bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)
Authors: Castilla-LLorente Cristina , Iwata Mineo , Mielcarek Marco , Abrams V. Kraig , Hwang Billanna , Nash Richard A. , Torok-Storb Beverly
Published: April 2020

Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.
Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis
Authors: Tibullo Daniele , Giallongo Cesarina , La Cava Piera , Guagliardo Provvidenza , Cavalli Maide , Romano Alessandra , Conticello Concetta , Chiarenza Annalisa , Palumbo Giuseppe A. , Di Raimondo Francesco
Published: April 2020

It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization).

Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression.

SM DA NI OM DA + OM NI + OM SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis. RUNX2 1,59 0,20 2,09 0,16 4,2 0,31 2,86 0,25 4,41 0,41 4,18 0,24 OCN 2,57 0,28 3,2 0,14 3,14 0,09 3,59 0,17 3,6 0,28 3,62 0,25 BMP-2 1,55 0,19 2,27 0,17 4,16 0,27 2,84 0,28 4,43 0,30 4,21 0,30
LSD1-Mediated Epigenetic Modification Is Important for TAL1 Function
Authors: Hu Xin , Li Xingguo , Ybarra River , Valverde Kristell , Fu Xueqi , Noguchi Constance , Qiu Yi , Huang Suming
Published: April 2020

TAL1/SCL is critical for normal and abnormal hematopoiesis by regulating hematopoietic stem/progenitor cell growth and differentiation. However, it is still unclear how its transcriptional activities are controlled during hematopoiesis. Here, we undertook the biochemical isolation of TAL1-associated protein complexes in erythroleukemia cells and showed that TAL1 interacts with histone demethylase LSD1 complexes containing LSD1, CoREST, HDAC1 and HDAC2. Interestingly, although TAL1 specifically colocalizes with LSD1 at the target gene promoter p4.2 in undifferentiated MEL cells, the recruitment of LSD1 is decreased at the p4.2 promoter upon induced MEL differentiation indicating that LSD1 may differentially regulate TAL1 target genes during differentiation. The siRNA-mediated knockdown of LSD1 in MEL and ES cells resulted in the derepression of p4.2 by increasing dimeH3K4 at their promoter region, respectively. Finally, we demonstrated that TAL1-associated LSD1 complexes, H3K4 demethylase, and histone deacetylase activities are coordinately regulated during erythroid cell differentiation. Thus, the data suggest that LSD1 mediated epigenetic modification may affect hematopoiesis and leukemogenesis through its association with the lineage-specific transcription factor TAL1.
A New Animal Model for Anemia Induced by Environmental Low-Temperature: Physiology of Erythrocyte Production and Circulation
Authors: Maekawa Shun , Iemura Hitomi , Kuramochi Yuko , Kosaka-Nogawa Nami , Nishikawa Hironori , Aizawa Youichi , Kato Takashi
Published: April 2020

To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.
Excessive Hematopoietic Stem Cell Proliferation Leads to Chromosomal Instability
Authors: Souza Liliana , Hawk Natalyn , Sengupta Sweta , Cabrera Carlos , McLemore Morgan L.
Published: April 2020

Truncation mutations in the granulocyte colony stimulating factor receptor (G-CSFR), common in severe congenital neutropenia (SCN), lead to excessive stem cell proliferation in response to G-CSF. These G-CSFR mutants are (at least indirectly) implicated in the progression of these patients to acute leukemia. Since SCN patients require continuous G-CSF treatment throughout their lifespan, we hypothesize that excessive stem cell proliferation can lead to DNA damage. Stem cells are relatively quiescent and rarely enter the cell cycle under normal conditions. During the cell cycle cells generate approximately 5000 single strand DNA lesions per nucleus (Vilenchik and Knudson, 2003). Approximately 1% of these lesions are ultimately converted to double strand DNA breaks (DSBs). Hematopoietic stem cells are found within the Sca+ ckit+ Lin- (KLS) population. Wild type and mice bearing a mutant G-CSFR similar to that found in patients with SCN were treated with G-CSF. After 21 days of treatment with G-CSF (10 ug/kg/day), the KLS population in the bone marrow increased four-fold in wild type mice and eight-fold in mutant mice. We isolated Lin-Sca+ bone marrow cells from these G-CSF treated mice and evaluated for the presence of double stranded DNA breaks by staining with anti-phospho-H2AX by immunofluorescence. H2AX is a histone whose phosphorylated form localizes to the site of double stranded DNA breaks. The results showed that there is an 8-fold increase in the DSB in wild type Lin-Sca+ and 10-fold in mutant Lin-Sca+ when compared to cells from untreated mice. This data suggests that excessive proliferation can contribute to an increase in DSBs in hematopoietic stem cells. Investigation of potential mechanisms contributing to DSB formation are ongoing. Understanding the causes and trends of chromosomal instability would improve our understanding of leukemogenesis and potentially reveal novel treatment strategies.
Quantification of Mouse Lymphohematopoietic Stem CellsUsing IR-Spectroscopy
Authors: Kapelushnik Joseph , Dror Ziv , Iskovich Sevtlana , Mordechai Shaul , Zelig Udi
Published: April 2020

