bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiphospholipid Antibody Syndrome: The Flow Cytometric Annexin A5 Competition Assay as a Diagnostic Tool.
Authors: Tomer Aaron , Bar-Lev Shira , Fleisher Stela , Shenkman Boris , Friger Michael , Abu-Shakra Mahmoud
Published: October 2019

The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient’s aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73∴3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72∴2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97∴0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.
Differential Procoagulant Phenotype of Pancreatic and Breast Cancer Cells Related to Different Tissue Factor Activity.
Authors: Gerotziafas Grigoris T. , Elalamy Ismail , Roman Marie-Paule , Prengel Claudine , Verdy Elisabeth , Bernaudin Jean-François
Published: October 2019

Tissue factor (TF) expressed by some cancer cells is implicated in metastasis and angiogenesis. The influence of cancer cells on blood coagulation has not been adequately studied. We evaluated the procoagulant potential of pancreatic and breast cancer cells (BXPC3 and MCF7 cell lines respectively) when they are in contact with human platelet-poor plasma (PPP). At 40% and 90% confluence, adhesive cultures of BXPC3 and MCF7 cells were treated with trypsine according to standardized procedure and cancer cells were suspended in normal human platelet poor plasma (PPP) at increasing concentrations. Coagulation was triggered by CaCl2 addition and thrombin generation (TG) was monitored using the Calibrated Automated Thrombogram-Thrombinoscope® (Biodis-France). In some experiments, cancer cells were incubated for 30 min with a polyclonal specific anti-TF antibody (American Diagnostics). Cancer cells accelerated TG by decreasing significantly lag-time, and time to Peak of thrombin (ttPeak) but they did not significantly influence the endogenous thrombin potential. BXPC3 had significantly more potent procoagulant activity compared to MCF7 cells. Incubation of cancer cells with anti-TF antibody resulted in a concentration dependent inhibition of their procoagulant effect. The IC50 of the anti-TF antibody for TG induced by BXPC3 was about 10-fold higher to that for MCF7. Pancreatic cancer cells (BXPC3) and breast cancer cells (MCF7) accelerate thrombin generation of human plasma in a TF-dependent manner. BXPC3 have more potent procoagulant activity than MCF7 probably due to increased TF expression.
Combined Antithrombotic Therapy but Not Single-Agent Therapy with Low-Dose Aspirin Deteriorated Platelet Activation in Patients with Antiphospholipid Syndrome (APS).
Authors: Yamazaki Masahide , Kadohira Yasuko , Morishita Eriko , Asakura Hidesaku , Nakao Shinji
Published: October 2019

Antiphospholipid syndrome (APS) is one of autoimmune disorders characterized with recurrent thromboses. It is considered that plasma levels of D-dimer and prothrombin fragment 1+2 (F1+2) could reflect hypercoagulable state in APS patients and those monitoring may be useful for a predictor of venous thromboses. In contrast, the estimation of platelet activation is difficult. Recently, plasma levels of platelet-derived microparticle (PDMP), which released from activated platelets, could be measured using ELISA supplied from JIMRO Co. JAPN (“JIMRO test PDMP”). We previously presented that plasma levels of PDMP were significantly elevated in APS-patients with arterial thromboses and that could reflect platelet activated condition in vivo. To evaluate the preventory effect of thromboses and the inhibitory effects for platelet activation by several kinds of antithrombotic agents in APS, 30 APS patients with cerebral infarctions were randomly treated with low-dose aspirin alone (Group A, n=10), aspirin plus warfarin (Group B, n=10), or aspirin plus cirostazole (Group C, n=10). Their plasma PDMP levels were measured using “JIMRO test PDMP” (JIMRO Co., JAPAN) before and after antithromboitic treatment on a regular schedule, and MRI brain scans were also underwent in every six months. Plasma PDMP levels in group B and group C significantly decreased by combined antithrombotic therapy (18.0 ± 7.1 to 13.0 ± 8.2 U/mL, 17.8 ± 6.7 to 12.4 ± 7.8 U/mL, p < 0.03 and < 0.01, respectively) but not in Group A (18.2 ± 8.1 to 17.9 ± 9.1, NS). Brain MRI revealed worsening of lacuna infarctions in only one patient in Group A. No serious adverse effects were shown in any patients. These findings suggest that single use of aspirin could be enough for prevention of arterial thrombosis in APS patients and that plasma PDMP levels may be useful for an indicator of antiplatelet therapy.
Gestational Age Above 12 Weeks Predicts Favorable Obstetric Outcome in Women with Antiphospholipid Syndrome (APS) Associated with First-Trimester Recurrent Pregnancy Loss (RPL) Treated with Enoxaparin and Low Dose Aspirin.
Authors: Noble Luis S. , Herrada Juan , Kutteh William H.
Published: October 2019

Background: Pregnant women with APS associated with RPL treated with enoxaparin and low dose aspirin (ASA) resulted in an 84% viable infant delivery (Fertil Steril. 2005 Mar;83(3):684–90).

Design and Methods: Retrospective analysis. 30 patients (pts) were enrolled. Mean chronological age was 33.4 years. All pts had 2 or more first trimester losses and had a complete evaluation for RPL including anatomical, hormonal, infectious, chromosomal, and immunological. A standard ELISA was employed to detect the presence of IgG, IgM, and IgA serum antibodies against the phospholipids cardiolipin, inositol, serine, and ethanolamine. All pts received ASA 81 mg orally and enoxaparin 40 mg subcutaneously daily as soon as the serum pregnancy test became positive. Pts were monitored at monthly intervals.

