bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted Replacement of the Murine Thrombomodulin Gene with Human Thrombomodulin Coding Sequence Results in Decreased Protein C Activation and Enhanced Thrombotic Response to Photochemical Injury.
Authors: Raife Thomas J. , Erger Rochelle A. , Wilson Katina M. , Fernandez Jose A. , Alternburg Jennifer , Kim Hyung-Suk , Griffin John H. , Maeda Nobuyo , Lentz Steven R. 
Published: April 2020

Abstract
Thrombomodulin is an endothelial transmembrane glycoprotein that regulates hemostasis and inflammation. Deletion of the thrombomodulin gene in mice results in embryonic lethality. To examine the function of human thrombomodulin in a murine model, we used gene targeting to replace the entire coding sequence of the intronless murine thrombomodulin gene with a human thrombomodulin cDNA. Mice derived from successfully targeted 129/SvEv embryonic stem cells were maintained on either a 129/SvEv background or a mixed (129/B6) background. Mice homozygous for the modified allele (hthm/hthm) were viable, and the total amount of thrombomodulin mRNA (measured by real time PCR) did not differ between hthm/hthm and wild-type (+/+) mice (p&gt;0.05). In heterozygous (hthm/+) mice, the ratio of human to murine thrombomodulin mRNA was similar in lung, heart, and kidney, and expression of murine thrombomodulin transcripts was 50% of that in wild-type (+/+) mice. Expression of human thrombomodulin protein was detected in endothelium by immunohistochemistry and in lung lysates by enzyme linked immunoassay. Thrombomodulin cofactor activity, measured in a protein C activation assay with human protein C and thrombin, was decreased in the aorta of hthm/hthm mice (1.1±0.5 nmol/mm) compared with +/+ mice (3.6±0.3 nmol/mm; p&lt;0.001). Thrombomodulin activity also was lower in lung lysates of hthm/hthm mice (0.7±0.1 nmol/mg protein) than in +/+ mice (2.5±0.5 nmol/mg protein; p&lt;0.01). The relative difference in lung thrombomodulin activity between hthm/hthm and +/+ mice was even greater when measured with murine protein C and thrombin (0.7±0.6 vs. 5.2±0.9 nmol/mg protein; p&lt;0.001). Hthm/+ mice had intermediate levels of cofactor activity with either human or murine reagents. Time to thrombotic occlusion of the carotid artery after photochemical injury (rose bengal and green laser) was shorter in hthm/hthm mice than in +/+ mice (10.4±3.0 vs. 34.6±10.6 minutes; p&lt;0.04). We conclude that the humanized thrombomodulin gene is expressed normally in mice but produces a phenotype with markedly diminished anticoagulant activity. The increased susceptibility to experimental thrombosis in hthm/hthm mice suggests that this animal model may be useful for investigation of human thrombomodulin function in vivo.
Development of a Human Acute Myeloid Leukaemia Screening Panel and Identification of Novel Gene Mutations.
Authors: Fitzgibbon Jude , Smith Matthew , Arch Rachael , Smith Lan-Lan , Bainton Nigel , Neat Michael , Bonnet Dominique , Cavenagh Jamie , Lister T. Andrew 
Published: April 2020

Abstract
Mutation studies in Acute Myeloid Leukaemia (AML) are complicated by the existence of distinct morphological and cytogenetic subtypes; consequently although mutations in any one gene may occur in only 5% of AML, the frequency of mutation may differ both between the different FAB-types and cytogenetic risk groups studied. It is important therefore not only to validate the screening methodology used but also the suitability of the patient panel tested. A screening panel was developed allowing detection of novel recurring gene mutations within samples derived from patients with AML. Mutation analysis of 6 previously described genes (RUNX1, FLT3, KIT, CEBPA, PTPN11, NRAS) and 2 candidate genes (CCND3, FES) were carried out in a cohort of 175 AML samples representing all FAB types (except M3) and cytogenetic risk groups using a combination of SSCP, DHPLC and sequence analysis. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with 2 mutations, and 2 patients (1%) with 3 mutations. Fifty-five out of 88 (63%) patients with normal karyotype AML had at least one mutation. There was was a weak negative association between FLT3 ITD and loop mutation (p = 0.095), a positive association between KIT mutation and favourable risk cytogenetics (p = 0.001), CEBPA mutation and intermediate risk/normal cytogenetics (p = 0.045) and PTPN11 mutation and poor risk disease (p = 0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 (Y589D, D839G, Y842H) were detected in 4 patients that would have been overlooked by conventional gel electrophoresis techniques. A single in frame 51bp deletion of nucleotide 939 – 990, resulting in a deletion of 17 amino acids at the carboxyl-terminus of the cyclin D3 protein was identified in a single patient. Overall, both the pattern and mutation frequencies reported in this cohort are similar to those in the literature supporting its further use as an investigational tool in the evaluation of candidate genes in the genesis of myeloid malignancy.
WT1 Overexpression after Induction Therapy in Children AML Is Associated with Higher Risk of Relapse.
Authors: Blaise Annick , Leroy Hugues , Auvrignon Anne , Ballerini Paola , Lapillonne Helene , Perot Christine , Mazingue Francoise , Fasola Sylvie , Flamant Cyril , Douay Luc , Leverger Guy , Preudhomme Claude , Landman-Parker Judith 
Published: April 2020

