α PANOVA=0.954. n=5). While at 12 hour, 24 hour or 48 hour time points, among the 4 groups, the index value of the IL-1β, IL-6 and TNF-α decreased along with the increase of the tirofiban concentration (PANOVA<0.01. n=5).

Conclusion: In vivo, full dosage tirofiban could inhibit PAG(M), decrease the production of rabbit plasma IL-6, IL-1β and TNF-α. While little dosage tirofiban (≤3.125mg/L) had no effect on PAG(M), could not decrease the production of rabbit plasma IL-6, IL-1β and TNF-α. GP IIb/IIIa antagonist could reduce the inflammatory factors in full dosage.
Disequilibrium of Human Platelet Antigen 1 (HPA-1) in U.S. Population
Authors: Hutchinson Tyler Davi d , Song Yuhua , Trainor Kevin , Hashmi Ghazala
Published: April 2020

Background: Alloimmunization against Human Platelet Antigens (HPA) is associated with Neonatal Alloimmune Thrombocytopenia (NAIT), post-transfusion purpura and refractoriness for platelet transfusion. A flexible BeadChip™ design was developed to simultaneously detect 22 platelet antigens, including HPA-1, and used to assay over 1,000 random blood donors from across the United States.

Methods: Samples from 19 labs/centers from across the country were assayed for 11 HPA loci (HPA-1 through 9, 11 and 15) using the BioArray Solutions HPA Assay. Each locus was independently assessed for Hardy-Weinberg Equilibrium.

Results: Allele and genotype frequencies for each locus were reported. Platelet antigens HPA-2 through HPA-9, HPA-11 and HPA-15 were all found to be in Hardy-Weinberg Equilibrium with a Chi-Squared value of <3.84 (1 degree of freedom, 5% confidence interval). HPA-1, however, did not exhibit Hardy-Weinberg Equilibrium yielding a Chi-Squared value of 43.4.

Conclusions: After reaffirming there was no sampling preference by inclusion of a second blinded random group, it was acknowledged that HPA-1 did not conform to a Mendelian distribution of alleles. The lower incidence of heterozygote HPA-1 individuals may lend credence to the recent finding by Ivanov et al (Akush Ginekol, 2007) linking the polymorphism in GPIIIa that is responsible for the HPA-1 antigen with embryo implantation failure. Further research may help elucidate the causes behind the HPA-1 disequilibrium and how much implantation failure impacts HPA-1 frequencies.
Distinct Gab2-Mediated Signaling Pathways Are Essential for Myeloid or Lymphoid Transformation and Leukemogenesis by BCR-ABL
Authors: Mohi M. Golam , Chan Wayne W. , Li Shaoguang , Neel Benjamin , Van Etten Richard A.
Published: April 2020