Results:

Conclusions: We did not observe miscarriages in pregnant women with APS associated with first trimester RPL treated with enoxaparin and low dose aspirin after the 12th week of pregnancy.
Study of In Vitro Proliferation Potential of CD133 Positive Cells Derived from Umbilical Cord Blood.
Authors: Zhang Ri , Gao Wenjin , Sun Yuanyuan , Miao Jingcheng , Zhang Xueguang
Published: October 2019

Transforming growth factor-beta 1 (TGF-β1) is known to maintain primitive human hematopoietic stem/progenitor cells with polyfunctional role in a quiescent state and CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. To investigate the specific effect of TGF-β1 on proliferation and differentiation of CD133 positive cells derived from umbilical cord blood (UCB) during short-term culture in vitro, CD133 positive cells from 20 fresh UCB samples were selected using Miltenyi Biotec’s CliniMACS separation device and were cultured in IMDM medium with 20% FCS in the presence of a cytokine combination of SCF, IL-6, thrombopoietin, IL-3 and Flt3-ligand for up to 2 weeks and TGF-β1 with low concentration was also added to the mediumon day 4. The proliferative response was assessed at day 7, day 10 and day 14 by evaluating the following parameters: nucleated cells (NC), clonogenic progenitors (CFU-GEMM,CFU-GM and BFU-E), and immunophenotypes (CD133 and CD34). The results showed that efficacious expansion of various hematopoietic stem/progenitor cells was constantly observed during the culture. The fold expansion of NC on day7, day10 and day14 expansion were 33.59,224.26 and 613.48, respectively. The fold expansion of CFU-GEMM, CFU-GM and BFU-E on day 10 were 24.89, 41.62 and 49.28, respectively, obviously higher than that without ex vivo expansion (P<0.05). The expansions of CD133+, CD133+CD34+ and CD34+ subpopulation on day 14 were up to 25.83-fold, 16.16-fold and 60.54-fold, respectively. Furthermore the expansion systems with TGF-β1 showed more CD133+ cells than control at every time points. Our datas suggested that the CD133+ cells from human UCB have great expansion potential for ex-vivo expansion. The low concentration of TGF-β1 may delay over-differentiation of hematopoietic stem/progenitor cells.
Inhibition of p38 MAPK Reduces Ionizing Radiation (IR)-Induced Hematopoietic Suppression by Preventing Hematopoietic Stem/Progenitor Cell Senescence.
Authors: Liu Lingbo , Wang Yong , Zhou Daohong
Published: October 2019

Bone marrow (BM) suppression is the most common dose-limiting side effect of conventional cancer therapy, particularly with certain alkylating agents and/or ionizing radiation (IR). It has been shown that p38 mitogen-activated protein kinase (p38 MAPK) plays a critical role in regulation of hematopoiesis and its activation mediates IR- and chemotherapy-induced cell injury and oxidative stress-induced hematopoietic stem cell (HSC) exhaustion. Therefore, we examined the role of p38 MAPK in radiation-induced BM injury. First, we monitored the activation of p38 MAPK in BM hematopoietic cells at different time after exposure to IR using our well-established long-term BM cell culture (LTBMC) model system. The activation of p38 MAPK was detected within 24 hours after BM hematopoietic cells were exposed to 4 Gy IR and this activation sustained up to 5 weeks after radiation. Inhibition of p38 MAPK activity with a p38 MAPK specific inhibitor (SB202190) attenuated IR-induced suppression of the hematopoietic function of BM hematopoietic cells in an in vitro colony forming cell (CFC) assay. Moreover, the number of hematopoietic progenitor cells produced by SB202190-treated BM cells was significantly greater than that by the cells without SB202190 treatment after exposure to IR and followed by a five-week LTBMC. Interestingly, p38 MAPK inhibition showed no effect on IR-induced apoptosis in both HSCs and hematopoietic progenitor cells (HPCs), whereas the radioprotection effect of SB202190 was associated with a significantly reduction of p16INK4a expression and senescence-associated β-galactosidase (SA-β-Gal) activity in irradiated BM cells after five weeks of LTBMC. These findings suggest that activation of p38 MAPK may mediate IR-induced hematopoietic suppression by induction of hematopoietic stem/progenitor cell senescence and pharmacological inhibition of the p38 MAPK pathway may have the potential to be developed as an innovatively therapeutic strategy to ameliorate IR- and chemotherapy-induced BM toxicity.
Gadd45 Genes Participate in Myeloid Cell Fate Determination.
Authors: Gibbs John D. , Mohamed-Hadley Alisha , Liebermann Dan A. , Hoffman Barbara
Published: October 2019

