bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Sensitivity Monitoring of Hematopoietic Chimerism after Double Umbilical Cord Blood Transplantation by Real-Time Quantitative Polymerase Chain Reaction.
Authors: Marty-Grès Sophie , Moussiere Marielle , Avinens Odile , Andary Monique , Ramounau-Pigot Annie , Ceballos Patrice , Meniane Jean-Côme , Margueritte Geneviève , Fegueux Nathalie , Rossi Jean-Francois , Eliaou Jean-François 
Published: October 2019

Abstract
Double umbilical cord blood transplantation (DUCBT) has recently emerged as a treatment of hematological malignancies. Chimerism after DUCBT typically shows a single unit engrafting when it relies on polymerase chain reaction (PCR) fluorescent amplification of short tandem repeat-markers analyzed by capillary electrophoresis (STR-CE). As real-time quantitative PCR (RQ-PCR) of short insertion and deletion polymorphisms stands for a more sensitive method of detection especially for minor cell populations, the goal of our study was to assess by RQ-PCR the potential persistence of the unit not detected by STR-CE, defined as a nanochimerism. Between July 2005 and April 2008, 26 patients underwent DUCBT with a reduced-intensity regimen in our institution, as described by Barker JN et al. (fludarabine, cyclophosphamide, total body irradiation 2 Gy). Their median age was 54.5 years (range 11–67). Most patients had advanced hematological malignancies. They all received 4-6/6 HLA-matched cord units. Numbers of infused nucleated and CD34+ cells were higher than 2x107/kg and 1x105/kg, respectively. Monitoring of hematopoietic chimerism was routinely performed on DNA from total peripheral blood mononuclear cells (PBMC) and from blood T cells, weekly during the first month, then twice monthly for 2 months, and monthly, using STR-CE. All available patients with one cord unit chimerism were studied by RQ-PCR. The median follow-up time was 6.5 months (range 1–35). Twenty-three of 26 patients had a full donor chimerism from one cord unit by STR-CE, at day 14 (N=12), day 21 (N=4), day 30 (N=4), day 60 (N=1) or day 200 (N=2) on PBMC and on T cells. Among them, 15 patients were analyzed by RQ-PCR. Discrepancies between both methods appeared in 14 of 15 cases for PBMC and in 8 of 12 available DNAs from blood T cells. RQ-PCR could still detect the minor cord unit at least 21 days after its “disappearance” assessed by STR-CE on PBMC (N=7/14) and on T cells (N=4/8). These data suggested that evaluation of chimerism by RQ-PCR after DUCBT lowers the detection threshold (sensitivity 0.01%) and allows to quantify a nanochimerism that cannot be detected by STR-CE (sensitivity 1–5%). This is the first series ever published on the monitoring of chimerism after DUCBT using RQ-PCR. Clinical implications of this nanochimerism will have to be unraveled in larger series.
Low Dose Alemtuzumab Achieves Long-Term Engraftment with Low Level Mixed Chimerism in Related Haemopoietic Stem Cell Transplantation for Haemoglobinopathies
Authors: de la Fuente Josu , O’Boyle Farah , Killeen Natalie , Tso Allison , New Helen , Atra Ayad , Dokal Inderjeet , Vulliamy Tom , Davis John , Foroni Letizia , Szydlo Richard M. , Roberts Irene 
Published: October 2019