The BCR-ABL oncogene encodes an activated fusion tyrosine kinase that causes chronic myelogenous leukemia (CML) and B-lymphoid acute lymphoblastic leukemia (B-ALL) in humans. An autophosphorylation site at Tyr 177 of BCR-ABL recruits Grb2 via its SH2 domain, and is required for efficient induction of CML-like myeloproliferative disease by BCR-ABL in a mouse BM retroviral transduction/transplantation model. We showed previously (Sattler et al., Cancer Cell2002;1:479) that the scaffolding/adapter protein Gab2 is recruited to Y177 of BCR-ABL via a Grb2/Gab2 complex, and in vitro transformation of primary myeloid and lymphoid progenitors by BCR-ABL was impaired in bone marrow from mice with homozygous null mutations in the Gab2 gene (Gab2−/− mice), coincident with decreased activation of the ERK and Akt signaling pathways. Here, we demonstrate an essential requirement for Gab2 in myeloid and lymphoid leukemogenesis by BCRABL. Whereas recipients of BCR-ABL-transduced Gab2+/+ BM develop fatal CML-like myeloproliferative disease within 4 weeks of transplantation, recipients of BCR-ABLtransduced Gab2−/− BM fail to develop CML but succumb after a long latent period to T-cell acute lymphoblastic leukemia, phenocopying the disease induced by the BCR-ABL Y177F mutant. These results suggest that the Y177F and Gab2 mutations have an epistatic relationship, and that the critical transforming signals from Tyr177 of BCR-ABL are transmitted through Gab2. Co-expression of Gab2 with BCR-ABL in Gab2−/− BM restored efficient induction of CML-like leukemia, but mutants of Gab2 that lacked either the pleckstrin homology domain or Tyr binding sites for the SH2 domains of the downstream Gab2 effector molecules SHP2 or p85 PI3K failed to rescue myeloid leukemogenesis by BCR-ABL, although the mutant Gab2 proteins were expressed in circulating myeloid cells. Gab2 deficiency attenuated B-lymphoid transformation by BCR-ABL in vitro, and significantly prolonged the latency of B-ALL induced by BCR-ABL in mice. In contrast to CML, induction of B-ALL in Gab2−/− BM was rescued by either WT Gab2 or the Gab2 mutant defective in p85 binding. These results demonstrate that BCR-ABL absolutely requires signaling via Gab2 to both SHP2 and PI3K to cause CML, while a Gab2-SHP2 signaling pathway contributes to the pathogenesis of BCR-ABL+ B-ALL.
GSK-3β Inhibition Activates WNT, Delays Division and Preserves the Function in Hematopoietic Stem Cell
Authors: Dolnikov Alla , Ko Kap-hyoun , Song Emma , Holmes Tiffany , O’Brien Tracey A
Published: April 2020

Wingless (Wnt) signaling appears to play an integral role in expansion and quiescence of stem and progenitor hematopoietic cell populations. To better understand this dichotomous role and aid in the translation of therapeutic goals including cellular therapies, greater understanding of the molecular mechanisms that regulate Wnt/β-catenin in the bone marrow niche are needed. We explored the impact of Wnt signalling in the context of the human HSC niche by using co-culture of human cord blood or bone marrow derived HSC with mesenchymal stem cells (MSCs) together with pharmacological inhibition of GSK-3β (iGSK-3β). We have previously shown that activation of the Wnt/β-catenin pathway using pharmacological inhibition of GSK-3β (iGSK-3β) in human cord blood derived HSCs acts to preserve their primitive status, often lost during the ex-vivo expansion process (1). Here we show using cell division tracking that iGSK-3β delays stem cell divisions induced by cytokines in co-culture with MSCs. Functional analysis of iGSK-3β-treated cells revealed an increased proportion of primitive cells producing mixed Colony Forming Units (CFU-GEMMs) formed in the methylcellulose cultures and Cobblestone Area Forming Cells identified in long-term co-cultures with the bone marrow stroma MS5 cells. In addition, iGSK-3β-treated cells exhibited a higher frequency of SCID Repopulating Cells. We also show that the iGSK-3β effect, at least in part, is mediated through VLA4 integrin engagement by fibronectin. Importantly, iGSK-3β also induced niche-independent delay in stem cell divisions seen in cytokine supported suspension cultures. Up-regulation of HoxB4 and p21Waf1 and down-regulation of cyclin D1 was observed in HSCs treated with iGSK-3β. These findings confirm that iGSK-3β– regulated Wnt/β-catenin activity is crucial for the maintenance of HSC quiescence, both in the MSC niche and in a niche-independent environment and that iGSK-3β can circumvent the requirement of exogenous Wnt ligand for the induction of Wnt/β-catenin signalling in MSC niche. Our results are consistent recent data showing that blocking of canonical Wnt signaling, by over-expression of dickkopf1 (Dkk1) increases the number of proliferating HSCs in the murine marrow microenvironment but reduces the ability to reconstitute the hematopoietic system of irradiated recipient transgenic mice (2). These findings contribute to a better understanding of the mechanisms regulating the balance between self renewal and quiescence and help to facilitate progress towards clinical application of Wnts as mediators of ex-vivo stem cell expansion or interruption of the Wnt pathway as potential anti-leukemic therapeutic strategy.
In Myeloma, the Prognostic Impact of Hyperdiploidy Is Mainly Related to the Gain of Chromosome 5
Authors: Loiseau Herve Avet , Li Cheng , Magrangeas Florence , Guerin Catherine , Gouraud Wilfried , Harousseau Jean-Luc , Attal Michel , Marit Gerald , Mathiot Claire , Facon Thierry , Moreau Philippe , Anderson Kenneth C , Campion Loic , Munshi Nikhil C. , Minvielle Stephane
Published: April 2020