The gadd45 family of genes is rapidly induced by different stressors, including differentiation-inducing cytokines, and there is a large body of evidence that their cognate proteins are key players in cellular stress responses. Gadd45 gene(s) were found to be primary response gene(s) to IL-6 mediated terminal differentiation of M1 monocytic leukemia cells, as well as to G-CSF mediated terminal differentiation of 32Dcl3 cells; furthermore, gadd45 gene(s) were induced by myeloid differentiation inducing cytokines such as IL-3 and GM-CSF in primary murine bone marrow (BM) cells (Zhang et al., Oncogene18:4899–4907, 1999; Abdollahi et al, Oncogene6:165–167, 1990). Induction of gadd45 genes at the onset of myeloid differentiation suggested that Gadd45 protein(s) play a role in hematopoiesis, yet no apparent abnormalities were observed in either the BM or peripheral blood compartments of mice deficient for either gadd45a or gadd45b. Both BM and Hoxb8 immortalized macrophage progenitors induced to differentiate in vitro revealed that deficiency in either gadd45a or gadd45b resulted in loss of macrophages and an increase in neutrophils compared to cells derived from wild type mice. How Gadd45a and Gadd45b influence the expression and activation of transcription factors that regulate myeloid cell determination will be discussed.
Effects of Temperature on the Change of HSP and the Viability of Mobilized Hematopoietic Stem Cells.
Authors: Kim Hawk , Min Young Joo , Baek Jin Ho , Shin Su Jin , Noh Eui-Kyu , Lee Eun-Hee , Park Sang-Kyu , Lee Jung Lim , Park Jae-Hoo
Published: October 2019

It is well known that HSP is influenced by temperature. Here we searched the effects of temperatures on preservation of hematopoietic stem cells (HSC) and the changes of HSP according to the temperatures and cryopreserving agents. The G-CSF-mobilized peripheral blood HSCs were collected from a healthy donor. DMSO, EG and GLY were used for cryopreserving agents. HSP were measured by real-time quantitative PCR, and cell viability were measured by trypan blue stain and MTS assay, respectively. Testing temperatures were 4°C, room temperature (RT, 20°C) and 37°C. Samples were HSCs stored according to testing temperatures without cryopreserving agents (cycle 0), HSCs thawed after first cryopreservation with various cryopreserving agents (cycle 1). The measure was done daily from D0 to D5 at cycle 0, and in 30 minutes interval (0, 30, 60, 90, 120 min) at cycle 1. Whereas more than 98% of cells were viable till 5 days after the harvest of cycle 0 at 4°C, RT and 37°C showed rapid decrease of viable HSCs. By day 3, less than 10% cells were viable at 37°C. At cycle 1, cell viability was rapidly decreased in both DMSO and GLY after 120 min stay at all temperatures. In case of EG, the same patterns were found at RT and 37°C. However, cell viability remained relatively well at 4°C. The expression of HSP 27 were over-expressed by day 2 and suppressed thereafter at all temperatures of cycle 0. At 4°C of cycle 0, the over-expression of HSP 27 was greater than those at RT and 37°C. The expression of HSP 70 was mostly suppressed along times. The suppression at 37°C was greater than those of other temperatures. In case of HSP 90, the overall expression was suppressed except for day 2 at 4°C and RT. At all cases of cycle 0, the expressions at 37°C were lower than those at 4°C and RT. At 4°C of cycle 1, DMSO showed decreased expression of HSP27 and HSP70. In contrast to DMSO, the expression of HSP27 and HSP70 was increased in case of EG and GLY at 4°C of cycle 1. The over-expression of HSP90 at 4°C of cycle 1 in EG and GLY was very low compared with those in DMSO. Overall expressions of HSPs in GLY were lower than in DMSO or EG at RT of cycle 1 except for HSP27. The expression of HSP27, HSP70, HSP90 were all decreased at 37°C of cycle 1 except for HSP70 in EG and HSP90 in DMSO. In conclusion, we should keep cool when storing HSCs without cryopreserving agent. The rapid apoptosis at 37°C could be caused by changed expression of anti-apoptotic HSPs. The expression of HSPs influenced by temperatures and cryopreserving agents may affect the viability of HSCs through the changes of HSPs.
ISWI ATPase Snf2h Is Required for Both Heterochromatin and Euchromatin Structure in ES Cells.
Authors: Podskocova Jarmila , Vargova Karin , Kokavec Juraj , Skoultchi Arthur I. , Stopka Tomas
Published: October 2019

Snf2h is a chromatin remodeling ATPase which along with associated factors has the ability to assemble and slide nucleosomes. Snf2h levels are substantially increased in acute leukemia cells and in aggressive solid tumors, suggesting a role for Snf2h in regulating chromatin structure in malignancy. Snf2h-null mice exhibit early embryonic lethality and Snf2h heterozygous mutant mice are growth-retarded (Stopka 2003, Chong 2007). In this study we have used Snf2h hemizygous (+/−) embryonic stem (ES) cells and mice to examine the consequences of reduced Snf2h stoichiometry on chromatin structure. Using indirect immunofluorescence and confocal microscopy we found that Snf2h in ES cells is localized both in euchromatin and to a lesser extent in heterochromatin. Its expression is markedly reduced in Sn2h +/− cells. Chromatin of Snf2h +/− ES cells exhibited multiple defects including decreased amounts of heterochromatin and a significant decrease of euchromatic marks including histone H3K79 and H4K16 acetylation. In contrast, histone H3K9 acetylation and the level of another chromatin remodeling ATPase, Brg-1, was not altered in Snf2h +/− ES cells as compared to wild-type ES cells. Thus in ES cells Snf2h may be involved in formation of both heterochromatin as well as transcriptionaly active euchromatin. Our data support the role of Snf2h in regulation of chromatin structure in leukemia, currently tested using Snf2h heterozygous animals.
Safety and Antitumor Efficacy of the Proteasome Inhibitor Carfilzomib (PR-171) Dosed for Five Consecutive Days in Hematologic Malignancies: Phase 1 Results.
Authors: Orlowski Robert Z. , Stewart Keith , Vallone Marcy , Molineaux Chris , Kunkel Lori , Gericitano John , O’Connor Owen A.
Published: October 2019

Introduction: Carfilzomib (CFZ) is a structurally- and mechanistically-novel proteasome inhibitor (PI) of the peptide epoxyketone class that exhibits a high level of selectivity for the unique N-terminal threonine active sites within the proteasome. CFZ is similar to bortezomib (BTZ; VELCADE®), in that it is a potent inhibitor of the chymotrypsin-like activity, but unlike BTZ, CFZ has shown minimal cross-reactivity with the other catalytic sites within the proteasome or across other protease classes. Preclinical human tumor xenograft models indicate that consecutive day (D1, D2) dosing resulting in 48 hours of proteasome suppression was superior to split dosing (D1, D4).