Abstract
Haemopoietic stem cell transplantation (HSCT) is the only proven curative treatment available for haemoglobinopathies. To reduce transplant-related mortality and morbidity associated with graft-versus-host disease (GvHD) and facilitate donor engraftment we have used low-dose alemtuzumab in 43 consecutive patients (36 with thalassaemia and 7 with sickle cell disease; median age 6 years; range 2 to 16 years) transplanted since 2000. Donors were HLA-matched family donors in all cases. All patients were conditioned with oral busulfan 14 mg/kg (days -9 to -6), cyclophosphamide 200 mg/kg (days -5 to -2) and alemtuzumab 0.3 mg/kg (days -8 to -6) with post-HSCT methotrexate (10 mg/m2 days +4 and +7) and ciclosporin for six months. Hypertransfusion was initiated six weeks before conditioning and maintained until day +28 to suppress endogenous haemopoiesis and minimise graft rejection. Patients with thalassaemia were Pesaro class I or II. Patients with sickle cell disease were transplanted for stroke or recurrent vasooclusive crisis and/or acute chest syndrome not responding to hydroxycarbamide. The source of stem cells was bone marrow in all cases except one patient who received PBSC with a median cell dose of 3.08 x 108 TNC/kg (range 1.69 – 10.90 x 108 TNC/kg). Median follow up was 25.8 months (range 6.5 – 94.3 months). Chimerism studies were performed on peripheral blood genomic DNA using 8 microsatellites markers by genescanning. All patients are alive (43/43), 42 with long-term transfusion-independence and donor haematological values (5 years DFS 97.7%). One patient failed to engraft and was re-infused with autologous cells. Donor haemopoiesis was ≥ 95% on day +28 in all 42 patients who engrafted and was maintained at ≥ 95% in the majority of patients at 12 months post-HSCT. Eleven patients (11/42; 31%) have stable mixed chimerism at ≥ 12 months post-HSCT: donor haemopoiesis 90–94% (n=4); <90% (n=7; median 85%; range 45 – 89%). There have been no late relapses and no patients had falling donor haemopoiesis <90% after day +90. Acute GvHD ≥ grade 2 occurred in 5 patients (11.6%) and was steroid-responsive in all cases except the patient who received PBSC. Limited chronic GvHD was present in 2 patients (4.6%) >18 months post-HSCT (on no treatment) and Karnofsky score is 100% in all engrafted patients (42/42). In conclusion, addition of low-dose alemtuzumab (total dose 0.3 mg/kg) achieved long-term engraftment in almost all children undergoing HLA-matched family donor HSCT for haemoglobinopathies with minimal chronic GvHD and excellent DFS (97.7%).
Discovery of a New Molecular Marker, Prohibitin, and Development of Candidates for New Chemotherapeutic Agents Targeting Prohibitin in Primary AML Cells.
Authors: Kim Hye-Ran , Shin Myung-Geun , Lee Sun-Woo , Kim Hee-Nam , Lee Il-Kwon , Kim Hyeoung-Joon , Kook Hoon , Shin Jong- Hee , Suh Soon-Pal , Ryang Dong-Wook 
Published: October 2019

Abstract
In light of the recent studies that showed significant causal relationship between mitochondrial genetic mutation and apoptosis in pathology of various tumors and degenerative diseases, mitochondrial proteins have been interesting targets for the study of apoptosis in leukemia and other malignancies. This observation prompted us to analyze mitochondrial proteins and develop new anti-proliferative agents targeting them. Mitochondria were isolated from AML cells by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. We identified 48 spots corresponding to 38 proteins in primary AML cells using 2-DE and mass spectrometry (MALDI-TOF/TOF technology), the expression of which were altered significantly compared to that of normal hematopoietic cells. Out of these deregulated proteins, 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (PHB) (gi4505773) was highly expressed in primary AML cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary AML bone marrow cells and sections. To assess the functional significance of aberrant prohibitin expression, we applied siRNA delivery for silencing of prohibitin. Transduction with a siRNA 11867 construct resulted in 75% decrease of AML cells as compared to the nonsilencing control construct after two days. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized in our laboratory. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in leukemic cell lines including THP-1, K-562 and Kasumi-1. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 – 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB in K-562 cell line. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in primary AML cells and cell lines. By analyzing AML cell mitochondrial protein we discovered a new molecular marker, PHB, characteristically overexpressed in AML cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in AML cells.
ChIP-Chip in Primary AML Blasts Identifies Peroxiredoxin-II as An Epigenetically Silenced Tumor Suppressor.
Authors: Agrawal-Singh Shuchi , Wiechmann Markus , Doths Sandra , Nolte Christina , Thoennissen Nils Heinrich , Klein Hans-Ulrich , Dugas Martin , Berdel Wolfgang E. , Koschmieder Steffen , Serve Hubert , Mueller-Tidow Carsten 
Published: October 2019

