bluuebunny/crossref_metadata_2025 huggingface dataset:
Student participation in private supplementary education: A comparative analysis of Japan, Korea, Shanghai, and the USA
Authors: Park Hyunjoon , Lim Youngshin
Published: April 2020

Private supplementary education, which refers to private lessons and learning outside of formal schooling purchased by families, has been widely practiced in East Asia. Its demand has grown even beyond East Asia, however, as educational competition for social mobility has intensified in many parts of the world. This global trend makes it important to determine who has greater access to private supplementary education and address the implications of the differential access for educational inequality. The current study compares how family socioeconomic status (SES) and students’ prior academic performance are related to their participation in private supplementary education in three East Asian societies—Japan, Korea, and Shanghai (China)—and the USA. Private supplementary education has existed on a substantial scale in Japan and Korea. The public and scholars have increased their concerns regarding the growing prevalence of private supplementary education in China. Although it has not been a major educational strategy, the demand for private supplementary education is rising in the USA as well, which can offer a useful insight into the global application of private supplementary education in contemporary educational systems. The focused comparisons across a small number of societies allow analyses of detailed patterns in each society in comparative perspective, thus moving beyond both single-country research and large-scale cross-national studies. The data for the current study come from 15-year-old students who participated in an international survey of student achievement, Programme for International Student Assessment (PISA) 2012. With a clearly defined measure of private supplementary education, PISA 2012 provides an exceptional opportunity for comparison. Drawing on data for 15-year-old students in four societies from PISA 2012, the linear probability models highlight heterogeneity among three East Asian societies. Japan is distinctive from Korea and Shanghai in terms of the influences of family SES on student participation in private supplementary education. Once school-fixed effects are taken into account, the strength of the relationship between family SES and private supplementary education in Japan is similar to the strength in the USA, which is weaker than the strength in Korea and Shanghai. Japan and the USA are also similar in that family structure is not significantly associated with private supplementary education, while students in non-two-parent families are significantly less likely to receive private supplementary education in Korea and Shanghai. The school-fixed-effects models also show that the within-school relationship between students’ prior academic performance and their participation in private supplementary education is generally negative. Japan, Shanghai, and the USA show a significantly negative relationship, while only Korea shows no significant relationship. Future research directions are suggested in the conclusion.
“How Do We Stop Trying to Do It All?” Application of the Family Business Parallel Planning Process
Authors: Davidson Amber , Berka Gregory , Meador Cami
Published: April 2020

Keith Lang, a then college senior, and his mother, Lori, launched Global Logistics & Shipping Partners (GLS) overnight when an opportunity presented itself to secure a number of clients. From Week 1, they were focused on exceptional client support and service without any infrastructure in place. Holly Primrose, Keith’s long-time girlfriend, who still had a year left in college, jumped in to help. Now 15 years later, the business grew to 22 employees, while experiencing many revenue challenges. Keith and Holly worked tirelessly for 15 years straight with an equal focus on clients, ethics, and GLS’s employees. After having a family and investing in other endeavors, Keith and Holly found themselves in a position where they needed to make decisions regarding the future vision for GLS and themselves. They desired to remain engaged with GLS but also to spend as much time as possible with family and personal interests. The case provides information that allows students to apply Carlock and Ward's family business parellel planning process to evaluate a family business' goals, evaluate the owners' personal goals, and find synergies across professional and personal goals and visions.
Is the Proposed World Health Organization (WHO) Classification for Chronic Myeloid Leukemia (CML) of Clinical Value in the Imatinib Era?.
Authors: Cortes Jorge , O’Brien Susan , Garcia-Manero Guillermo , Faderl Stefan , Shan Jenny , Giles Francis , Verstovsek Srdan , Rios Mary Beth , Talpaz Moshe , Kantarjian Hagop
Published: April 2020

The criteria to define chronic (CP), accelerated (AP) and blast phase (BP) CML vary in the literature. The WHO recently proposed, based on the literature and collective experience of the clinical advisory committee, new criteria for myeloid malignancies including CML in an attempt to provide more uniform criteria (Blood2002; 100:2292). Imatinib is the current standard therapy for CML, and the criteria used in most studies using imatinib are shown below in contrast to the WHO proposal:

Phase Criteria Imatinib WHO *Unrelated to therapy (Rx), **Unresponsive to Rx, Plts=platelets, BM=bone marrow AP Blasts 15–29% 10–19% Blasts + promyelocytes ≥ 30% NA Basophils ≥ 20% ≥ 20% Plts <100 x 109/L* Yes Yes Plts >1000 x 109/L** No Yes Increasing spleen size and WBC** No Yes CE At any time Not at diagnosis (Dx) BP Blasts ≥ 30% ≥ 20% Extramedullary disease Yes Yes Large clusters of blasts in BM NA Yes