Methods: This phase 1 sequential dose escalation trial tested an intensive proteasome inhibition schedule with CFZ administered as an intravenous push (IVP) daily (QD) for 5 consecutive days on a 14 day cycle. The doses administered ranged from 1.2 mg/m2 to 20 mg/m2. Multiple myeloma (MM), non-Hodgkin’s lymphoma (NHL) that included mantle cell lymphoma (MCL), Hodgkin’s disease (HD), or Waldenström’s macroglobulinemia (WM) were eligible if they had relapsed after 2 or more prior therapies.

Results: A total of 29 subjects have been treated. The minimal effective dose (MED) was 11 mg/m2. A maximum of 80% proteasome inhibition was achieved in peripheral blood and mononuclear cells at ≥15 mg/m2. Five responses have occurred in 14 patients (6 MM, 3 MCL, 1 WM, 4 NHL) enrolled at ≥ the MED: 1 MM partial response (PR), 2 MM minimal responses (MR), 1 WM MR and one macroscopic CR in a patient with gastrointestinal MCL. Responses occurred in patients whose disease had failed therapy with BTZ, immunomodulatory agents, and stem cell transplant. Four NHL patients have had stable disease and remained on treatment for up to 7 months. Dose limiting toxicity reported at 20 mg/m2 included febrile neutropenia (reversible) and grade 4 thrombocytopenia. Grade 3 (25%) or grade 4 events (8%) were primarily hematologic events. Most withdrawals occurred due to the inconvenience of frequent visits, or progressive disease.

Conclusions: This study demonstrates that intensive CFZ dosing leading to sustained proteasome inhibition is well tolerated. The selectivity of the epoxyketone class of PI may promote a favorable safety profile. Furthermore, antitumor responses in heavily pretreated patients suggest non-cross resistant activity. Phase 2 trials in MM and solid tumors are proceeding with an alternate IV schedule. An oral agent suitable for frequent dosing schedules is under development.
Switch to Full-Length of XAF1 mRNA Expression in Myeloma RPMI8226 Cells by DNA Methylation Inhibitor 5-Azacytidine.
Authors: Chen Guanghua , Wu De Pei , Yang Ming Zhen , Tang Xiao Wen , Sun Ai-ning
Published: October 2019

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspase-3, -7, -9, while the XIAP-associated factor1 (XAF1), a zinc finger protein, antagonizes XIAP activities, thereby promotes apoptosis.The aberrant silencing of the XAF1 gene has recently been found in various types of cancer cells, which is suggested to be one of the potential mechanisms underlying survival advantages of malignant cells. We investigated the XAF1 expression in myeloma RPMI8226 cells. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and western blot methods were used for detecting the expressions of XAF1, XIAP, SURVIVIN, β-actin genes in RPMI8226 cells at different time points after treatment with 2.5 μM or 5 μM 5-azacytidine. The apoptosis of RPMI8226 cells was simultaneously detected using flow cytometric assay. Methylation-specific PCR was used to assay methylation status of the CpG sites in the XAF1 promoter. Compared with normal bone marrow mononuclear cells where only a full-length of XAF1 mRNA is detected, RPMI8226 cell expressed a full-length form of XAF1 transcript and a short form of XAF1 transcript. Only a full-length form of XAF1 transcript was detected after RPMI8226 cells treated with 5 μM 5-azacytidine for 120h. More percentages of RPMI8226 cells progressed to early apoptosis after 5 μM 5-azacytidine treatment for 120h. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human myeloma. DNA methylation inhibitor 5-azacytidine treatment can increase myeloma RPMI8226 cell XAF1 gene expression and promote apoptosis.
Pegylated Recombinant Human Arginase, Rharg-Peg5,000MW, Targets Cell Cycle Machinery in Multiple Myeloma Cells.
Authors: Liu Delong , Lin Xianghua , Lu Quanyi , Leung Thomas , Cheng Paul N.M. , Chiao Jen-Wei
Published: October 2019

Arginase has been shown to inhibit growth of human hepatocellular carcinoma by depletion of arginine. We have studied the effects of the pegylated human recombinant arginase (BCT-100, rhArg-peg5,000MW) on RPMI8226 cells, a multiple myeloma cell line. This study showed that three day exposure of the myeloma cells to pegylated rhArg at a concentration of 0.08 IU/ml and 0.48 IU/ml resulted in growth suppression of 10% and 70% respectively, as compared to untreated control. Cell cycle analysis revealed significant decreases in the proportion of cells in both S- and G2M-phase and a concomitant increase of cells in G1-phase in a time- and concentration- dependent manner. We further studied the mechanisms of cell cycle arrest induced by the pegylated rhArg. The pegylated rhArg inhibited both cyclin-dependent kinases CDK2 and CDK4, enhanced the expression of the CDK inhibitor p21, and reduced the expression of cyclinD1, D2, and E. The level of phosphorylated Rb protein was also found to be significantly decreased. The regulators of cell cycle have thus been revealed as targets of pegylated rhArg for myeloma growth arrest. The pegylated rhArg may serve as a novel antitumor agent for multiple myeloma.
Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.
Authors: Cokic Vladan P. , Beleslin-Cokic Bojana B. , Noguchi Constance Tom , Schechter Alan N.
Published: October 2019