Multiple myeloma is characterized by multiple chromosomal abnormalities, and virtually 100% of the patients are cytogenetically abnormal. Among the few recurrent changes described, hyperdiploidy is one of the most frequent. In cytogenetic series, hyperdiploidy is reported in 50 to 60% of the cases. It has been associated with prolonged survival in some series, whereas other series concluded to a neutral prognostic impact. These conflicting data might be related to the bias of cytogenetic analyses, which are abnormal in only 10 to 30% of the cases. In order to circumvent the pitfall of the low proliferation of plasma cells, we conducted a study based on DNA microarray. A series of 192 patients presenting multiple myeloma at diagnosis and treated homogeneously in tandem transplantation IFM trials were analyzed using the 500K Affymetrix SNP-arrays. In agreement with cytogenetic series, hyperdiploidy (> 47 chromosomes) was observed in 99 patients (53%). As previously reported, the most frequently gained chromosomes were chromosomes 3, 5, 7, 9, 11, 15, 19, 21, and X in female patients. We did perform a prognostic analysis. Hyperdiploid patients displayed a clear survival advantage as compared with non-hyperdiploid patients: p=0.006 and p=0.0008 for event free survival (EFS) and overall survival (OS), respectively. When adding all the chromosomal abnormalities observed on the SNP-arrays, 3 chromosomal lesions were independently associated with prognosis : gain of 1q, loss of 12p, and gain of chromosome 5. If serum b2- microglobulin (sb2m) level and t(4;14) were entered in the model, only sb2m, del(12p) and gain of chromosome 5, kept an independent prognostic value. These data suggest that most of the prognostic impact of hyperdiploidy could be linked to the gain of chromosome 5 (p=0.0001 and p<0.0001, for EFS and OS, respectively). In order to test this hypothesis, we did compare the survival of hyperdiploid patients with gain of chromosome 5 to that of hyperdiploid patients lacking chromosome 5 gain. As expected, patients with chromosome 5 gain displayed a significantly longer EFS and OS than those lacking this gain (p=0.005 and p=0.001 for EFS and OS, respectively). In conclusion, this study suggests that most of the prognostic value of hyperdiploidy is actually borne by chromosome 5.
Prospective Validation of the Peds Clot Clinical Decision Rule [Pdcr] in Hospital-Acquired Venous Thromboembolism: An Interim Analysis
Authors: Schultz Rachael F , Kwon Soyang , Sharathkumar Anjali , Bhat Rukhmi
Published: April 2020

Background: Venous thromboembolism (VTE) is an emerging hospital acquired complication in pediatric hospitals. Substantial efforts have been made to identify high-risk population based on exposure to risk-factors for VTE. Our group developed the PedsClot Clinical Decision Rule (Sharathkumar et al Journal of Thrombosis and Haemostasis, 10: 1326-1334) to identify this high-risk population based on a case-control study but this has not been validated yet.

Objective: To validate thePedsClot Clinical Decision Rule [PDCR] through prospective collection of data in a tertiary pediatric hospital. This study reports our findings of interim analyses for process evaluation.

Methods: This prospective data collection was performed using the Lurie Children's hospital automatic data import facility through the Enterprise Data Warehouse[study period: 02/01/2012-12/31/14]. Real time data was added to a research database of consecutive admissions based on following inclusion criteria: > 48 hour stay in the Intensive Care (Pediatric, Cardiac, Neonatal), Hematology Oncology and Infectious Disease units. Following variables were included in the dataset: age, sex, ethnicity, date of admission and discharge, ICU admission, central venous catheter (CVC), blood stream infection, immobilization, oral contraceptives, mechanical ventilation> 12hrs, and length of stay. Risk factors were weighed and scored as per PDCR rule. Chi-square tests were used to examine the association between the potential risk factors and VTE. PDCR model performance was evaluated by reporting the sensitivity and specificity.