We investigated the clinical validity of the WHO proposal among 809 patients (pts) with CML in all stages treated with imatinib at MDACC since 1999. According to the imatinib classification, 537 (66%) pts were in CP, 196 (24%) in AP, and 76 (9%) in BP. When analyzing the specific subsets of pts where the classifications differ, major findings are: 1) Pts with CE at Dx (n=14) have similar cytogenetic (CG) complete remission (CR) rate (86%) than pts (n=477) with imatinib CP (75%, p=.53), but it is lower when CE develops after Dx (34/64, 53%; p=.0005). Similar results are found for overall survival (OS) and progression-free survival (PFS). 2) CG CR rate among pts with 20%–29% blasts (4/19, 21%) is more similar to that of AP (37/99, 37%)(p=0.19) than to pts with ≥30% blasts (5/76, p=0.07). This trend is more significant for OS and PFS. 3) Pts with increasing WBC and spleen, or plts >1000 x109/L unresponsive to Rx have a CG CR rate (9/42, 21%; p=.07) lower than others with AP, but there is no difference in OS or PFS. 4) Pts with plts >1000 x109/L without prior Rx have similar outcome as those with CE developing on therapy. In conclusion, based on results imatinib therapy, AP can be defined as blasts ≥10%, basophils ≥20%, plt <100 x109/L (unrelated to Rx) or >1000 x109/L (unresponsive to Rx), increasing WBC and spleen (unresponsive to Rx), and CE not at diagnosis; BP is defined by blasts ≥30% or extramedullary disease; pts who develop CE during the course of Rx of with plts >1000 x109/L not related to Rx constitute an intermediate group between CP and AP; all others are CP.
Frequency of Blast Crisis after Achieving Complete Cytogenetic Remission in First Chronic Phase CML Patients Who Recieved Imatinib Therapy within Six Months of Diagnosis.
Authors: Laurence Arian , Marin David , Clark Richard , Shepherd4 Patricia , Mackinnon Stephen
Published: April 2020

Stem cell transplantation (SCT) remains the only proven curative therapy for chronic myeloid leukemia (CML) but is limited by significant procedure related morbidity. Imatinib is the first of a new generation of specifically targeted drugs for this disorder based on the ubiquitous presence of the BCR-ABL kinase. The success of Imatinib therapy to induce a cytogenetic remission (CCyR) raises questions over the suitability and timing of SCT as a treatment for CML. Of importance is the degree of protection CCyR infers from developing a blast crisis. We report a total of 68 patients from five centres all of whom started Imatinib within six months of diagnosis of 1st chronic phase CML and subsequently achieved a complete cytogenetic remission. In this group, five cases spontaneously developed blast crisis despite achieving a CCyR. In the affected patients CCyR was achieved between 97 and 254 days (mean 148 days) and BC developed between 177 and 875 days (mean 446 days). The BC was myeloid in three cases and lymphoid in the remaining two. Statistical analysis of this group predicts a 91% freedom from blast crisis at two years (figure 1). The development of acute leukemia is assumed to derive from further mutations within the BCR-ABL expressing stem cell pool. The success of SCT therapy in curing CML drops significantly if the patient has previously progressed to a blast crisis. The finding that cytogenetic remission is no guarantee of protection from developing a blast crisis suggests that for young patients with a sibling match, stem cell transplantation may be considered as an early therapy in the management of CML.
The Resusciatative Fluid You Chose May Potentiate Bleeding.
Authors: Brummel-Ziedins Kathleen E. , Whelihan Matthew F. , Ziedins Eduards G. , Mann Kenneth G.
Published: April 2020

Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation to be used (dilutions ranging up to 66%) in hemorrhagic shock for restoring hemodynamic stability. Trauma results in massive exposure of tissue factor to the circulation and explosive amounts of thrombin being generated. We studied the effect of various resuscitative formulas to blood coagulation, specifically thrombin generation and fibrin formation, in a controlled setting using corn trypsin inhibited whole blood initiated with a 5pM stimulus of tissue factor. Thrombin generation measured as its complex with antithrombin III (TAT) was evaluated periodically over a time course of 20 min. Fibrin clots were collected and weighed. We investigated four diluents (0.9% NaCl (NS), lactated Ringer’s solution (LR), 6% hydroxyethyl starch (HES) and 3% NaCl (HS)) each at a 0,10, 20, 30, 40, 50 and 60% blood dilution. At a 10% dilution TAT generation was in the order of LR (−4%) < HES (−8%)< NS and HS (−12%). Diluting by 20% resulted in further decreases of TAT formation. The fibrin clot mass decreased dramatically with a 20% dilution for NS (−42%) and HES (−30%). Conversely, HS produced no change in fibrin mass but effected the largest change in thrombin generation rate (−56%). At a 50% dilution, comparable thrombin generation profiles were obtained for LR, HES and NS (~35% decrease). However, the fibrin masses decreased by 27% with LR, 46% with NS and 74% with HES. No clot formation or thrombin generation was seen with HS at > 20% dilution. This in vitro study shows that: 1) LR has the least effect on thrombin generation and gave higher than anticipated clot weights; 2) HES reduced the fibrin clot mass at higher dilutions; 3) HS abolishes coagulation after a 20% dilution. Overall, both the extent and nature of hemodilution cause profound alterations in the hemostatic mechanism.
The Predictive Value of hs-C-Reactive Protein Is Based on the Presence of Antiphospholipid Antibodies.
Authors: Miesbach Wolfgang A. , Boehm Martina , Claus Detlef , Scharrer Inge
Published: April 2020

High-sensitive C-reactive protein (hs-CRP) is a marker of inflammation which has been shown in several prospective studies to predict independently myocardial infarction, stroke or peripheral artery disease. Patients with antiphospholipid antibodies (aPL) are at increased risk of recurrent thromboembolic events but the possibility to predict such risk seems rather limited. Recently, similarities were found in the pathology of thrombosis between elevated levels of hs-CRP and the presence of aPL.We studied the predictive role of hs-CRP levels in patients with the presence of aPL of a cohort of patients with neurological manifestations compared to those where aPL could be excluded.

Patients A follow-up investigation was done in 55 aPL-positive and 61 aPL -negative, sex- and age matched patients of the same cohort of patients with acute manifestations of neurological diseases. Hs-CRP levels were measured in all patients at enrollment and were related to the outcome of the patients after a median time of 32 months.