Abstract
Epigenetic silencing of tumor suppressors is a frequent event in leukemogenesis. We performed ChIP-Chip to identify genes with altered Histone H3 acetylation in primary AML specimens (n=115) and normal CD34+ progenitor cells (n=21). Interestingly, promoters of several members of the peroxiredoxin family (PrdxII and Prdx IV) were identified to be significantly Histone H3 hypoacetylated in primary AML blasts. Peroxiredoxins (Prdx) are antioxidant enzymes that regulate the amount of reactive oxygen species in the cell. Prdx scavenge H2O2 and protect cells from oxidative damage to cellular DNA, lipid and proteins. Confirmation by quantitative PCR revealed that the PrdxII promoter was hypoacetylated in AML, and mRNA expression was 10-fold induced in U937 cells upon exposure to the demethylating agent 5-Azadeoxycytidine (Aza). Demethylation of U937 cells by Aza resulted in increased Histone H3 acetylation at the PrdxII promoter. DNA hypermethylation was frequent in primary AML blasts (18/103) but not in control samples (0/40) as assessed by methylation-specific PCR. Hypoacetylation of Histone H3 and DNA hypermethylation was associated with repression of PrdxII mRNA and protein levels in primary AML samples. Decreased PrdxII protein expression as assessed by immunohistochemistry in a tissue microarray was associated with a poor prognosis in AML patients. On the functional level, PrdxII inhibited the growth of hematopoietic progenitor cells in colony assays and negatively influenced receptor tyrosine kinase signaling by inhibition of ERK and AKT activation upon overexpression. Conversely, PrdxII knock-down by shRNA in myeloid progenitor cell lines and in murine primary bone marrow cells led to enhanced growth and tyrosine kinase signaling. A decrease in PrdxII levels was associated with an increase in reactive oxygen species and enhanced phosphorylation of STAT5, AKT and ERK. Taken together, these findings suggest that chromatin modifications in AML suppress expression of PrdxII. Our data also revealed that PrdxII is a novel putative tumor suppressor in AML.
Rac GTPases Are Required for MLL-AF9-Induced Mixed Lineage Leukemia.
Authors: Wei Junping , Wunderlich Mark , Harris Chad , Mizukawa Benjamin , Zheng Yi , Williams David A. , Mulloy James C 
Published: October 2019

Abstract
The t(9;11) translocation fusion gene MLL-AF9 (MA9) is commonly found in acute myeloid and lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. It has recently been reported that MA9 leukemia cells express higher levels of the small GTPase protein Rac and CDC42 when compared to in vitro MA9 immortal cells in a murine model. To determine the importance of Rac GTPase signaling in MA9-induced transformation, we used an MA9 leukemia model we recently established involving MA9 fusion gene expression in human CD34+ cells. Treatment with the Rac specific inhibitor NSC23766, or transient knockdown of Rac expression by RNAi, induced rapid apoptosis in MA9 cells but not in normal cord blood or t(8;21) translocation fusion gene AML-ETO expressing cells. These data demonstrate that the Rac signaling pathway plays a critical role in the growth and survival of MA9 leukemia cells. To extend this work to an in vivo genetic model, we used mice deficient in Rac2 (Rac2-KO) or with floxed alleles of Rac1 in mice transgenic for Mx-Cre. Leukemia development was compared in mice transplanted with MA9-transduced wild type, Rac1 or Rac2 deficient low density bone marrow cells. Poly I:C injections were performed 2 weeks after transplantation to delete Rac1. Rac deletion was confirmed by PCR and western blot analysis. Mice that received either WT or Rac1−/ − MA9 expressing cells uniformly developed AML and died at 3 to 5 months. Mice transplanted with Rac2-KO cells expressing MA9 showed a decreased incidence and increased latency (6 to 10 months) of AML development despite the persistent engraftment of MA9-expressing cells. All three groups of mice maintained MA9 EGFP+ cells in peripheral blood over the entire experiment, and eventually gave rise to similar end stage AML with a Gr-1+/Mac-1+/Kit+/B220-/CD3- phenotype and myelomonocytic blast morphology. Combined with our observation in human CD34+ cord blood cells transduced with MA9, these in vitro and in vivo data indicate that MA9-mediated transformation and survival requires Rac and their downstream effectors. Rac2 signaling appears to be particularly important in the murine MA9 AML model. Therapeutic targeting of Rac could be a unique and important approach to treating MLL leukemia.
Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.
Authors: Kuang Shaoqing , Zweidler-McKay Patrick , Yang Hui , Fang Zhi Hong , Tong Weigang , Garcia-Manero Guillermo 
Published: October 2019

Abstract
The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5’-aza-2’-deoxycytidine resulted in HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.
The Anti Von Willebrand Factor Aptamer ARC1779 Prevents the Desmopressin-Induced Thrombocytopenia in VWD Type 2B.
Authors: Paulinska Petra , Jilma Bernd , Firbas Christa , Schaub Robert G , Gilbert James C. , Knoebl Paul 
Published: October 2019