Stem cells and in particular, Hematopoietic stem cells (HSCs) therapy is valued for its potential to restore damaged or degenerating tissues. Transplantation of HSCs is already a routine procedure for treatment of blood and immune system disorders. Till now complicated and expensive procedures were needed for identification and isolation of HSC. In the present research we propose a simple and reagent-free procedure for identifying and quantifying lymphohematopoietic stem cells (LHSC) using Fourier transform infrared (FTIR) spectroscopy, which is able to monitor content and conformation of all bio-molecules in cells simultaneously. B6D2F1 mice, 6 to 12 weeks of age were used for this study. Bone marrow (BM) from male B6D2F1 mice was drawn. Putative LHSC were isolated from the BM by common methods according to cell size, markers and high levels of aldehyde dehydrogenase. After isolation of LHSC, various dilutions of LHSC among BM cells were performed and measured under FTIR-microscope. Using spectral and statistical analysis, we identified several spectral markers which characterize and distinct LHSC from pure BM. These markers refer to nucleic acids which increase in LHSC due to their extensive proliferation and differentiation. Furthermore, we achieved high sensitivity quantification of LHSC from BM. (about one LHSC per thousand BM cells) Thus, we conclude that the proposed method of FTIR spectroscopy identification and quantification of LHSC may significantly contribute to stem cells research and hearten the use of stem cells in medicine.
Circulating Endothelial Cells: A Potential Parameter of Organ Damage in Sickle Cell Anemia?
Authors: Strijbos Michiel , Landburg Precious , Nur Erfan , Biemond Bart J. , Leebeek Frank , Gratama Jan-Willem , Duits Ashley , Schnog John-John
Published: April 2020

Traditional laboratory markers of disease severity (Hb concentrations, HbF%, LDH concentrations and leukocyte counts) are not powerful enough to identify individuals with sickle cell disease (SCD) at high risk of complications. In several disease states, the number of circulating endothelial cells (CEC) is indicative of the extent of vascular disease and several small studies employing cumbersome techniques have shown increased CEC to occur in SCD as well. In this pilot study we determined if, by employing the CellSearch system, elevated CEC can be detected in SCD and whether the CEC count is related to the presence of organ damage, painful crisis frequency, traditional laboratory markers of disease severity as well as markers of endothelial activation. Peripheral blood was collected from 15 sickle cell patients and 15 healthy sex, age and race matched HbAA controls. Cells positive for CD146 (present on endothelial cells) are immuno-magnetically isolated and stained with nuclear DAPI and anti-CD105 (present on endothelial cells) and anti-CD45 (pan-leukocyte marker). Within the CD146 enriched cell suspension, cells meeting the DAPI+/CD146+/CD105+/CD45− criteria after image cytometry are designated CEC. CEC counts were significantly higher in sickle cell patients (12 cells/mL, IQR 8–29) as compared to controls (4 cells/mL, 3–10) (p=0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (p=0.02), and increased with the presence of number of affected organs as well (0–4 involved organs, p=0.03), with the number of affected organs being the most important determinant of increased CEC numbers in SCD (R2=0.72, P=0.001). No statistically significant correlation was detected between CEC counts and markers of endothelial activation (serum sVCAM-1 and plasma VWF:Ag levels). CEC counts did not differ between patients on hydroxyurea and those who did not use hydroxyurea. No statistically significant correlations were detected between CEC counts and painful crisis frequency, Hb, HbF%, leukocyte counts, LDH concentrations. In conclusion, elevated CEC’s can be demonstrated in the clinically asymptomatic state in patients with sickle cell anemia with a validated and automated technique and, for the first time, a relation of CEC counts to SCD related organ damage is demonstrated. Furthermore, the extent of EC activation (as assessed by sVCAM-1 and VWF:Ag levels) is not necessarily related to the extent of EC damage (as assessed by CEC counts).
The Plasma Concentration of the Antithrombotic Protein Annexin A5 Is Elevated in Sickle Cell Disease and Rises Further during Painful Crisis
Authors: van Tits Berry , van Heerde Waander , Landburg Precious , Muskiet Frits , Duits Ashley , Schnog John-John
Published: April 2020