Methods Lupus anticoagulants were detected according to the SSC of the ISTH. Anticardiolipin tests were performed by a ß2-glycoprotein I-dependent enzyme-linked immunsorbent assay (Pharmacia ELISA). Hs-CRP was measured by latex enhanced turbidometry (dimension RXL, Dade Behring).

Results Cerebral infarctions and transient ischemic attacks were the most common cerebral manifestations of the patients. In APS patients elevated levels of hs-CRP could be measured significantly more frequently than in patients where aPL could be excluded (44 % vs. 16 %, p<0.005). The rate of recurrences or severe residual symptoms was higher in patients with aPL (45 %) compared to 32 % in aPL-negative patients. In non APS patients hs-CRP levels were not associated to the occurrence of future neurological events. In patients with aPL elevated levels of hs-CRP were highly associated to an increased rate of recurrent or residual symptoms (OR, 12.5; 95 % CI, 3.72 to 41.94) and not related to other risk factors, except of smoking (p<0.05).

Conclusion Elevated levels of hs-CRP were associated to the presence of aPL and related to the risk of recurrences in these patients but not in patients where the presence of aPL could be excluded. In patients with APS elevated levels of hs-CRP may identify a group of patients at high risk of recurrent or residual neurological symptoms who may benefit from a more careful follow-up and antithrombotic therapy.
Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.
Authors: Kentner Taryn A. , Berry Leslie R. , Chan Anthony K.C.
Published: April 2020

Prothrombinase is the surface-bound complex in which factor Xa (Xa) converts prothrombin to thrombin in vivo. Studies have shown that Xa within prothrombinase is resistant to inhibition by antithrombin + heparin (AT+H). Previously we found that, unlike AT+H, a covalent conjugate of AT and H (ATH) was able to neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa in prothrombinase. Mixtures of CaCl2, PCPS vesicles, factor Va (Va) and prothrombin in TSP buffer were combined with either AT+H or ATH. Following addition of Xa, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to those in corresponding experiments with Xa alone. Inhibition of Xa in prothrombinase by AT+H had a k2 (x 108 M−1min−1) of 0.688 +/− 0.030. Conversely, free Xa neutralization by AT+H was significantly more rapid (k2 = 1.53 +/− 0.35, p = 0.041). ATH reaction with prothrombinase Xa proceeded at a rate that was insignificantly different from that with Xa alone (2.36 +/− 0.31 and 2.83 +/− 0.83, respectively) and similar to AT+H reaction with free Xa. Varying concentrations of prothrombinase components showed similar effects. We conclude that covalently linked complexes of AT + H undergo unhindered reaction with Xa in prothrombinase, while non-covalently linked AT+H encounters resistance. It is possible that ATH may effectively prevent plasma thrombin generation by neutralization of Xa in prothrombinase and thus may prohibit feedback activation of coagulation.
Minimal Residual Disease Detection Identifies Differences between the Risk Groups Defined by the ALL IC-BFM 2002 and the ALL-BFM 2000 Protocols.
Authors: Fronkova Eva , Reznickova Leona , Muzikova Katerina , Mejstrikova Ester , Hrusak Ondrej , Schrappe Martin , Stary Jan , Trka Jan
Published: April 2020

Minimal residual disease (MRD) testing based on a unique Ig/TCR gene rearrangement pattern of each patient’s leukaemia turned out to be an independent tool to determine treatment response and the risk of relapse in paediatric acute lymphoblastic leukaemia (ALL). Since 07/2000, MRD information at week 5 and 12 of therapy has been used for stratification in ALL-BFM 2000 trial. In parallel, ALL IC-BFM 2002 has been designed by the International-BFM Group to test the morphological assessment of the early treatment response. Patients are stratified according to the blast proportion in peripheral blood (PB) at day 8 and in bone marrow (BM) at day 15 and 33 of therapy, age, initial WBC and the presence of BCR/ABL and MLL/AF4 fusion. One of the goals of the study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. In the Czech Republic, 73 patients were treated according to ALL IC-BFM 2002 protocol from 11/2002 to 12/2003, 29 in the standard-risk (SR), 35 in the intermediate-risk (IR) and 9 in the high-risk (HR) group. The SR, HR and all T-cell ALL patients were examined for clonal Ig/TCR rearrangements. RQ-PCR patient-specific systems were designed for each of these patients according to the ESG-MRD-ALL criteria. For 39 of the 40 patients tested (97.5%) at least one target with minimal sensitivity of 10(−4) was identified. MRD was evaluated in BM samples from 34 patients at several time points inclusive of the mandatory 5 and 12 week ones. Simultaneously the PB specimens of the T-ALL patients were tested. In total, 205 BM and 64 PB specimens were included. In 7 patients of 24 in the SR group, MRD positivity at week 5 and/or at week 12 was observed (ranging between 9.7x10(−4) and 1.5x10(−2)), thus identifying patients who would not qualify to the MRD-based SR group in ALL-BFM 2000 despite the identical induction regimen. In T-ALL patients, PB-MRD levels paralleled those in BM. MRD results showed no separation of MRD levels between IR- and HR-stratified T-ALL patients. These preliminary findings reveal a significant difference between the stratification results of ALL IC-BFM 2002 and ALL-BFM 2000. A fast response as measured by the morphology criterion (M1 or M2 bone marrow at day 15) together with other low-risk features does not necessarily correspond with rapid MRD clearance. The complete analysis of MRD is planned for the international consortium participating in the ALL IC-BFM 2002 protocol.
IRTA2 Protein on Lymphoma Cell Lines and Hairy Cell Leukemia Cells Detected by Novel Monoclonal Antibodies.
Authors: Ise Tomoko , Nagata Satoshi , Maeda Hiroshi , Kreitman Robert , Stetler-Stevenson Maryalice , Pastan Ira
Published: April 2020

The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of the mRNA in B cell-linage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the function and expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. We successfully made 36 MAbs from a mouse immunized with IRTA2-encoding plasmid DNA. All the MAbs reacted with IRTA2-extracellular domain-Fc fusion protein in ELISA and 293T cells expressing IRTA2 in a fluorescence-activated cell sorter (FACS) analysis. Binding of each MAb to a series of deletion mutants of IRTA2-Fc fusion protein and mutual competition of the MAbs to the binding to the whole extracellular domain of IRTA2-Fc fusion protein revealed that the panel of MAbs recognizes more than 10 epitopes on IRTA2. There are at least 1, 1, 2, 4, and 2 epitopes on extracellular domain 1 (ED1), ED3, ED1-3, ED4-9, and whole extracellular domain, respectively. Twenty five out of the 36 MAbs are specific to IRTA2 but 11 others showed cross reactivity to other members of the IRTA family. Among 36 MAbs produced, 3 MAbs (F25, F56 and F119) were used for further analysis based on their specific reactivity with recombinant IRTA2 expressed on 293T cells and lack of cross reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression on 6/7 B-cell non-Hodgkin’s lymphoma and 1/6 Burkitt’s lymphoma cell lines. Reverse transcriptase-polymerase chain reaction experiments and Western blotting using MAb F25 confirmed the expression profile. We also analyzed blood samples of 11 patients with hairy cell leukemia (HCL), 11 patients with chronic lymphocytic leukemia (CLL), and 4 patients with follicular lymphomas (FL) by FACS. We detected IRTA2 expression on 100% HCL (11/11), 55% CLL (6/11) and 0% of FL cells (0/4). Our results provide the first evidence that the IRTA2 is expressed on the surface of HCL and CLL cells as well as some human lymphoma cell lines. We expect IRTA2 will be established as a new target for immunotherapy.
Array CGH Analysis for Target Genes at 6p21 Amplification Region in Diffuse Large B-Cell Lymphoma.
Authors: Kasugai Yumiko , Tagawa Hiroyuki , Kameoka Yoshihiro , Karnan Sivasundaram , Suzuki Ritsuro , Tsuzuki Shinobu , Hosokawa Yoshitaka , Seto Masao
Published: April 2020

Amplification of 6p21 has been detected in various solid tumor and hematological neoplasms. These include hepatoma, osteosarcoma, pancreatic cancer, bladder cancer, and mantle cell lymphoma . However, no candidate target genes in this amplified region have been identified. In diffuse large B-cell lymphoma (DLBCL), no amplification has been reported. Recently, we established genome-wide array comparative genomic hybridization (array CGH) consisting of 2300 BAC clones that can survey a whole genome at the density of an average size of 1.3 Mb. The array CGH detected amplification at 6p21 in 12 of 70 (24%) DLBCL patients (Tagawa et al., Cancer Res. in press) and one DLBCL cell line, SUDHL-9. We next made high density array glass for 6p21 region with 23 BAC clones covering 3 Mb. We found that the minimal common region of amplification was approximately 2Mb in size. In this region, there are 27 known genes including cyclin D3. The cyclin D3 has been reported to be overexpressed in a case of DLBCL with t(6;14)(p21.1;q32.3). We have analyzed expression level of those genes in the minimal common region of amplification, and found that five genes, BYSL, cyclin D3, TBN, KIAA0240, and TBCC were overexpressed. These results indicate that not only cyclin D3 but also other genes are also possible target genes for the 6p21 amplification.
Critical and Complementary Role of FLT3 and Interleukin 7-Receptor Alpha Signaling in T Lymphocyte Development.
Authors: Buza-Vidas Natalija , Ahlenius Henrik , Cilio Corrado M. , Svensson Marcus , Agace William , Jacobsen Sten Eirik W. , Sitnicka Ewa
Published: April 2020

We recently demonstrated that signaling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor a (IL-7Ra) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). These receptors are also implicated to be important in regulation of T cell development, but their potential interdependence remains unexplored. We recently showed that flt3 ligand (FL)-deficient mice have reduced levels of early thymic progenitors as well as the common lymphoid progenitor (CLP) (Sitnicka et al., Immunity, 17:463, 2002). In the present study we investigated T cell development in mice deficient in FL and IL-7Ra expression. Strikingly, when compared to FL−/− and IL-7Ra−/− mice, FL−/−xIL-7Ra−/− (double deficient) mice (8-10 week old) lack visible lymph nodes and Peyer’s Patches. Thymic cellularity was dramatically reduced to only 0.3% of FL−/− and wild type (WT) controls and to only 4% of IL-7Ra−/− mice. In agreement with previous studies, IL-7Ra−/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−xIL-7Ra−/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. Thus, severe reductions in early thymocyte development in FL−/−xIL-7Ra−/− mice support a similar role for cross talk between these two signaling pathways in T cell development as recently demonstrated for B cell genesis.
Hematopoiesis in the Ts65Dn Mouse Model of Down Syndrome (DS) Recapitulates Many Features of Human DS.
Authors: Mundschau Gina , Jilani Sarah , Le Beau Michelle , Crispino John
Published: April 2020

Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL) is a complex malignancy that evolves in hematopoietic progenitors with trisomy 21 that acquire a somatic mutation in the blood transcription factor GATA1. The mechanistic relationship between these two genetic factors that leads to leukemia is poorly understood. In order to study the interplay between trisomy 21 and GATA1 mutations, we are developing a mouse model of this malignancy. The Ts65Dn mouse, which contains a segmental trisomy for mouse chromosome 16, homologous to human 21, has been reported to display several of the cognitive and craniofacial phenotypes seen in humans with DS, but the hematopoietic system has not been assessed as a model for blood development in humans with DS. We have evaluated adult hematopoiesis in the Ts65Dn strain by comparing monthly complete blood counts (CBC) of peripheral blood from 14 trisomic and 20 disomic littermates. Similar to humans with DS, Ts65Dn trisomic mice display persistent erythrocyte macrocytosis, with values at the high end of the normal range. Trisomic mice also harbor decreased numbers of red blood cells, mildly elevated platelet counts, a higher percentage of monocytes and a lower hemoglobin concentration. Interestingly infants with DS frequently display thrombocytosis. In addition, we have characterized fetal liver hematopoiesis in the Ts65Dn strain by FACS analysis of hematopoietic precursors and by performing colony assays. In general, we did not detect any significant differences in erythroid, myeloid, or megakaryocytic colony formation between trisomic or disomic fetuses. Likewise flow cytometry for CD34, TER119, and CD41 demonstrated overall similar numbers of cells in these compartments for Ts65Dn mice and disomic littermates. However, one of seven trisomic embryos displayed a significant increase in the proportion of CD34+ cells with concomitant decrease in both Ter119+ and CD41+ populations. In addition, cells from this fetal liver gave rise to seven-fold and three-fold increases in BFU-E and CFU-Mk colonies respectively, with no change in the CFU-GM. Although the sample size is small, these findings suggest that a subset of Ts65Dn trisomic fetuses exhibit aberrant hematopoiesis. Taken together, our study indicates that the Ts65Dn trisomic mouse may be an excellent model to study human DS hematopoiesis.
Evaluation of Existing Limited Sampling Models for Therapeutic Drug Monitoring of Busulphan in Children and Adults Undergoing SCT.
Authors: Hassan Zuzana , Sandström Marie , Hassan Moustapha
Published: April 2020

Busulphan (Bu) is used in high dose conditioning regimen prior to stem cell transplantation. Bu has a narrow therapeutic window and over- and under-dosing may have a fatal outcome. Bu pharmacokinetics and pharmacodynamics were extensively studied and wide inter- and intra-individual variation was found. Several limited sampling models (LSM) have been developed for Bu administered orally to simplify therapeutic drug monitoring and consequently dose adjustment. The aim of this study was to evaluate the existing LSM in adults and children undergoing conditioning regimen before SCT. Seventy-four patients (62 adults and 12 children) with malignant and non-malignant diseases were analysed. Plasma was sampled at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5 and 6 hours after the first dose of Bu. Bu was determined using gas chromatography with electron capture detection. The area under the plasma concentration-time curve (AUC) for time interval 0 to 6 hours was determined using Winnonlin program and trapezoidal rule. Results were compared to the estimated AUCs using LSMs (Vassal 1992, Schuler 1994, Hassan 1996, Chatergoon 1997). The best correlation between the AUCs determined using trapezoidal rule and 3-points model by Schuler was found (R²=0.95 for all patients, R²=0.97 for children and R²=0.94 for adults). In children, a correlation between AUCs determined with trapezoidal rule and following LSMs was found: 2-points LSM by Schuler (R²=0.94), LSM by Hassan (R²=0.94), LSM by Vassal (R²=0.81) and 3 of 5 LSMs by Chatergoon (R²=0.85, 0.88 and 0.87, resp.). AUCs in children determined using Winnonlin showed good correlation with both Schuler’s models, model by Hassan and one of 4-points models by Chatergoon. However, the correlation between the AUCs determined using trapezoidal rule and Winnonlin was good in children (R²=0.98), but not in adults (R²=0.65). Thus, several limited sampling models are suitable for AUC estimation in children, while there is only one suitable model for adults. This conclusion is made with reservation that even the trapezoidal rule may underestimate the real AUC dependent on sampling density.
High Expression of Multidrug Resistance-Associated Proteins Predicts Prognosis in Acute Lymphoblastic Leukemia in Children and Adults.
Authors: Plasschaert Sabine L.A. , de Bont Eveline S.J.M. , Boezen Marike , van der Kolk Dorina M. , Daenen Simon M.J.G. , Faber Klaas Nico , Kamps Willem A. , de Vries Elisabeth G.E. , Vellenga Edo
Published: April 2020

Background: Patients with acute lymphoblastic leukemia (ALL) are treated with different chemotherapeutic drugs, which can be transported by ABC-transporters, including six multidrug resistance-associated proteins (MRPs). New insights suggest that a drug resistance profile, in which multiple transporters act in concert, is more likely to predict prognosis than one single transporter. The aim of this study was to investigate the expression profile of MRP1-6 in ALL in both children and adults and determine its impact on prognosis.

Methods: mRNA expression of MRP1-6 was analyzed by real-time PCR in human de novo ALL samples from 56 children and 49 adults. Of these patients, 32 showed a T-lineage and 73 a B-lineage immunophenotype.

Results: Adults showed a significantly higher expression of MRP1 (median value 1.08 vs. 0.69), MRP2 (0.96 vs. 0.53) and MRP3 (0.31 vs. 0.07) than children. However, no difference in expression levels was observed between children and adults who developed a relapse. High expression of one MRP in individual patients was correlated with increased expression of multiple MRPs. The ability to discriminate between patients who will remain in continuous complete remission and patients who will relapse was determined by receiver operating characteristic (ROC) curves. When children and adults were analyzed together, the area under the ROC curves showed that MRP1 (AUC 0.72, p=0.002), MRP2 (AUC 0.71, p=0.002), MRP3 (AUC 0.72, p=0.001), MRP5 (AUC=0.70, p=0.004) and MRP6 (AUC=0.68, p=0.009) predicted relapse. Using median values as cutoff for high versus low expression, high expression of all six MRP genes, except MRP4, was significantly associated with reduced event-free survival in children and adults (Kaplan-Meier curves with Log-rank tests).

Conclusion: Our results suggest that a profile with high mRNA expression of MRP1, MRP2, MRP3, MRP5 and MRP6 is involved in drug resistance in ALL. This unfavorable profile is more often observed in adults, and is associated with reduced event-free survival.
Expression of the Human ATP-Binding Cassette Transporter Superfamily in Acute Myeloid Leukemia.
Authors: Ho Maria , Hogge Donna E. , Ling Victor
Published: April 2020

Members of the ATP-Binding-Cassette (ABC) transporter superfamily of proteins are involved in resistance to multiple chemotherapeutic drugs ( Multidrug Resistance or MDR) in a variety of malignant cells including leukemic blasts. Overexpression of some ABC transporters has been demonstrated in acute myeloid leukemia (AML) and is associated with clinical MDR and failure of conventional chemotherapy, which occurs frequently in this leukemia. Recent studies have also demonstrated ABC transporter expression in primitive normal hematopoietic cells, including progenitors which may give rise to AML after malignant transformation. In this study we used quantitative Real-Time PCR to assess and compare the expression level of all 47 known human ABC transporters in AML blasts and normal peripheral blood. Peripheral blood blasts from 17 patients with newly-diagnosed AML who subsequently received conventional remission induction therapy with cytosine arabinoside and daunorubicin were studied; 11 of these subsequently achieved complete remission of their leukemia while the remaining 6 had chemotherapy refractory disease. Contrary to expectations, no consistent difference in mRNA levels was found between the chemotherapy responsive and refractory groups of patient samples for any ABC transporter, including known MDR-related members such as MDR-1 and BCRP. Profiling of the 47 ABC transporters in 12 normal peripheral blood samples (6 mobilized with G-CSF, 6 non-mobilized) showed that TAP1 and MRP3 were 3.3-fold (P = 0.032) and 24-fold (P = 0.012), respectively, higher in normal donors as compared to AML patients. ABCA7, ABCB8, MRP3, MRP7, ALDP, PMP70 and PMP69 were greater than 3-fold higher (P < 0.05) in G-CSF-mobilized as compared to steady state normal blood. These results suggest that levels of ABC transporter mRNA expression in AML blasts prior to chemotherapy are not predictive of treatment response. This raises questions regarding the role of ABC transporters in intrinsic as opposed to induced or acquired chemotherapy drug resistance, which in turn has important implications in clinical usage of ABC-reversal agents. In addition, we identified expression of a variety of ABC transporters in both AML blasts and normal blood cells suggesting that this class of transporter proteins may have importance in both normal and malignant hematopoiesis.
Glucosylceramide Synthetase Inhibitors Sensitise B-CLL Cells to Cytotoxic Agents without Reversing P-gp Functional Activity.
Authors: Gerrard Gareth , Butters Terry D. , Mehta Atul B. , Hoffbrand A. Victor , Hughes Derryln , Burgess Michael , Ganeshaguru Kanagasabai
Published: April 2020

Malignant B-cells from a high proportion of chronic lymphocytic leukaemia (B-CLL) patients over express the multidrug resistance (MDR) -1 gene encoded transmembrane efflux pump P-glycoprotein (P-gp). Inhibition of glucosylceramide synthesis has been shown to correlate with the expression and function of P-gp and sensitise cells to cytotoxic agents. We analysed the ability of glucosylceramide synthetase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1, 500μM) and N-nonyl-deoxygalactonojirimycin (OGB-2, 100μM) to sensitise B-CLL cells to conventional cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the in vitro cytotoxicity MTT assay. The effect on P-gp activity was also analysed using the calcein-AM accumulation assay and the results expressed as multidrug activity factor (MAF), where a MAF of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. GCS inhibitors were cultured with B-CLL cells for 24-48h before the assays were performed. The P-gp negative cell line CEM-CCRF had no MAF activity with an IC50 for vincristine (a known P-gp substrate) of <1ng/ml. The P-gp over expressing cell line CEM-VLB showed a MAF value of 96.4 with zosuquidar trihydrochloride (Z.3HCL), a specific inhibitor of P-gp, 15.7 with OGB-1 and 45.9 with OGB-2. The IC50 for vincristine was reduced from >10ug/ml to 55.5ng/ml in the presence of OGB-2. In peripheral blood mononuclear cells from 3 normal volunteers (all P-gp +ve), the mean MAF value for Z.3HCL was 23.86 and for OGB-2 was 16.2. In 9/13 B-CLL samples there was P-gp functional activity in the presence of Z.3HCL with a mean MAF value of 22.15 (range 11.27–37.3). P-gp was over expressed in10/13 B-CLL samples. However, when available samples from this cohort were assessed with OGB-1 (n=4) and OGB-2 (n=13) the MAF value was <10. Nevertheless, sensitisation of B-CLL cells was observed by a reduction in the IC50 in the presence of OGB-1 with CdA in 3/4 (to 40% in the presence of cytotoxic drug alone), Chl in 3/4 (39%), FdR in 2/4 (26%) and in the presence of OGB-2 with CdA in 8/13 (42%), Chl in 5/13 (40%) and FdR in 7/13 (34%). Although GCS inhibitors sensitize B-CLL cells to cytotoxic drugs in some B-CLL patients, they do not appear to have any effect on P-gp functional activity.
Alpha-1 Antitrypsin Is a Potent Inhibitor of Cytokine-Induced Hematopoietic Stem Cell Mobilization.
Authors: van Pel Melissa , van Os Ronald , Velders Gerjo A. , Hagoort Henny , Lindley Ivan J. , Willemze Roelof , Fibbe Willem E.
Published: April 2020