Abstract
The Wilms’ s tumor gene (WT1) is a tumor suppressor gene highly expressed in most acute leukemias. To determine whether WT1 gene expression is a valuable and informative marker for minimal residual disease in pediatric AML, we quantified WT1 transcript amount by RQ-PCR in 92 de novo AML and 20 normal controls. The WT1 transcripts obtained were normalized with respect to the number of TBP transcripts and expressed as WT1 copy numbers by the ratio WT1/TBPx1000. The WT1 levels were extremely low in normal controls, and the median number of WT1 copies was 10 (range 4–30 ). A level above 50 copies was considered as significant. All the patients (aged 2 months-18 years, median age:5.9 years, male/female ratio 0.87) were treated for de novo AML between 1995 and 2003 in two French institutions and enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: MO 5.4%, M1 4.3%, M2 18.5%, M3 14.1%, M4 and M4Eo 12%, M5 33.6 %, M7 10.9% and unclassified 1%. Cytogenetics features according to the MRC classification were favourable, intermediate or poor in 27% (23/83), 59% (49/83) and 13% (11/83) respectively. With a median follow up of 24 months (range 8–97months) OS was 75± 8% and EFS 60 ± 6%. At diagnosis WT1 overexpression was detected in 78.3% (72/92) with a median copy number of 2231 (range 50-429200). The WT1 values were significantly higher (p=0.02) in M2-FAB subtype and lower (p=0.01) in M5-FAB subtype while no correlation was found with WBC count or cytogenetic abnormalities. WT1 quantification for MRD was evaluable in 41/72 pts and positive in 9/32 at D40-50, 5/25 at M3-M5, 5/7at M6-M8. At least one analysis above 50 copies after induction therapy is associated with a significant risk of subsequent relapse 8/11 vs 8/30 ( p=0.007) RR=22 (IC 95%:46–118) and death 10/14 vs 3/30 (p=0.001) RR=7.3 (IC95%: 1–34). Although retrospective analysis may include bias, we conclude that WT1 is a useful and informative molecular marker for MRD in pediatric AML and performed as prospective analysis in ELAM02 protocol.
NUP98 Is Fused to PRRX2, a New Homeobox Partner Gene in t(9;11)(q34;p15), in a Therapy-Related Acute Myeloid Leukemia.
Authors: Gervais Carine , Mauvieux Laurent , Perrusson Nathalie , Helias Catherine , Struski Stephanie , Leymarie Vincent , Lioure Bruno , Lessard Michel 
Published: April 2020