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles plays an important role in the etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells, exhibits high affinity for PS and has been shown to inhibit microparticle-induced PS-mediated thrombin generation. We determined plasma annexin A5 levels and microparticles in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty five HbAA blood donors served as controls. Both annexin A5 levels and microparticle numbers were highest in HbSS patients (5.7 ng/mL IQR 3.7–7.6 and 37.9 nM, IQR 31.9–69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7–7.6 and 20.9 nM IQR10.9–29.6) and healthy controls (2.5 ng/mL, IQR 1.4–4.4 and 13.1 nM, IQR 9.5–18.5) (p=0.01 and p<0.001, respectively). At presentation with a painful crisis, annexin V levels and microparticle numbers increased significantly in 16 of 17 patients (p=0.001) and 16 of 17 patients (p<0.001), respectively. Annexin V/microparticle ratio’s were lower at presentation with a painful crisis in 9 patients. In conclusion, sickle cell patients have elevated plasma concentrations of annexin V. The potential role of annexin V in modulating PS related pathophysiological processes in SCD is subject of further investigation.
Cardiopulmonary Testing and Pulmonary Hypertension in Patients with Sickle Cell Anemia
Authors: Chapdelaine Hugo , Soulieres Denis , Poirier Claude
Published: April 2020

Objectives: Sickle cell disease (SCD) is responsible for multiple complications that can lead to dyspnea, including pulmonary hypertension (PAH). PAH is a common cause of death among these patients and represents a prognosis feature, but can be clinically difficult to detect. We reviewed the charts of patients with SCD and analyse correlation between hemoglobin level, EKG, chest X-ray, pulmonary function tests, and cardiopulmonary exercise test (CPET) with echocardiography.

Methods: We performed a retrospective chart review of 25 patients with SCD (11 patients with SC, 12 patients with SS and 2 patients with S-β thalassemia haplotype). All patients had pulmonary functions testing, CPET and resting echocardiography in the same period. PAH was defined as tricuspid regurgitant jet velocity (TRJV) >2,5 m/s on cardiac echography. Subjects underwent symptom-limited CPET using cycle ergometry and individualized ramp protocol. Exercise was stopped when subject showed criteria of maximality or inability to keep the pace. Breath-by-breath analysis was done.

Results: Only 3 patients had PAH. All these patients were SS and suffered numerous acute thoracic syndrome. No correlation were made between lower hemoglobin level and higher TRJV; no patients had EKG or CXR anomaly. DLCO/VA appeared to be a independent risk factor: DLCO/VA < 75 % correlated with a TRJV > 2,5. CPET was abnormal in all patients, but were limited by patient fatigue and patterns were consistent with peripheral muscle disease except for one patient who had PAH; this patient showed a higher VE/VCO2 and a slower pulse kinetics, compatible with pulmonary vasculopathy.

Conclusions: Decrease DLCO/VA may indicate a sickle pulmonary vasculopathy. and this factor appears to play a significant role in detecting PAH in SCD patients. CPET was a safe procedure and gave details about global aerobic performance and exercise tolerance. Because of peripheral muscle limitations, CPET still an unreliable diagnostic test for PAH. Peripheral muscle dysfunction could explain a large part of exercise intolerance, but physiopathologic mechanisms still underexplored.
Differential Effect of the Histone Deacetylase Inhibitors Vorinostat and MGCD0103 in Cell-Free and Cell-Based Assays
Authors: Buglio Daniela , Khaskhely Noor M , Georgakis George V , Younes Anas
Published: April 2020

Recent clinical trials demonstrated a promising clinical activity of a variety of Histone deacetylase inhibitors (HDACi) in patients with relapsed non-Hodgkin and Hodgkin lymphoma (HL). In this study, we examined the in vitro activity of the pan- DAC inhibitor vorinostat and the isotype selective HDAC inhibitor MGCD0103. Using a cell free fluorogenic peptide-based substrate assay, both MGCD0103 and vorinostat were potent inhibitors of class I HDACs, with vorinostat also effectively inhibiting HDAC6. Subsequently, we examined the effect of these two compounds on three HL-derived cell lines (HD-LM2, L-428, and KM-H2). Although these cell lines had a similar expression level of class-I HDAC proteins, as determined by western blot analysis, they were more sensitive to MGCD0103 in short term culture. After 72 hours of incubation, the IC50 of MGCD0103 was consistently <0.5 μM compared with >1.5 μM for vorinostat. This differential antiproliferative effect was also observed at the molecular level. Submicromolar concentrations (as low as 0.1 μM) of MGCD0103 effectively induced the expression of p21 and the acetylation of H3 histones, whereas higher concentration of vorinostat were required to achieve a similar effect. Furthermore, we examined the effect of equimolar concentrations of MGCD0103 and vorinostat on cytokine and chemokine secretion, using a multiplex cytokine array (34 cytokines/chemokines), on a selected panel of gene expression using pathway PCR array, and on selected protein expression using pathway protein array (phospho kinases and apoptosis pathways) and multicolor FACS experiments. While vorinostat and MGCD0103 shared similar effects on a variety of these elements, MGCD0103 was more effective in upregulating p21 expression, activating the intrinsic caspase pathway, and favoring the secretion of Th1-type cytokines. Given the recent reported results from two independent phase-II studies in patients with relapsed HL that demonstrated higher response rate achieved with MGCD0103 compared with vorinostat, our results suggest that cell-based assays may have a better predictor value of the potential clinical activity of HDACi in HL compared with the cell-free assays.
Incidence and Prognostic Value of FLT-3 and NPM1 Genes Abnormalities in Acute Myeloid Leukemia in the Côte D’or Population, France
Authors: Maynadié Marc , Courtois Martine , Mounier Morgane , Janoray-Manivet Ines , Lafon Ingrid , Guy Julien , Carli Paule-Marie , Girodon François
Published: April 2020

Context: In acute myeloid leukemia (AML), the recently described FLT-3 and NPM1 genes abnormalities were found to have a prognostic value in AML with normal karyotype and a specific therapeutic strategy was proposed according to these abnormalities. We look for the incidence and prognostic value of these abnormalities in cases diagnosed on a well defined population.

Material and Methods: AML diagnosed according to WHO classification between 01/01/2001 and 31/12/2006 in the population of the Côte d’Or department, were included. Karyotype analyses were performed in 81% of the cases. The FLT3 D835 mutation, the FLT3 internal duplication (ITD) and the NPM1 mutation were systematically studied on the biological material of the diagnosis cryopreserved in the Ferdinand Cabanne Biobank of Burgundy, by PCR and DNA sequencing techniques. The vital status of the patients was update on 31/10/2007. The relative survival was calculated with the STATA (V9) software.

Results: 100 de novo AML and 47 secondary AML (sAML) were registered (72 females and 75 males). The world standardized incidence rate was 2.4 in men and 1.5 in women for de novo AML instead of what it was respectively of 1.1 and 0.6 in sAML. The urban predominance was present in both type of AML. The karyotype was normal in 38% (45/119) of cases (35% of de novo AML and 21% of sAML). It was abnormal in 62% of cases (74/119)(51% of de novo AML and 49% of sAML). Molecular analyses were performed in 78 de novo AML and in 24 sAML. FLT3 ITD was present in 19% (15/78) de novo AMl and in any sAML. The FLT3 D835 mutation was present in 6.5% of de novo AML and in 8% of sAML. NPM1 was mutated respectively in 26% and 4% of the cases. There was a significantly higher WBC count and proportion of blast cells in peripheral blood in FLT3 ITD cases. Overall and relative survivals of FLT3 ITD cases were decreased compared to FLT3 wild type cases. No difference according to NPM1 status was found.

Conclusions: These data confirm the bad prognostic value of FLT3 ITD status in AML observed in clinical series. Furthermore their particular interest lies in the fact that they are the first molecular data in AML produced on a population-based series indicating the feasibility of such epidemiological studies.
Clonal Profile Analysis of Leukemic Progenitors and Diagnosis Blast Cells in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia
Authors: Veltroni Marinella , Paganin Maddalena , Frasson Chiara , Fabbri Giulia , Marzollo Antonio , Seganfreddo Elena , Giarin Emanuela , Fortunato Elena , Aricó Maurizio , Basso Giuseppe
Published: April 2020