Previously, we have shown that IL-8 and G-CSF-induced hematopoietic stem cell (HSC) mobilization is inhibited in mice that underwent low dose (0.5 Gy) total body irradiation (TBI), whereas the number of progenitor cells in the bone marrow remained similar in all groups. The mechanism underlying this inhibition remains unknown. Since the release of granular proteases by neutrophils is well known to play a role in HSC mobilization, we also considered a possible role for serine protease inhibitors in the induction of HSC mobilization. Serine proteases, such as elastase and cathepsin G, are irreversibly inhibited by serine protease inhibitors including alpha-1 antitrypsin (alpha-1 AT) and alpha2-macroglobulin. In-vitro tests revealed that addition of bone marrow extracellular extracts, that were obtained from murine femurs 24 hours following low dose (0.5 Gy) TBI, inhibited the activity of exogenous elastase in a chromogenic substrate conversion assay up to 78.1 % compared to extracts obtained from sham irradiated controls (p<0.05). Since elastase inhibition by alpha2-macroglobulin cannot be detected in a chromogenic substrate conversion assay, alpha-1 AT was considered as the primary candidate serine protease inhibitor to inhibit elastase activity in our in-vitro system. Quantitative PCR of total bone marrow cells revealed that alpha-1 AT mRNA was 20-fold increased relative to the housekeeping gene ß-actin and 7-fold relative to the housekeeping genes HPRT and GAPDH at 24 hours following low dose (0.5 Gy) TBI. In addition, Western blot analysis indicated that alpha-1 AT protein concentrations were significantly (p<0.01) increased in bone marrow extracellular extracts derived from low dose (0.5 Gy) irradiated mice, compared to extracts obtained from sham-irradiated controls (5.1 ± 0.6 scanning units [SU] vs. 3.9 ± 0.7 SU for 0.5 Gy;n=8 vs. 0 Gy; n=6 respectively). To further substantiate a possible in-vivo role of alpha-1 AT in the inhibition of HSC mobilization, we administered alpha-1 AT (300 μg/mouse i.p.) at 2 hours and at 5 minutes prior to IL-8 injection (30 μg/mouse i.p.). Administration of alpha-1 AT prior to IL-8 injection completely (p<0.05) inhibited IL-8-induced HSC mobilization (472.9 ± 289.5 CFU-GM per ml blood for IL-8; n=5 vs. 44.8 ± 35.5 CFU-GM per ml blood for alpha-1 AT/IL-8; n =11). These results indicate that 1) alpha-1 AT is a potent inhibitor of IL-8-induced HSC mobilization and 2) in-vivo induced alpha-1 AT contributes to the inhibition of HSC mobilization after low-dose (0.5 Gy) TBI. We hypothesize that a critical balance between serine proteases and serine protease inhibitors plays an important role in cytokine-induced HSC mobilization.
Intrathymic and Extrathymic Notch-Dependent T Lineage Checkpoints during Normal Development and after Bone Marrow Transplantation.
Authors: Maillard Ivan , Sambandan Arivazhagan , Zediak Valerie P. , Schwarz Benjamin , Xu Lanwei , Aster Jon C. , Pear Warren S. , Bhandoola Avinash
Published: April 2020

The essential role of Notch for T lineage commitment is well established, but it remains unclear where and on which progenitor subset(s) Notch signals act. Addressing these questions is critical to understand the regulation of lymphopoiesis in normal and lymphopenic settings, such as after bone marrow transplantation (BMT).