Abstract
The nucleoporin gene NUP98 is known to be rearranged in several recurrent translocations occurring in de novo and therapy-related myelodysplastic syndrome and acute leukemia, in children or adults. All these abnormalities seem to have bad prognosis, thus, it is important to identify NUP98 rearrangements. As other genes (MLL, ETV6, RUNX1, ...), NUP98 may fuse to different partners, often homeobox genes such as HOXA, HOXC, HOXD, PMX1, but also non homeobox genes such as RAP1GSD1, NSD1, NSD3, LEDGF, ADD3, DDX10, and TOP1. The fusion transcript always juxtapose the N-terminal FG repeats of NUP98, required for its docking function, to the C-terminus of the partner gene, which contains the homeodomain in the case of a homeobox partner gene. We identified a t(9;11)(q34.1;p15.5) by conventional cytogenetic analysis, in a 65 years old Caucasian female who developed a t-AML four years after lymphoma treatment. The involvement of NUP98 gene was confirmed by FISH analysis using BAC RP11-120E20 and PAC RP11-1173K1. 3′RACE analysis allowed to identified a novel partner gene, the class II homeobox gene PRRX2 (Paired Related homeobox 2), located on 9q34. The breakpoint occurred in an infrequent breaking region in exon 11 of NUP98, and in exon 2 of PRRX2. The NUP98-PRRX2 fusion transcript was cloned and sequenced, and confirmed by RT-PCR. As its homologue PRRX1 (PMX1), PRRX2 is a DNA-binding transcription factor that is essential for fetal development. PRRX1 has been described to be fused with NUP98 in t(1;11)(q23;p15), but it is the first time PRRX2 is implicated in leukemia, and even in malignancy. Further studies are necessary to confirm the recurrence of this translocation and its prognosis. Furthermore, transformation assays in cells lines and transgenic mice studies would be interesting to understand the leukemogenicity induced by the NUP98-PRRX2 fusion transcript.
Identification of a Second Acute Promyelocytic Leukemia (APL) Patient with the STAT5b-RARα Fusion Gene among PML-RARα-Negative Eastern Cooperative Oncology Group (ECOG) APL Protocol Registrants.
Authors: Gallagher Robert E. , Mak Serena , Paietta Elisabeth , Cooper Brenda , Ehmann W. Christopher , Tallman Martin S. 
Published: April 2020

Abstract
In addition to the PML-RARα fusion gene, which accounts for &gt;98% of APL cases, a common segment of 5′-truncated RARα has been found to fuse with the following 4 alternative genes: promyelocytic leukemia zinc finger (PLZF-RARα), nucleophosmin (NPM-RARα), nuclear mitotic apparatus protein (NUMA-RARα) and signal transducer and activator of transcription 5b (STAT5b-RARα). While recurrent cases of PLZF-RARα and NPM-RARα have been documented, there are only single case reports of NUMA-RARα and STAT5b-RARα. In two Phase III clinical trials, E2491 and C9710, the ECOG registered 10 and 12 presumptive APL patients, respectively, based on initial clinical and cytological assessment, which were negative for PML-RARα by reverse transcriptase-polymerase chain reaction (RT-PCR) testing. In this study, we tested 20/22 of these PML-RARα-negative protocol patients for the presence of the 4 alternative fusion genes by RT-PCR, using the corresponding normal transcripts for the rearranged genes as RNA transcription controls. All specimens were positive for the control gene, while 1 case each was positive for PLZF-RARα or STAT5b-RARα. The karyotypes for these 2 cases, respectively, were 46, XY, t(11:17)(q23;q21) and 46, XY, t(10;11)(q22:q25), i(17)(q10). None of the X-RARα-negative cases had abnormalities of chromosome 17. Immunophenotypically, the PLZF-RARα case was consistent with APL; the STAT5b-RARα case had all features of APL (negative for CD11a, CD18, CD34, HLA-DR) except for weak CD133 expression. These findings imply that it is the RARα rearrangement that prompts the APL immunophenotype. Sequence analysis of the STAT5b-RARα mRNA disclosed the same break/fusion point of the STAT5b gene as the only previously-reported case of STAT5b-RARα-positive APL (Arnould, et al, Hum Mol Genet8, 1741, 1999). Also like the first case (67 years), our STAT5b-RARα patient was an elderly male (57 years). These results suggest that X-RARα fusion genes will rarely be found in the absence of cytogenetic evidence of chromosome 17 abnormalities and establish that STAT5b-RARα, although rare, is a recurrent pathogenetic factor in APL.
The Presence of FLT3/ITD Mutations Is an Independent Prognostic Factor in Acute Myeloid Leukemia Patients with Normal Karyotype.
Authors: Kim Yeo-Kyeoung , Lee Je-Jung , Lee Yu-Ra , Lee Il-Kwon , Kim Hee-Nam , Kim Nan-Young , Park Kyeong-Soo , Yang Deok-Hwan , Park Moo-Rim , Kim Hyeoung-Joon 
Published: April 2020