We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.
3D Grown Human Placental-Derived Mesenchymal Stromal Cells (PLX-I) Support and Potentiate the Engraftment of Human Cord Blood Unfractionated (UFCBC) and Fractionated CD34+ Cells (hCD34+) in Irradiated NOD/SCID Mice.
Authors: Burger Ora , Jacov Osnat M. , Carmi Nirit D. , Ashtamker Galit F. , Pinzur Lena , Mizrachi Einat , Rusanovsky Marina , Bercovich Noam , Moshe Ofer B. , Lior Limor , Meiron Moran , Prather William R. , Meretzki Shai , Treves Avi J. , Kadouri Avinoam , Rowe Jacob M. , Horwitz Edwin M. , Nagler Arnon
Published: October 2019

The use of human umbilical cord blood (hUCB) for transplantation (CBT) in adults is limited due to the low cell number in each unit. Engraftment rates are lower and the time to neutrophils and platelet recovery is longer following CBT compared to bone marrow (BM) transplantation (28 vs 14 days, respectively). Co-transplantation of human hematopoietic stem/progenitor cells (hHSC) with mesenchymal stromal cells in an irradiated NOD/SCID mice model has been demonstrated to promote HSC engraftment. In a typical clinical setting of CBT, unfractionated cord blood cells (UFCBC) are usually used. On the other hand, in the irradiated NOD/SCID mice model, the CD34+ enriched fraction is usually used. Therfore, performing pre-clinical studies using UFCBC would better reflect the outcome in the clinical setting. Using PluriX technology, we expanded human placental derived mesenchymal stromal cells on 3D carriers in a bioreactor (PLX-I) and evaluated their potential to promote the engraftment of UFCBC or hUCB derived fractionated CD34+ cells (hCD34+) in NOD/SCID mice. UFCBC (107) or hCD34+ (5×104) were injected into the tail vein of 7–8 week old NOD/SCID mice along with 0.5×106 or 1×106 PLX-I cells. Following 5–6 weeks, FACS analysis of % human CD45+ cells in mice BM demonstrated that in the PLX-I untreated mice, the 107 UFCBC engrafted in a lower rate compare to 5*104 hCD34+ (6.28% vs. 14.43% respectively, n=10). Furthermore, in both settings, hCD34+ and UFCBC, the addition of PLX-I enhanced the hHSC engraftment 1.7 and 2 fold respectively compared to PLX-I untreated mice (24.13% vs. 14.43% in hCD34+ setting and 12.85% vs. 6.28% in UFCBC setting, n=10). We conclude that UFCBC can be used for hHSC engraftment in NOD/SCID mice model and co-transplantation of these cells with PLX-I may serve as a promising approach for the improvement of the hitherto delayed engraftment following cord blood transplantation.
Molecular Mechanisms of Cell Density-Dependent Growth in Myeloid and Lymphoid Leukemia Cell Lines.
Authors: Murohashi Ikuo , Ihara Noriko
Published: October 2019

Normal hematopoietic stem cells have been shown to be maintained through interaction with their environmental niches, such as osteoblastic and endothelial ones. The growth of leukemia cells has been shown to be stimulated by environmental niches (paracrine growth) or by cell-to-cell interaction and/or excreted factors of leukemia cells (autocrine growth). The growth of myeloid (MO7-E and HL-60) and lymphoid (Raji, U-266, Daudi and RPMI-1788) leukemia cell lines cultured at various cell densities in serum free medium (Sigma H 4281) with 1% BSA was evaluated. The cells cultured at higher cell densities (cultured cell densities of more than 105/ml) showed logarithmic linear increases in cell number, whereas those at lower cell densities (cultured cell densities of less than 104/ml) ceased increasing cell number. Supernatants of myeloid leukemia cells stimulated the growth of autologous clonogenic cells, but not those of lymphoid leukemia cells. Neutralizing antibodies (Abs) against various hematopoietic growth factors failed to inhibit cell growth except for anti-VEGF Ab, which significantly decreased HL-60 leukemia cell growth. In contrast, anti-TNF-α Ab significantly stimulated the growth of the HL-60 cells. To clarify the nature of the cultured cell density on the growth of leukemia cells, leukemia cells were cultured at higher cell densities (group H, cultured cell densities of 106/ml) or at lower cell densities (group L, cultured cell densities of 104/ml). After culture of 3-, 6-, 10-, and 24-hr, cells were serially harvested and total cellular RNA was extracted. Gene transcript levels were determined by using Real-Time PCR. Gene transcripts examined in the present study were as follows: Jagged-1, -1, Notch-1, -2, -3, Ang-1, -2, Tie-1, -2, Wnd3a, Wnd5a, β-Catenin, γ-Catenin, N-Cadherin, Cyclin D1, p16, p21, HOXA6, HOXA7, HOXA10, HOXB4, and Mef2c. At 24-hr cultures, transcripts of myeloid leukemia cell lines for Bmi-1, Wnt-3a, β-Catenin and γ-Catenin were higher, and those of lymphoid leukemia cell lines for Notch 1, 2, and 3 were higher in group H compared with group L. Transcript levels for Wnt5a were higher at 10-hr culture (HL-60 and Raji), those for HOXA7 at 30–10-hr (MO-7E, U-266 and Raji), and those for Mef2c at 3-hr (MO-7E, U-266 and Raji) in group H compared with group L. Taken together, our present results favor the conclusions that genes related to transcription factors and growth factors are sequentially and differentially expressed through cell-to-cell interaction of leukemia cells. The nature of the leukemia cell-to-cell interacrtion, which is related to the growth advantages of leukemia stem cells over normal hematopoietic stem cells, remains to be further clarified.
FISH Analysis of IgH Rearrangement by 4 Kinds of Probes Involving IgH Genes in Multiple Myeloma: Improved Sensitivity for the Detection by Use of IGH Break-Apart Probe.
Authors: Chung Hye Yoon , Kim Miyoung , See Cha Ja , Min Hyun Jung , Yoon Sung-Soo , Cho Han Ik , Lee Dong Soon
Published: October 2019