Recent studies suggest that the majority of malignant cells found in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) arise from a rare population of leukemic progenitors. Little information is available on the presence of clonal rearrangements in cells at the stage of early precursor. To address this issue we analyzed clonality profile of early leukemic precursors sorted by flow-cytometry. Leukemic cells were obtained from bone marrow samples collected at diagnosis from 6 patients with childhood BCP-ALL. Furthermore, bone marrow cells were collected from 3 healthy children who were harvested for bone marrow donation. Three subpopulations of leukemic cells were investigated: total unsorted blasts, the sorted CD34+/CD38−/CD19+, and the sorted CD34+/CD38−/CD19− cells. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were screened by polymerase chain reaction (PCR) in the sorted populations and in the bulk leukemic cells in order to identify molecular markers of clonal evolution. Sequence analysis was then performed on the N-region. Overall, a total of 38 different Ig/TCR gene rearrangements were identified in the 3 cell populations under study (total blasts, CD34+/CD38−/CD19+, and CD34+/CD38−/CD19−). Of them, 13 (34%) were found in the three populations; 12 (31%) were found in two of the three populations: 7 in total blasts and CD19+ subset, 3 in total blasts and CD19−, 2 in CD19− and CD19+; finally, 13 were found only in one subpopulation: 4 in total blast cell, 5 in CD19+, 4 in CD19−. In all the six patients studied, BCP-ALL progenitors CD34+/CD38−/CD19− and CD19+ and the bulk tumor blasts shared at least one Ig/TCR gene clonal rearrangement with the same N-region. In 5 out of 6 patients at least one rearrangement detected in the BCP-ALL progenitors was undetectable in total blasts. Conversely, in 3 patients the clonal rearrangement observed in the bulk leukemic cells was not identified in any of the two sorted ALL precursor populations. Clonal rearrangement was never detected in the samples from healthy bone marrow donors. Our findings confirm that clonal rearrangement may be detected at the stage of early B-lineage precursor CD34+/CD38−/CD19−, suggesting that leukemic transformation may occur at this stage or even before in BCP-ALL. We plan to extend this observation by repopulating studies in NOD/SCID mice.
Down-Regulation of MicroRNA-143 and -145 in Childhood B-Lineage Acute Lymphoblastic Leukemia at Initial Diagnosis and in Relapse but up-Regulated When in Remission
Authors: Yang Yung-Li , Jou Shiann-Tarng , Lin Shu-Wha , Lin Dong-Tsamn , Hu Chung-Yi , Chang Sheng-Kai , Yen Ching-Tzu , Chiou Rong-Jing , Lin Kai-Hsin , Lu Meng-Yao , Chang Hsiu-Hao , Lin Shu-Rung
Published: April 2020

Inappropriate expression of microRNAs (miRNAs) is strongly associated with leukemogenesis. miRNAs (miRs)-143 and -145, previously shown to be reduced in colon cancers, have been demonstrated recently to be down-regulated in B-cell malignancies including chronic lymphoblastic leukemia, B-cell lymphoma, Burkitt’s lymphoma. In this study, we determined the role of miR-143 and miR-145 in childhood leukemia treated by the standard TPOG (Taiwan Pediatric Oncology Group)-2002 protocols approved for treatment of patients in Taiwan. Ten patients with childhood B-lineage ALL and four ALL leukemia cells lines including REH (CRL-8286TM), CCRF-SB (CCL-120TM), RS4 (CRL-1873TM), and SUP-B15 (CRL-1929) were measured for the miRNA expression using a TagMan quantitative RT-PCR method. Our results showed that miR-143 and miR-145 were both down-regulated 0.09 and 0.19 times individually (n= 13, p <0.001 and p = 0.011) in mononuclear cells in bone marrow of newly-diagnosed and relapsed samples in comparison with the same cell types of remission samples (n=16). To examine possible suppressive functions of miR-143 and miR-145 with respect to cell growth, the REH cells were used for expression with precursor form and mature form miR-143 and miR-145, and subsequently measured for cell growth rate. Preliminary results showed that the two miRNAs did not alter the growth rate of the REH over-expressing either miRNA. Taken together, we have identified miR-143 and miR-145 as biomarkers that may differentiate malignant and normal B cells. miR-143 and miR-145 may contribute to leukemogenesis in childhood B-lineage ALL by new yet to be defined mechanisms.
Complete Loss of the RUNX1 Gene: A Review of the Implications for Leukemogenesis and Patient Management
Authors: Stephenson Christine F. , Valenzuela Martha , Iyer Uma , Richmond Lioudmila
Published: April 2020

The RUNX1 (AML1) gene is a transcription factor that regulates expression of genes involved in hematopoietic cell differentiation. It is a gene located on chromosome 21 at q22. Genetic alterations of RUNX1 whether through loss-of-function point mutations, translocation or amplification are known to impact myeloid differentiation and trigger leukemic transformation in particular with respect to myelodysplasia and acute myeloid leukemia. However, while there are many articles describing the impact of these types of RUNX1 genetic alterations, there is a paucity of information regarding loss of the entire RUNX1 gene. The case in this abstract highlights the significance of understanding loss of the RUNX1 gene. An 87 year old patient presented for evaluation for anemia and leukopenia. Flow cytometric evaluation revealed 26% myeloid blasts and confirmed a diagnosis of acute myeloid leukemia (AML). The cytogenetic findings demonstrated a translocation between chromosomes 17 and 21 −t(17;21)(q11.2;q22). The dilemma then was to determine if this was a variant of the traditional t(15;17) associated with acute promyelocytic leukemia or a variant of the t(8;21) associated with the M2 subtype of AML. FISH studies determined that there was no involvement of the RARA gene and no evidence of a RUNX1/ETO rearrangement. However, there was a complete loss of RUNX1 on the abnormal chromosome 21. Thus, what appeared to be a balanced translocation included a cryptic loss of RUNX1. While this may be an interesting case presentation the more pertinent question is what is the impact of the RUNX1 loss? This case prompted a review of the data on complete loss of the RUNX1 gene which while limited suggests that RUNX1 loss on its own is not leukemogenic. This poster presents the data and implication of complete loss of RUNX1, the role of this loss in leukemogenesis and patient management.
Erythropoietin and Cyclophosphamide Combined Treatment Additively Enhances Immunoglobulin Production in Mice
Authors: Lifshitz Lilach , Berger Assaf , Avneon Maayan , Mittelman Moshe , Neumann Drorit
Published: April 2020

Erythropoietin (EPO) is an important component in the treatment of cancer patients for improvement of cancer related anemia. EPO treatment for cancer related anemia is usually combined with chemotherapy. Cyclophosphamide (CP) is a known cytotoxic alkylating agent widely used in cancer chemotherapy. While at high doses it functions as an immunosuppressive agent, the anti-neoplastic activities of CP at low doses are attributed to enhancement of cellular and humoral immunity e.g. (Berd et al., Cancer Res; 1984). We have previously shown that EPO displays anti-neoplastic activities (Mittelman, 2001, 2004) and that EPO treatment is associated with enhancement of both the humoral and cellular immune responses (Prutchi-Sagiv 2006, Katz 2007). Here we focused on a murine model of DNP-KLH-injection, used to assess the humoral response in mice. Recently we demonstrated that administration of high doses of EPO (180U×3) to DNP-KLH-injected C57BL mice resulted in an increase in anti-DNP immunoglobulin G1 (IgG1) production. The present study was designed to examine the effect of combining low dose CP (12.5mg/kg ×2) used to achieve an anti-neoplastic activity, with a lower dose of recombinant human EPO (rHuEPO; 90U×3) on the humoral immune response of the DNP-KLH-injected mice, thus simulating the conditions of patient care. Hence, we compared anti-DNP Ig serum levels in DNP-KLH-injected C57BL mice that were treated with either EPO or CP alone, or the combination of CP and EPO (CP-EPO). CP treatment alone resulted in increased levels of serum anti-DNP IgG1 (O.D.(CP) = 0.38±0.06 vs O.D.(Non treated) = 0.18±0.064). In contrast, EPO treatment alone enhanced serum levels of IgG2 (O.D.(EPO) = 0.47±0.09 vs O.D.(Non treated) = 0.18±0.069). CP or EPO alone did not affect the total levels of anti-DNP total Ig (O.D.(EPO) = 0.37±0.07 vs O.D.(Non treated) = 0.28±0.04). Yet, the combined CP-EPO treatment resulted in increased levels of anti-DNP total Ig (O.D.(EPO+CP) = 0.48±0.05 vs O.D.(EPO or CP) = 0.37±0.04), maintaining the higher levels of IgG1 (O.D.(EPO+CP) = 0.38±0.06) and IgG2 (O.D.(EPO+CP) = 0.49±0.1). In summary, the combined CP-EPO treatment additively improved immunoglobulin production, compared to treatment with CP or EPO alone. We thus demonstrate that in context of chemotherapy treatment as usually administered in the clinic, EPO can enhance humoral immunity alongside its erythropoietic activities. Our findings emphasize the role of EPO as an immunomodulator, particularly when given as treatment in a combined therapeutic panel
Proteinase 3 Mediates Direct Inhibition of CD4 and CD8 T Lymphocyte Proliferation by Inducing Enzyme-Independent G0/G1 Cell Cycle Arrest
Authors: Yang Tian-Hui , Quintanilla Kathryn E , Cortez Amy M , Molldrem Jeffrey J.
Published: April 2020

Proteinase 3 (P3), a serine protease found in primary granules in granulocytes, is the target of T cell- and B cell-mediated autoimmunity in Wegener’s granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). Although aberrant P3 and neutrophil elasase (NE) expression in leukemia increases susceptibility to PR1-CTL-mediated killing, overexpression of P3 also induces apoptosis of the high affinity PR1-CTL leading to deletional tolerance and leukemia outgrowth. Because expression of P3 and NE in sera from leukemia patients is increased by 5-fold compared to healthy controls, we sought to determine whether such overexpression of P3 or NE impairs PR1-CTL immunity to leukemia by a direct affect on T lymphocytes. To study this, T cells from healthy donors were activated by anti-CD3 and anti-CD28 and exposed to increasing concentration of P3 or NE over one to five days, and the percentage of apoptotic cells and cell proliferation were determined by flow cytometry using PI, anti-Ki-67, and CFSE. P3, but not NE, induced dose-dependent apoptosis of up to 30% of T cells, and in the non-apoptotic cells, a 50% inhibition of CD4 and CD8 T cell proliferation at 1 μg/ml and 100% at 10 μg/ml compared to untreated cells. This effect was not enzyme-mediated since prior exposure of P3 to 56°C or co-incubation with the serine protease inhibitors Elafin and alpha-1 antitrypsin showed no affect on apoptosis or cell proliferation. P3 induced a cell cycle arrest at the G0/G1 interface, determined with PI and Ki-67 staining of healthy donor T cells that were exposed to P3 for up to 3 days. In contrast, at protein concentrations up to 25 μg/ml, NE showed no such inhibitory effect on apoptosis or cell proliferation. In addition to its role as a leukemia-associated antigen, P3 is also targeted by the cANCA antibody in patients with WG and the serum titer correlates with disease activity. Therefore, we hypothesized that the effect of P3 on T cell proliferation might also be affected by humoral immunity during circumstances of systemic autoimmunity. Co-incubation of P3 with a molar excess of cANCA reversed P3- mediated inhibition of both CD4 and CD8 T cells, consistent with a role of this antibody and the P3 target antigen in controlling T cell autoreactivity. Taken together, this data shows a new role for P3 in regulating T cell proliferation, which occurs only at high P3 concentration, similar to P3 in sera from leukemia patients, which is not enzymedependent. This supports a direct role for P3 in regulating both anti-leukemia immunity and autoimmunity. This data will need to be considered for effective immunotherapy targeting P3 in leukemia patients and these inhibitory effects also suggest a role for P3 in regulating autoimmunity at sites of inflammation, such as in patients with WG.
Mono-Chlorinated 3-Carboxy-7-Hydroxycoumarin: A Highly Fluorescent and Water-Soluble Violet Excitable Dye for Cell Analysis
Authors: Abrams Barny
Published: April 2020

In our search for new violet excitable dyes with improved photochemical properties, we examined several halogen substituted 7-hydroxycoumarins and found that chlorinated derivatives could be as bright as their fluorinated analogs. In particular, mono-chlorinated 3-carboxy-7-hydroxycoumarin (V450) has a quantum yield equal to 3-carboxy-6,8- difluoro-7-hydroxycoumarin (Pacific Blue™). Antibodies specific to various cell surface markers (CD3, CD4, CD45) were labeled with V450 and tested in single color flow cytometry assays. Our results show that conjugates of V450 are brighter than the same clones labeled with Pacific Blue™. Additional studies were performed in which V450-antibody conjugates were included in 8-color cocktails, along with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 conjugates, aimed at subsetting lymphocytes and myeloid cells. These cocktails met all standards of performance for the particular applications for which they were designed, including stability, lack of interaction between components, and brightness. The new V450 fluorochrome proved to be compatible with both FACSLyse and PharmLyse processing of EDTA anti-coagulated whole blood, and showed no signs of adverse effects on unfixed cells.
Antitumour Effects of Exosomes in Combination with CTX and Poly I:C
Authors: Chang Chunkang , Guo Fang , Fan Huahua , Li Xiao
Published: April 2020

Exosomes are vesicles that are approximately 30–100 nm in diameter and are produced by different cell types. Exosomes derived from dendritic cells (DCs) expressing major histocompatibility complex class I/II molecules and T-cell costimulatory molecules are useful in antitumour therapy. Here we examined the antitumour effects of cyclophosphamide (CTX), polyinosinic-polycytidylic acid sodium salt (poly I:C) and exosomes. Although exosomes could be used to treat tumour, the therapy effect was unsatisfied. In order to enhance the therapy effect, we used Poly I:C and CTX. Poly I:C had been proved to enhance DC maturation. CTX could suppress Treg cells. Tumour growth and survival time were monitored. We have established that treatment with exosomes prolongs the survival time,. Treatment with the combination of CTX + exosomes + poly I:C not only suppressed tumour growth but also prolonged survival time. Unexpectedly, poly I:C could not suppress tumour growth and prolong survival time in the group treated with CTX + poly I:C. Because we did not study the group treated with poly I:C + exosomes, we could not determine whether poly I:C provides beneficial effects in this combination. We have established that exosomes can prolong survival time And therapy with exosomes in combination with CTX and poly I:C not only suppressed tumour growth but also prolonged survival time.
Survival Benefit of Post Induction Consolidation Therapy in MCL (Mantle Cell Lymphoma): a Polish Lymphoma Research Group (PLRG) Retrospective Multicenter Analysis
Authors: Jurczak Wojciech , Giza Agnieszka , Krochmalczyk Dorota , Zimowska-Curylo Dagmara , Sobocinski Marcin , Stella-Holowiecka Beata , Holowiecka-Goral Aleksandra , Boguradzki Piotr , Kisiel Elzbieta , Mazur Grzegorz , Wrobel Tomasz , Knopinska-Posluszny Wanda , Warzocha Ewa Kalinka - , Skotnicki Aleksander B.
Published: April 2020

Background: In MCL, early intensification and consolidation of the first line therapy by ASCT is the treatment of choice. Elderly age and co-existing co-morbidities makes it however feasible for less than a third of patients.