Abstract
An internal tandem duplication of the FLT3 gene (FLT3/ITD) has been detected in approximately 20–30% of patients with acute myeloid leukemia (AML). These are frequently associated with poor outcome in AML patients, but it is still a matter of debate whether the FLT3/ITD mutations play a role in the prognosis of AML patients independently or not. We investigated the presence of FLT3/ITD mutation in 165 patients with de novo AML, except acute promyelocytic leukemia (APL), to evaluate its clinical and prognostic significance. The FLT3/ITD mutations were studied on bone marrow samples at diagnosis using PCR assay. Of the patients, 58 patients (35.2%) demonstrated the aberrant FLT3/ITD mutations. The patients with FLT3/ITD had significantly higher WBC counts at presentation compared with patients without FLT3/ITD (52.9 ± 66.9 ×109/L vs. 32.4 ± 41.8 ×109/L, p &lt; 0.05). However, there was no statistically significant difference in age, gender, hemoglobin level, platelet count, percentage of peripheral or bone marrow blasts, or the presence of molecular abnormalities between the patients with FLT3/ITD and the patients without FLT3/ITD. To analyze the response to or outcome of therapy, we evaluated 118 patients who received intensive induction chemotherapy. In univariate analysis, there was no significant difference in complete response rate (p = 0.21), in median duration of overall survival (13.9±3.8 ms. vs. 16.5±0 ms., p = 0.07), or in median duration of leukemic-free survival (LFS) (9.8±3.5 ms. vs. 34.6±17.9 ms., p = 0.09) between the patients with FLT3/ITD and the patients without FLT3/ITD. However, the presence of FLT3/ITD was associated with lower LFS in the patients with a cytogenetically intermediate-risk group (p &lt; 0.05). Furthermore, in multivariate analysis, FLT3/ITD mutations were an independent prognostic factor in LFS in AML patients with normal karyotype (p &lt; 0.05). In conclusion, this study demonstrates that the presence of FLT3/ITD mutations is a significantly poor prognostic factor for leukemic free survival in non-APL patients with a cytogenetically intermediate-risk group, especially normal karyotype.
FAS Promoter Polymorphism and Etiology and Outcome of Childhood Acute Myeloid Leukemia.
Authors: Mehta Parinda A. , Alonzo Todd A. , Elliot James S. , Gerbing Robert B. , Wilke Tiffany A. , Perentesis John P. , Lange Beverly J. , Davies Stella M. 
Published: April 2020

Abstract
FAS (TNFRSF6/CD95/APO-1) is a cell surface receptor involved in apoptotic signal transmission. Dysregulation of this pathway is believed to result in down regulation of apoptosis and subsequent persistence of a malignant clone. A single nucleotide polymorphism resulting in guanine-to-adenine transition in the FAS promoter region occurs at position −1377, affecting an SP1 transcription factor binding site; an adenine residue at position −1377 of FAS promoter significantly reduces SP1 binding compared to guanine residue causing a decrease in FAS expression. Recent data in adult leukemia indicate that a variant allele at this site increases susceptibility to AML (Morgan et al; Cancer Research 63, 4327–4330). We hypothesized that FAS genotype would also increase risk of childhood AML and, by altering susceptibility to apoptosis might impact outcome of AML therapy. Methods: 442 children treated for AML on CCG studies 2941 and 2961 (intensive induction with daunorubicin, idarubicin, Ara-C, 6-thioguanine, etoposide, dexamethasone, +/− fludarabine, Ara-C, idarubicin; consolidation with high-dose Ara-C and L-asparaginase or HLA-matched sibling BMT) were genotyped for the −1377 FAS promoter polymorphism using mismatch amplification coupled with real time PCR (MAMA assay). Genotype frequencies were compared with published normal control frequencies. In addition patient outcomes were analyzed according to genotype.
Results: Comparison of gene frequencies in AML patients and reported normal controls showed similar frequencies (78.7% GG, 18.6% GA, 2.7% AA in patients vs. 78% GG, 20% GA and 2% AA in controls; p=0.6). Stratification of cases by age at diagnosis, white blood count at diagnosis, AML subtype, or cytogenetics revealed no difference in genotype frequencies. Outcome data showed no significant difference in OS (p=0.22), EFS (p=0.31), TRM (p=0.42) or relapse rate (p=0.57) between patients with 1377GG genotype vs. 1377GA/1377AA genotypes. However, there was trend towards improved survival (OS at 5 years 75% AA, 55% GA, 49% GG; p=0.16) and EFS (66% AA, 43% GA, 39% GG; p=0.19) in AA cases, but small numbers (n=12 AA) may have contributed to the results not being statistically significant. Conclusion: These data, representing the only data in pediatric AML, suggest that FAS genotype does not affect the etiology of childhood AML and that children with AML differ from adults in terms of biology of their disease. However, further studies are needed to evaluate a potentially important role of FAS genotype in outcome of AML therapy.
Heparin-Induced Thrombocytopenia Is a Thymus (T cell) Dependent Disorder.
Authors: Suvarna Shayela , McCracken Emily K.E. , Arepally Gowthami M. 
Published: April 2020