Background: IgH (immunoglobulin heavy chain) gene rearrangement is known to be the most frequent chromosome change in multiple myeloma. The detection of this genetic change is conveniently done by using fluorescence in situ hybridization (FISH) method recently. The aim of this study is to determine the utility of most commonly used probes, IGH/CCND1 dual color, dual fusion probe (Downers Grove, IL, USA), IGH/BCL2 dual color, dual fusion probe (Downers Grove, IL, USA), IGH/FGFR3 dual color, dual fusion probe (Downers Grove, IL, USA) and IGH dual color break apart rearrangement probe from Vysis Products (Downers Grove, IL, USA).

Methods: We applied four different probes of IgH FISH on 202 Korean patients with multiple myeloma for comparing the utility of four probes.

Results: 84 of 202 patients (41.6%) had the IgH gene rearrangement. 44 of 84 patients (52.4%) showed positive to all four probes, but 40 of 84 patients (47.6%) showed discrepancy. IGH dual color break apart rearrangement probe showed highly detectable rate (41.6%) compare to IGH/CCND1 (31.2%), IGH/BCL2 (27.7%) and IGH/FGFR3 (24.3%). Among patients who showed discrepancy between four probes, 20 of 40 patients (50.0%) were only positive to IGH dual color break apart rearrangement probe.

Conclusions: The IGH break-apart probe was qualitatively and quantitatively better than three other probes at initial diagnosis and during follow-up of myeloma. In conclusion, it would be most efficient and advisable to use the IGH break-apart probe at first, and then to identify the translocation partner in case of positive rearrangement of IgH.
Hypermethylation and Expression of Rassf1a in Childhood Acute Lymphoblastic Leukemia (ALL).
Authors: Yaniv Isaac , Betzer Hadar , Naumov Inna , Stark Batia , Avrahami Gali , Luria Drorit , Kodman Yona , Avigad Smadar
Published: October 2019

Epigenetic modifications including promoter hypermethylation and histone acetylation are emerging as one of the factors controlling the expression of many tumor suppressor genes. One such gene is the Ras effector RASSF1A (Ras association domain family 1). Promoter hypermethylation of Rassf1a is already well established and clinically relevant in many adult cancers and also many pediatric solid tumors. However not much is known about its status in childhood acute lypmphoblastic leukemia (ALL). The aim of this research was to study RASSF1A methylation and expression and also evaluate mutations in ras oncogene in childhood ALL and correlate it with clinical outcome. Bone marrow samples at diagnosis were obtained from 147 patients diagnosed and treated at the Schneider Children’s Medical Center of Israel. The cohort consisted of 101 and 46 B-lineage and T-cell ALL, respectively. The median age at diagnosis was 6 years (range 0.3–18.8 years). Nineteen (13%) of the patients responded poorly to steroids at day 8 and relapse occurred in 41 (28%) patients. Median follow up was 80.5 months (range 5–253 months). DNA from BM was bisulfite modified and subjected to methylation specific PCR (MSP) analysis using primers specific for RASSF1A. Direct sequencing was done for N, H and K ras mutations. RASSF1A expression was detected by RT-PCR. Methylation of RASSF1A was detected in 30% of patients. There was no correlation of gene methylation to known clinical or biological features of ALL. A total loss of expression of RASSF1A was seen only in 7/27 (26%) methylated samples. Furthermore 11/53 (21%) unmethylated samples also showed a loss of expression for RASSF1A.There could be two possible explanations for these findings; firstly unlike the scenario in solid tumors, RASSF1A hypermethylation may not be a major operant mechanism in childhood ALL. Secondly, a more precise correlation of RASSF1A methylation to its expression could be established by quantitative real time analysis. We also detected mutations in ras oncogene in 16 (11%) patients (14 in N-ras and 2 in K-ras). However no correlation between mutations and clinical parameters was found. Thus a more comprehensive epigenetic analysis using a wider panel of genes/genome wide study should be designed to generate a clinically relevant “Methylotype” for childhood ALL.
MiRNA Profiles in Acute Lymphoblastic Leukemia: Rather a Characteristic for the Leukemia Subtype Than a Reflection of Its Differentiation Status.
Authors: Schotte Diana , Broekhuis Mathilde J.C. , Jansen Mieke W.J.C. , Pieters Rob , Chen Chang-Zheng , Den Boer Monique L.
Published: October 2019

MicroRNAs (miRNAs) are noncoding RNAs that regulate processes involved in proliferation and differentiation. Since abnormal proliferation and differentiation is the hallmark of cancer, miRNAs may have a role in the pathogenesis of cancer. Here we examined the miRNA expression in various subtypes of Childhood Acute Lymphoblastic Leukemia (ALL) including MLL-rearranged ALL (n = 16), T-ALL (n = 10) and precursor B-ALL patients positive for TEL-AML1 (n = 10), BCR-ABL (n = 10), E2A-PBX (n = 8) or hyperdiploidy (more than 50 chromosomes, n = 10) and other precursor B-ALL patients that are negative for all major genetic abnormalities in ALL (n = 19). We have found that miR-196b is highly expressed only in MLL-rearranged ALL and its expression level is ∼400 to 700-fold (P < 0.05) higher than the levels in all other precursor B-ALL subtypes and T-ALL cases. Moreover, all precursor B-ALL subtypes including TEL-AML1, BCR-ABL, E2A-PBX and hyperdiploid positive cases have a 250- to 6500- fold upregulation (P ≤0.01) of miR-708 compared to MLL-rearranged ALL and T-ALL patients. Interestingly, it is known that MLL-translocated ALL arises in an earlier stage of B-lineage development compared to other precursor B-ALL subtypes negative for MLL rearrangements, thus subtype-specific expression of miR-196b and miR-708 could reflect a difference in maturation status. However, miR-196b and miR-708 expression levels do not seem to correlate with the differentiation status of the leukemia cells based on the immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements in case of precursor B-ALL and EGIL classification in case of T-ALL. Furthermore, miR-196b is located between the HOXA9 and HOXA10 genes, which are known to be upregulated in MLL-translocated patients. Here we find a positive correlation between miR-196b expression and HOXA9/HOXA10 expression within MLL- rearranged and T-ALL patients. Further research has to reveal whether high expression of miR-196b actively contributes to leukemogenesis like HOX genes or whether it is an epiphenomenon of HOXA upregulation in MLL-rearranged ALL patients. All together, our present study suggests that the expression level of miR-196b and miR-708 reflects differences between genetic subtypes rather than differences in differentiation status and emphasizes the need to further investigate the role of these miRNAs in leukemogenesis.
Identification of Genes Associated with Risk to Aplastic Anemia (AA) Using Array-Based Genotyping.
Authors: Lee Il-Kwon , Kim Hee Nam , Kim Yeo-Kyeoung , Park Kyeong-Soo , Yang Deok-Hwan , Lee Je-Jung , Shin Myung Geun , Choi Chan , Kook Hoon , Hwang Tae Ju , Kim Hyeoung-Joon
Published: October 2019

In this study we tested whether 3104 SNPs in 200 candidate genes were associated with risk to aplastic anemia (AA). Genes were considered potential candidates for their known or suspected roles in DNA repair system, pharmacogenomics and transcriptional regulation in hematopoiesis or putative pathways related to development of AA. Selection of SNPs was performed using dbSNP and HapMap project databases, with emphasis on non-synonymous SNPs or haplotype tagging SNPs. To discover potential SNPs responsible for AA susceptibility, a case-control study using Affymetrix targeted genotyping 3K array was designed. We applied 3K array to analyze the samples from 132 AA patients and 382 healthy controls. Genotyping were processed using GeneChip scanner 3000 TG(Affymetrix) and analyzed with GCOS software(Affymetrix). In total more than 160,000 SNPs were genotyped for this study. Samples with suboptimal call rates were excluded. Statistical testing were carried out using χ2, Cochran-Armitage trend, Fisher’s exact, odds ratio, haplotype estimation, LD block definition. Here we report that 19 SNPs in 14 genes are associated with elevated or reduced risk to AA. Three associated genes map to chromosome 11 and 16 respectively. Some of associated genes were transcription factors such as RARB and ZNF233. Among those associated SNPs, one was located in coding region while the rest of SNPs were located in introns of associated genes. In conclusion, we identified SNPs responsible for AA susceptibility by candidate gene-based SNP array approach. These promising data when supported by further molecular validation would greatly enhance the current understanding of AA predisposition and diseases progression. Implication of polymorphic variants in AA etiopathogenesis will be presented and discussed.
Autophagy Induction of Acute Myeloid Leukemia (AML) Cells after Treatment with Low Dose-Cytarabine (Ara-C) and Enhanced Cytocidal Effect on AML Cells by Combined Treatment with Ara-C Plus Lenograstim.
Authors: Miyazawa Keisuke , Toyotake Juri , Naito Munekazu , Itoh Masahiro , Ohyashiki Kazuma
Published: October 2019

Autophagy fundamentally refers to a process where cells enclose their own cell organelles with isolated membrane and deliver them to lysosomes for degradation, servicing as a nonspecific degradation mechanism of autologous proteins. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal to sustain metabolism by producing catabolites. In this context, autophagy acts as a cytoprotective mechanism for cells to protect themselves from extracellular stresses. However, in a culture using cells in which autophagy-related genes were suppressed by siRNA and to which an autophagy inhibitor, 3- methyladenine (3-MA), was added, cytotoxic effects of some anticancer agents on cancer cells were diminished. Based on these results, the concept of caspase-independent cell death via autophagy, which is designated as type II programmed cell death (PCD) in contrast to apoptosis as type I PCD, has been receiving increased attention. We here report that treatment of AML cells with low does-Ara-C induces autophagy and enhances its cytocidal effect on AML cells by combined treatment with G-CSF. Treatment of AML cell line U937 cells with 1 to 50 ng/ml of Ara-C induced cell growth inhibition in a dose-dependent manner. Exposure to the higher concentrations of Ara-C (more than 10 ng/ml) induced apoptosis in U937 cells along with formation of apoptotic bodies and caspase-3 activation. However, treatment with the lower concentration of Ara-C (2.5 to 7.5 ng/ml) induced autophagy as assessed by electron microcopy and the punctuated distribution pattern of cytoplasmic microtubule-associated protein 1 light chain 3B (LC3B) by fluorescent immunocytochemistry. Low dose-Ara-C inducing -cell death and -autophagy both were suppressed when the cells were cultured in the presence of 3-MA. This suggests that the autophagic cell death but not the cytoprotective autophagy was induced in response to Ara-C. Combined treatment with G-CSF (lenograstim; 100 ng/ml) plus 2.5 to 7.5 ng/ml of Ara-C, but not plus higher concentrations of Ara-C, resulted in the enhanced cytocidal effect in U937 cells in vitro. These data indicate that combined treatment of Ara-C plus G-CSF has a therapeutic benefit for the AML patients by enhanced cytocidal effect which appears to be mediated via induction of autophagy. In addition, our data appear to explain in part the high response rate of CAG therapy in refractory AML, which consists of low-dose Ara-C in combination with G-CSF and aclarubicin.
A Novel Target of Histone Deacetylase Inhibitor, Involves in Mitochondria Membrane Protein Ubiquitination and Regulates Apoptosis.
Authors: Wu Peng , Cao Yang , Tian Yuan , Hong Zhenya , Sun Lishi , Gui Lingli , Zhang Yicheng , Ma Ding , Zhou Jianfeng
Published: October 2019

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. we employed a genetic technique, named suppression of mortality by antisense rescue technique(SMART), to understand the molecular mechanism of apoptosis induced by HDACI and identify the genes that participate in this process. We identified several novel genes and demonstrated that HDACIs promote apoptosis through activation ubiquitin/proteasome by inducing ubiquitin B not ubiquitin C gene expression. According to degrading the mitochondria membrane protein Mcl-1 by activated ubiquitin/proteasome system, the mitochondria potential collapse and cell apoptosis. Further observation demonstrated that depletion of MCL-1 by RNA interference (RNAi) sensitizes Hela cells to TSA mediated mitochondria membrane potential collapse and apoptosis, and overexpression of MCL-1 confers apoptosis resistance.
Prospective Evaluation of the NIH Staging Criteria in Chronic GVHD and Correlation to Quality of Life - Results of a German Multicenter Validation Trial.
Authors: Wolff Daniel , Herzberg Philipp Y. , Heussner Pia , Mumm Friederike , von Harsdorf Stephanie , Rieger Katrin , Greinix Hildegard , Hahn Joachim , Holler Ernst , Freund Mathias
Published: October 2019

The NIH staging and response criteria offer for the first time the chance for uniform documentation and evaluation of physical functioning and quality of life aspects of chronic graft-versus-host disease (cGVHD) as well as its response to immunosuppressive treatment. Here we present the interim analysis of a prospective Germany multicenter study on the NIH staging criteria in cGVHD.

Methods: Seventy-six patients (median age 46 years, range 19–64) after allogeneic hematopoetic stem cell transplantation (HSCT) for hematologic malignancies were evaluated according to the NIH criteria based cGVHD activity assessment, the Lee Chronic GVHD Symptom-Scale, FACT-BMT, human activity profile (HAP), SF 36, Berliner Social Support Scale (BSSS), 24 item Adjective Measure (24 - AM), Hospital Anxiety and Depression Scale (HADS) and the NCCN-Distress-Thermometer. Enrolment occurred between day 100 and 1 year after HSCT or in the presence of active cGVHD also at later time points. Follow-up surveys were conducted at 1, 2, 3, 5, 8, 12 and 18 months after baseline survey. At all time points disease status, comorbidities and medication were documented. Additionally the NIH-Symptom-Scale was applied for clinician rating of cGVHD symptoms.

Results: Fifty-one patients had cGVHD (mild n=15, moderate n=32, severe n=4) while 25 patients did not have cGVHD. The cGVHD NIH consensus grading as well as the 10 point scale correlated with impairment of physical functioning (p<0.01, r=.48), the FACT-G total score (p=0.02, r=.33) and mental health (p<0.01, r=.41). The HAP maximum activity score correlated inversely with severity of cGVHD (p=0.028, r=.44), with the physical functioning (p<0.01, r=.53) and with role limitation due to physical problems (p<0.01, r=.43) evaluated by the SF36. In addition an impaired physical functioning correlated with role limitations due to emotional problems (p<0.01, r=.52) and with impaired mental health (p<0,01, r=.63). Moreover the HAP maximum activity score correlated with emotional well being as evaluated by the FACT (p<0.01, r=.39). Depression as measured by the HADS depression score correlated with pain (p<0,01, r=.35) and impaired physical functioning (p=0.03, r=.26) while the HADS anxiety score did not. The results demonstrate, that severity of cGVHD as assessed by the NIH consensus grading and the 10 point scale correlate with impairment of physical functioning as well as daily activities and quality of life. Moreover reduction of daily activities lead to impairment of emotional well being.