Abstract
Introduction: Von Willebrand Disease (VWD) Type 2B is characterized by increased affinity of VWF for the platelet glycoprotein receptor Ib (GPIb). Consequently, infusion of desmopressin, a standard of treatment for other forms of VWD, causes transient thrombocytopenia in patients with VWD-2B. The aim of the study was to test whether the novel anti-VWF aptamer ARC1779, which inhibits the interaction of VWF with GPIb, could abrogate the transient desmopressin-induced thrombocytopenia in VWD-2B.
Patients and Methods: This was part of a clinical trial using a double-blind, randomized, placebo-controlled, crossover design. A patient with VWD 2B received desmopressin (0.4 mcg/kg) alone, or desmopressin after pre-treatment with ARC1779 targeted to a plasma concentration of 10 mcg/mL. Multiple blood samples were obtained over 24 hours to measure von Willebrand Factor Antigen, Ristocetin Cofactor (RiCO), FVIII activity and multimers.
Results: Desmopressin alone (open circles) increased VWF:Ag by 38% (not shown), and VWF:RiCo 3.4-fold within 30 min, and profoundly decreased platelet counts by 87%. In striking contrast, ARC1779 (solid triangles) completely prevented the desmopressin induced fall in platelet counts (Figure), although the increase in vWF:Ag and vWF:RiCO was greater (2.7-fold and 12-fold, respectively).
Conclusions: This randomized, double-blind, placebo-controlled experiment shows that ARC1779 effectively prevents consumption of VWF and platelets in response to desmopressin in VWD Type 2B. It provides in vivo proof of concept that ARC1779 is a potent inhibitor of the VWF A1 domain interaction with GPIb.
Figure Figure
Subclinical Deep Venous Thrombosis Affects 10 % of Haemophiliacs Undergoing Major Orthopaedic Surgery.
Authors: Hermans Cedric R. , Lambert Catherine 
Published: October 2019

Abstract
The management of venous thromboembolism (VTE) in patients with haemophilia undergoing major orthopaedic surgery requiring intensive replacement therapy remains controversial. Information about the incidence of proven VTE in this setting is lacking. Despite the absence of data, half of haemophilia comprehensive centers in Europe recently reported the use of pharmacological antithrombotic prophylaxis in this setting.
We conducted a prospective study to evaluate the occurrence of deep venous thrombosis (DVT) of the lower limbs in all consecutive patients with haemophilia referred for major orthopaedic surgery at the Cliniques universitaires Saint-Luc, Brussels, Belgium. Screening for DVT by unilateral (13) or bilateral (16) compression ultrasonography (US) of the lower limbs was performed 5 to 12 days after 29 major orthopaedic surgeries (total hip replacement (5), total knee replacement (TKR) (15), ankle arthrodesis (4), decompressive laminectomy for lumbar stenosis (1), femoral osteosynthesis (4)) in 22 patients with severe (16), moderate (4) or mild (2) haemophilia A (20) or B (2), intensively treated with continuous infusion of clotting factor concentrate. None of the patients received pharmacological anti-thrombotic prophylaxis with LMWH. There was no case of clinical DVT or pulmonary embolism during the 3-months post-operative clinical surveillance. However distal DVT involving a single peroneal vein without proximal extension was identified in two patients with severe HA after TKR and in one patient with mild HB after decompressive laminectomy. The latter was efficiently treated with a short course of LMWH. The two others resolved spontaneously without antithrombotic therapy.
In conclusion, sub-clinical DVT affects up to 10 % of patients with haemophilia undergoing major orthopedic surgery. All thrombotic events were distal and resolved spontaneously or with a short course of LMWH without complications. This study suggests that routine screening for DVT by compressive US is indicated in patients with haemophilia undergoing major orthopaedic surgery and not receiving pharmacological thromboprophylaxis. Further studies are needed to determine if systematic pharmacological prevention of DVT is required in this setting as in patients without haemophilia.
Monocyte-Derived Microparticles Improve Hemostasis in Factor VIIIDeficient Mice: Exploring the Role of PSGL-1/P-Selectin.
Authors: Gross Peter L. , Vaezzadeh Nima , Ivicic Lori , Ni Ran , Esposito Bruno , Bai Xufang , Akram Ali 
Published: October 2019

Abstract
Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice.
Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss.
Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours.
Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not.
Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.