In normal mice, a small number of progenitors settle in the thymus from the blood and expand to generate a pool of early T lineage progenitors (ETPs). At the population level, ETPs can give rise to T cells, NK cells, B cells, DCs and myeloid cells. We found that ETPs could be subdivided based on expression of the cytokine receptor Flt3, which is expressed on about 10% of ETPs and is inversely correlated with expression of Notch target genes. B lineage potential was restricted to ETPFlt3 positive cells. The Notch targets Hes-1, Hes-5, and Deltex1 were present at low levels in ETPFlt3 positive and high levels in ETPFlt3 negative cells. Induction of Notch signaling resulted in the rapid downregulation of surface Flt3 expression. In contrast, culture of ETPFlt3 negative cells in the absence of Notch ligands resulted in upregulation of Flt3. Although both ETP subsets were efficient T lineage progenitors, ETPFlt3 negative cells had a more rapid differentiation kinetics resembling DN2 thymocytes, consistent with a more advanced state of T lineage commitment relative to the Flt3 positive subset. In mice reconstituted with HSCs transduced with the pan-Notch inhibitor DNMAML1 (Maillard et al., Blood 2004), no ETPFlt3 negative and very few ETPFlt3 positive cells were observed, indicating that the generation of ETPs is Notch-dependent. These observations position the physiological Notch checkpoint either very early after thymic seeding and/or in a prethymic location.To further investigate if Notch-dependent prethymic T lineage commitment occurs in adult mice, we studied lymphoid reconstitution early after BMT. ETPs were absent in the thymus of recipients until ≥ 6 weeks post-BMT, despite the presence of donor-derived pre-T cells and double positive thymocytes as early as 2–3 weeks post-BMT. Instead, cells with pre-T cell characteristics were present in the spleen of BMT recipients in the first month post-BMT. These cells were absent when DNMAML1-transduced bone marrow was used as the source of HSCs, indicating that generation of extrathymic pre-T cells is Notch-dependent. Thus, extrathymic sites may be important for efficient lymphoid reconstitution after BMT. Altogether, our results indicate that both intrathymic and extrathymic Notch-dependent checkpoints regulate T lineage commitment during normal development and in the post-BMT setting.
Erythroid-Specific Intergenic Transcripts Originate from the 200 Kb Region 5′ to the LCR in Erythroleukemia and Human Primary Cells.
Authors: Xiang Ping , Fang Xiangdong , Yin Wenxuan , Stamatoyannopoulos George , Li Qiliang
Published: April 2020

Intergenic transcripts have been discovered in globin and other multigene clusters. The role of this transcription activity in gene regulation is still unclear. We found aberrant globin gene transcripts that initiated from the regions 77 kb and 236 kb 5′ to the e gene in K562 and primary cells. The most abundant splicing form of the transcripts is a 500–600 bp RT-PCR product containing 36 bp RNA from the −240 kb region spliced to the upstream region starting at −270 bp of the e gene promoter. Several spliced transcripts also contain RNA fragments from the −77 kb region, or from the −12 kb region (between HS3 and HS2 of the LCR), or from the −8 kb region (between HS2 and HS1). A total of 17 different transcripts were found in K562 cells. Transient transfection assays showed that the −77 kb and −236 kb regions had promoter activity in K562 cells. DNase I hypersensitive sites were detected in these two regions in K562 cells, but not in Jurkat cells. The −236 promoter activity is located within the region encompassing a retrotransposon and the −77 region is located at 2 kb 5′ to an olfactory receptor gene. To test whether such aberrant transcripts occur in primary cells, we tested various human tissues. In fetal bone marrow (72 days) a spliced transcript was detected by RT-PCR and confirmed by DNA sequencing, which contained a 36 bp RNA from the −236 kb region and was spliced to the e gene promoter starting at −270 bp. Using a RNA RACE technique, we also found several aberrant globin gene transcripts in human placenta, adult bone marrow, and fetal liver (20–40 weeks, BD Biosciences). These transcripts contained 181 bp RNA from the −76 to −77 kb region 5′ and the sequence starting at −214 bp 5′ to the cap site of the e gene. While it is unclear whether these transepts or transcription itself has any biological function, this erythroid-specific transcription occurring in the 200 kb region 5′ to the LCR may play a yet-unknown role in globin gene regulation.
Hydroxamic Acids Derivatives Induce γ Globin Gene Expression in Vivo.
Authors: Cao Hua , Jung Manfred , Stamatoyannopoulos George
Published: April 2020

We have previously shown that four hydroxamic acids: butyric and propionic hydroxamic acids, subericbishydroxamic acid (SBHA) and suberoylanilide hydroxamic acid (SAHA) are potent inhibitors of histone deacetylase and strong inducers of fetal hemoglobin expression in vitro (Exp Hematol.31:197, 2003). In the present study we tested their effect on fetal hemoglobin synthesis in vivo. Transgenic mice carrying the human μLCR Aγ construct continue to express the human γ gene in the adult stage of development ( γ/α mRNA ratio ~ 5%, Blood.77:1326, 1991). These mice were crossed to mice heterozygous for a thalassemia gene due to β globin gene deletion (PNAS.92:11608, 1995). The β thalassemia/μLCR Aγ mice represent an appropriate moderately anemic animal model for testing the effects of Hb F inducers. Compounds were administered subcutaneously with a mini-osmotic pump continuously for 7days in a high and a low concentration. Concentrations were: for butyric hydroxamic acid: 500mg/kg/day/100mg/kg/day; for propionic hydroxamic acid: 500mg/kg/day/100mg/kg/day; for SAHA: 100mg/kg/day/20mg/kg/day; and for SBHA: 200mg/kg/day/40mg/kg/day. Two test groups were studied. In group 1, 70μL mice blood was drawn every other day up to 20 days; in group 2, 70μL mice blood was drawn only on days 0 and 21. Reticulocytes and F reticulocytes were measured using flow cytometry, while γ globin gene expression was quantitated by RNase protection assay. Butyric and propionate hydroxamic acids increased reticulocytes by 70.52% (from 13.96% to 23.81%) and 172.52% (from 10.34% to 28.20%) respectively. There was only small increase in reticulocytes in the mice treated with SAHA (from 13.33% to 15.36%), SBHA (from 14.24% to 16.27%) and the PBS control (11.06% to 14.11%). All the compounds increased the level of γ mRNA: butyric hydroxamate by 53.07%; propionic hydroxamate by 40.05%; SAHA by 49.87%, and SBHA by 34.05%. These results suggest first that all the hydroxamic acid derivatives we used increase fetal hemoglobin in vivo in the thalassemia animal model; second butyric and propionic hydroxanic acids are in addition inducers of in vivo erythropoiesis.