Results: A total of 1722 children were eligible for the interim analyses. The demographic and clinical features of the dataset analyses and the odds ratio [OR] are detailed in Table 1 and 2 respectively. Of 1722 patients, 57 (3.3%) were identified as VTE, 50% were admitted to the ICU and 51% had a CVC. VTE was associated with age ≥ 13 years (AOR=2.3; 95% CI=1.3, 4.0), ICU admission (AOR=2.4; 95% CI=1.3, 4.4), and CVC (AOR=1.9; 95% CI=1.1, 3.3). The model performance showed that at the cut off point of 3, the specificity of the PDCR in predicting VTE was 75% but had a low sensitivity (37%). Risk prediction variables such as assessment of immobilization, use of oral contraceptives and prediction of hospital stay required manual entry into the datasheet; this makes data-capture labor intensive, especially for larger datasets.

Conclusions: This study utilized technological advances and database warehouse facility to validate PDCR. This interim data analyses indicates that in real life, PDCR has a high specificity but poor sensitivity in identifying children with predisposition for VTE. We are currently working to refine the risk model which may lead to a better model performance.

Disclosures

Sharathkumar: Bayer, Baxter, CSL Behring: Consultancy.
A Single Genetic Origin for a Common Caucasian Risk Factor for Venous Thrombosis
Authors: Zivelin Ariella , Griffin John H. , Xu Xiao , Pabinger Ingrid , Samama Michel , Conard Jacqueline , Brenner Benjamin , Eldor Amiram , Seligsohn Uri
Published: April 2020

A common genetic risk factor for venous thrombosis among Caucasoid subpopulations is a polymorphism, nt G1691A, in blood coagulation factor V that replaces Arg506 with Gln and imparts resistance of factor Va to the anticoagulant, activated protein C. Haplotype analyses using six dimorphic sites in the factor V gene for 117 Caucasian subjects of Jewish, Arab, Austrian, and French origin who were homozygous for nt A1691 compared with 167 controls (nt G1691) support a single origin for this polymorphism. The nt G1691A mutation is estimated to have arisen circa 21,000 to 34,000 years ago, ie, after the evolutionary divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations.
Graft versus host disease prophylaxis with low-dose cyclosporine-A reduces the risk of relapse in children with acute leukemia given HLA-identical sibling bone marrow transplantation: results of a randomized trial
Authors: Locatelli Franco , Zecca Marco , Rondelli Roberto , Bonetti Federico , Dini Giorgio , Prete Arcangelo , Messina Chiara , Uderzo Cornelio , Ripaldi Mimmo , Porta Fulvio , Giorgiani Giovanna , Giraldi Eugenia , Pession Andrea
Published: April 2020

Leukemia relapse is a major cause of treatment failure for patients with acute leukemia given allogeneic bone marrow transplantation (BMT). This study evaluated whether a reduction of the dosage of cyclosporine-A (Cs-A) used for graft versus host disease (GVHD) prophylaxis could reduce relapse rate (RR) in children with acute leukemia given BMT. Fifty-nine children who had transplantation from HLA-identical siblings were randomized to receive Cs-A intravenously at a dosage of 1 mg/kg/d (Cs-A1) or of 3 mg/kg/d (Cs-A3) until patients were able to tolerate oral intake. Subsequently, both groups received Cs-A orally at a dosage of 6 mg/kg/d, with discontinuation 5 months after BMT. The probability of developing grade II-IV acute GVHD was 57% for the Cs-A1 group versus 38% for the Cs-A3 group (P = .06); the probability of developing chronic GVHD was 30% for the Cs-A1 group and 26% for the Cs-A3 group (P = NS). Three patients died of grade IV acute GVHD: 2 were in the Cs-A1 and the third in the Cs-A3 group. The RR was 15% for the Cs-A1 group and 41% for the Cs-A3 group (P = .034); 1-year transplant-related mortality estimates were 17% and 7%, respectively (P = NS). With a median observation time of 44 months from BMT, the 5-year event-free survival for children belonging to Cs-A1 and Cs-A3 groups was 70% and 51%, respectively (P = .15). Our data demonstrate that the use of low Cs-A doses is associated with a statistically significant reduction of leukemia relapse, probably due to an increased graft versus leukemia effect.
A microsimulation model to assess the economic impact of immunotherapy in non-small cell lung cancer
Authors: Legoupil Clémence , Debieuvre Didier , Marabelle Aurelien , Michiels Stefan , Kapso Raïssa , Besse Benjamin , Bonastre Julia
Published: April 2020

Introduction

Immunotherapy has become the standard of care in advanced non-small cell lung cancer (NSCLC). We aimed to quantify the economic impact, in France, of anti-PD-1 therapy for NSCLC.

Methods

We used patient-level data from the national ESCAP-2011-CPHG cohort study to estimate time to treatment failure and mean cost per patient for the four label indications approved by the European Medicines Agency (EMA) for NSCLC in May 2018. To compute the budget impact, we used a microsimulation model to estimate the target populations of anti-PD-1 therapy over a 3-year period, which were combined with the annual cost of treatment.

Results

Overall, 11 839 patients with NSCLC were estimated to be eligible for anti-PD-1 therapy 3 years after the introduction of anti-PD-1 therapies. The mean annual cost per patient in the control group ranged from €2671 (95% CI €2149–3194) to €6412 (95% CI €5920–6903) across the four indications. The mean annual cost of treatment for the four EMA-approved indications of anti-PD-1 therapy was estimated to be €48.7 million in the control group and at €421.8 million in the immunotherapy group. The overall budget impact in 2019 is expected to amount to €373.1 million. In the sensitivity analysis, flat doses and treatment effect had the greatest influence on the budget impact.

Conclusion

Anti-PD-1 agents for NSCLC treatment are associated with a substantial economic burden.
Prevalence and incidence of, and risk factors for chronic cough in the adult population: the Rotterdam Study
Authors: Arinze Johnmary T. , de Roos Emmely W. , Karimi Leila , Verhamme Katia M.C. , Stricker Bruno H. , Brusselle Guy G.
Published: April 2020

Chronic cough is a common complaint in the general population but there are no precise data on the incidence of, and prospectively examined risk factors for chronic cough in a population-based setting. Therefore, we investigated the period prevalence, incidence and risk factors for chronic cough in adult subjects.

In a prospective population-based cohort study among subjects aged ≥45 years, data on chronic cough were collected on two separate occasions using a standardised questionnaire. Chronic cough was defined as daily coughing for at least 3 months duration during the preceding 2 years. Potential risk factors were gathered by interview, physical examination and several investigations.

Of the 9824 participants in this study, 1073 (10.9%) subjects had chronic cough at baseline. The prevalence of chronic cough increased with age and peaked in the eighth decade. In subjects aged <70 years, chronic cough was more common in women. During an average follow-up of 6 years, 439 incident cases of chronic cough occurred with an overall incidence rate of 11.6 per 1000 person-years (95% CI 10.6–12.8). In current smokers, the incidence of chronic cough was higher in men. In the multivariable analysis, current smoking, gastro-oesophageal reflux disease (GORD), asthma and COPD were identified as risk factors for chronic cough.

Chronic cough is common among adults and highly prevalent in the older population. Current smoking, GORD, asthma and COPD are independent risk factors for chronic cough. Individuals at risk of developing chronic cough may benefit from smoking cessation and control of the underlying disease.
Short paired-end reads trump long single-end reads for expression analysis
Authors: Freedman Adam H. , Gaspar John M. , Sackton Timothy B.
Published: April 2020

Background

Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases.

Results

At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75.

Conclusion

Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level.