Methods: All MCL cases consulted in 8 PLRG centers within the last 5 years (n=140) were included in a retrospective analysis. Only 23% (n=32) were consolidated with ASCT, further 28% (n=40) by radioimmunotherapy (Ibritumomab), while in 49% (n=68) neither consolidation was performed. Rituximab was used in 36/72 patients subjected to consolidation and 25/68 treated without consolidation. There were no statistically significant differences in IPI, CS (clinical stage), frequency of extranodal manifestations and B symptoms between analyzed subgroups (Table 1) although patients subjected to ASCT were younger (median age 54 vs 62) and tend to have higher LDH (556 IU vs 473), while those who were not consolidated more frequently had a large tumor burden (defined as a mass > 7 cm, 24 vs 15%).

Results: There was a clear impact of consolidation on Overall and Progression Free Survival (OS,PFS): at 5 years OS 65 vs 20 % (p= 0.0003 in Gehan Wilcoxon test); and PFS 40 vs 0 % (p= 0.0003 in Gehan Wilcoxon test). Rituximab used in the first line therapy further increased it’s efficiency in terms of PFS, prolonging median time to progression from 15 to 26 months, however the OS benefit was seen only in consolidated patients (at 5 years 75% OS in those with Rituximab including induction followed by consolidation).

Conclusion: With all limitations of retrospective analysis, it strongly supports the necessity of post induction therapy in MCL patients. The role of ASCT is established in younger patients, radioimmunotherapy may prove to be a feasible approach for the elderly and unfit ones.

N IPI Age LDH WHO CS (IV) Extra-nodal B sympt. X (>7 cm) Radioimmunotherapy 40 2,49 61,7 542 1,43 83% 78% 63% 12% ASCT 32 2,62 54 556 1,24 80% 84% 68% 17% No Consolidation 68 2,98 62,8 473 1,74 85% 83% 68% 24% All cases 140 2,76 60,13 497 1,55 81% 81% 67% 20%
Treatment of Good Prognosis CD20+ Large-Cell Lymphomas in Patients Aged 65 to 80, with 3 Cycles of R-CHOP14 Followed by An Injection of 90Y Ibritumomab Tiuxetan
Authors: Frederic Peyrade , Gérard Lepeu , Provencio Mariano , Lauris Gastaud , Xavier Fontana , Jean-Marc Broglia , Patricia Carrier , Emmanuel Chamorey , Christophe Hebert , Thyss Antoine
Published: April 2020

Background : Treatment of newly diagnosed DLBCL in patients aged 65 to 80 is based on 6 to 8 cycles of R-CHOP at 14 to 21 day intervals. Recent data suggest that for patients with an IPI of 0 or 1, a shorter treatment may be considered. Furthermore, early negative PET-CT results after chemotherapy are an important favourable prognostic factor.

Patients and methods : We propose carrying out two cycles of R-CHOP 14 in patients aged 65 to 80 with newly diagnosed DLBCL and an IPI of 0 or 1. PET-CT will be performed on D28. If the results are normal, an R-CHOP cycle followed by an injection of ibritumomab tiuxetan 90Y will be administered. The anticipated total treatment period is 51 days. Ten patients were included from June 2007 to June 2008 (7 women and 3 men), the age-adjusted IPI was 0 and 1 in 6 and 4 cases, respectively. None of the patients had bone marrow involvement and the PS was 0 or 1 in every case.

Results : CT and PET-CT showed complete remission in 9 out of 10 patients, who therefore received the full protocol. The average age was 72 (range, 68–77). The average duration of treatment for these patients was 54 days. As regards to safety, hospitalisation or the prescription of antibiotics was not necessary in any case. No serious adverse event was reported. Mean platelet and leukocyte nadirs were 23,000/mm3 and 1,180/mm3, respectively. Two transfusions of red cell concentrates and one of platelet concentrate were necessary in one patient. To date, all patients are in complete remission.

Conclusion : The treatment regimen of 3 cycles of R-CHOP14 followed by an injection of 90Y ibritumomab tiuxetan appears to be tolerated by patients aged 65 to 80 with newly diagnosed DLBCL. Longer follow-up and the inclusion of more patients are necessary to confirm these preliminary data.