Abstract
Heparin-Induced Thrombocytopenia (HIT) is a drug-dependent immune disorder caused by autoantibodies to Platelet Factor 4 (PF4) and heparin. The immune basis of HIT is poorly understood. Recent studies describing transient antibody responses and absence of immunologic memory in HIT suggest that PF4/heparin autoantibodies may develop independently of T cell help. To investigate the cellular basis of the HIT immune response, we have developed a murine autoimmune model in which anti-murine PF4/heparin (anti-mP+H) arise de novo. Cohorts of BALB/c mice were immunized daily either intravenously (IV, n=10) or intraperitoneally (IP, n=10) for five days with complexes of murine (m) PF4/heparin (IV, n=5 or IP, n=5), heparin alone (IV, n= 2) or buffer (IV, n=3 or IP, n=5). Mice were screened for anti-mP+H for four weeks after immunization using a murine PF4/heparin ELISA. Peak antibody responses to antigen were seen at 11–15 days in 2/5 mice injected with IV mP+H (Day 11, mouse IV P+H #0 peak A450nm =0.34±0.01; Day15 mouse IV P+H #2 peak A450nm =0.69±0.01), and at days 22–25 in 2/5 mice injected by IP route (Day 22, mouse IP P+H #0 peak A450nm =0.37±0.01; Day 25 mouse IP P+H #2 peak A450nm =0.78±0.02). Anti-mP+H were not detected in mice injected with heparin alone or buffer alone at any time point (peak maximum IV A450nm= 0.1±0.001, Control #2; mouse IP P+H Control #2 A450nm =0.04±0.002). Serologically, murine autoantibodies were similar to anti-human (h) PF4/heparin. Anti-mP+H reactivity was specific for murine antigen (mouse IV P+H #2 A450nm=0.65±0.06), and was reduced with antigen in the presence of excess heparin (A450nm=0.38±0.01). Minimal reactivity was seen with wells coated with hP+H (A450nm=0.09±0.005), albumin (mouse IV P+H #2 A450nm=0.15±0.03), or PBS alone (mouse IV P+H #2 A450nm =0.16±0.01). Similar to human HIT antibodies, anti-mP+H were of IgG1 subclass. To determine if T cells are required for development of anti-mP+H, mice lacking T cell function (BIG:BALB/c-Nu, n=10) were injected IV with mP+H daily for five days. Unlike euthymic mice, nude mice did not manifest any antibody responses to IV injections of mP+H. In summary, we have developed a novel murine autoimmune model of anti-PF4/heparin that recapitulates many salient features of the human immune syndrome. Using this murine model, we demonstrate that T cells are essential for development PF4/heparin autoantibodies. Studies are currently underway to delineate mechanisms of T cell regulation and peripheral tolerance in HIT.
Comparison of Notoginsenoside R1 and TPO on Thrombopoiesis in Irradiated Mice.
Authors: Yang Mo , Lam T.L. , Li Karen Kwai Har , Leung L.K. , Lau KM , Cheng S.W. , Pong N.H. , Lau C.P. , Fung K.P. , Fok Tai Fai 
Published: April 2020

Abstract
The use of combination of Chinese herbs as a treatment for thrombocytopenia has been reported to be effective and safe. We have reported that Angelica Polysaccharide (extracts of Angelica Sinensis) has promoting effect on blood stem cells and megakaryocytopoiesis (Yang et al, Blood, 2002, 100 (11); 53a). Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been used for treatment of trauma and bleeding due to internal and external injuries. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice. NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays (n=5). Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 ± 0.7 vs 19 ± 0.38 colonies/2 x 106 cells, p=0.0034), CFU-MK (22 ± 1.9 vs 26 ± 3.8 colonies/2 x 105 cells, p=0.025), CFU-GM (26 ± 5.2 vs 37 ± 4.3 colonies/2 x 105 cells, p=0.002), and BFU-E (13 ± 2.9 vs 18 ± 1.9 colonies/2 x 105 cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0–50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (19 ± 2.2 vs 30 ± 6.8 colonies/2 x 105 cells, p=0.02, n=5). Furthermore, the effect of NR1 (5–50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 ± 3.7 vs 24 ± 1.8 colonies/2 x 106 cells, p=0.043, n=5). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Although TPO has been used as an agent for the recovery of platelet production after the onset of thrombocytopenia, long-term clinical usage of TPO may induce potential side effects such as thrombosis. Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice.