bluuebunny/crossref_metadata_2025 huggingface dataset:
Multigenic Dissection and Prognostic Modeling of Overt Stroke in Sickle Cell Anemia.
Authors: Sebastiani Paola , Ramoni Marco F. , Nolan Vikki , Baldwin Clinton T. , Steinberg Martin H.
Published: October 2019

Stroke is a major vascular complication of sickle cell anemia, more frequent in patients under the age of 20 years. Transcranial Doppler (TCD) flow studies can predict the likelihood of stroke in children with sickle cell anemia, but only 10% of individuals with abnormal TCD values will have stroke, and stroke will occur in some individuals with normal TCD. Therefore, more precise means of prognosis would be welcome so that prophylactic treatments like transfusions or hydroxyurea can be targeted to individuals at highest risk. Using Bayesian networks (BNs), we analyzed 235 single nucleotide polymorphisms (SNPs) in 80 candidate genes in 1398 unrelated subjects with sickle cell anemia enrolled in the Cooperative Study of Sickle Cell Disease. Bayesian networks are a novel generation of multivariate models that represent the complex structure of interactions between many variables by a network of interrelated modules. These modules can be learned from data and then can be used to describe how changes in some variables affect other variables and ultimately the risk for the phenotype of interest. Bayesian networks provide a coherent framework within which genotypes, phenotypes and environmental factors can be seamlessly integrated into a comprehensive genomic landscape. We found that 25 SNPs on 11 genes and 4 clinical variables - including α thalassemia and fetal hemoglobin - interact in a complex network of dependency to modulate the risk of stroke. This network of interactions includes three genes (TGFBR2, TGFBR3, BMP6) with a functional role in the TGF-beta pathway and one gene (SELP) already associated with stroke in the general population. We validated our results in a different population by predicting the occurrence of stroke in a set of 114 subjects not included in the original study: 7 stroke patients and 107 control patients, a proportion consistent with the phenotype distribution in the original cohort study. Our model predicted the correct outcome for all 7 stroke patients, and for 105 of 107 non-stroke patients, with 100% true positive rate and 98.14% true negative rate, and an overall predictive accuracy of 98.2%. Our results support the hypothesis that stroke in sickle cell anemia patients is a complex trait caused by the interaction of multiple genes, and the predictive accuracy of our model is a step toward the development of additional prognostic tests to allow us to more precisely identify sickle cell anemia patients at risk for stroke. The presence in our model of genes already associated with stroke, such as SELP, suggests that some genetic factors predisposing to stroke are shared by both sickle cell anemia patients and stroke victims in the general population, and that our model may offer some insights into the genetic basis of the third leading cause of death in the United States.
Growth after Hematopoietic Cell Transplantation for Sickle Cell Disease.
Authors: Walters Mark C. , Patience Melinda , Edwards Sandie , Robertson Shanda , Kesler Karen , McMurray Marsha , Donfield Sharyne , Eggleston Barry
Published: October 2019

Although allogeneic hematopoietic cell transplantation (HCT) has curative potential for sickle cell disease (SCD), the impact on growth in children treated by HCT is important to consider when counseling families about this therapeutic intervention. To investigate this, we compared linear growth after BMT to 3 cohorts of children with SCD. The comparison groups included 68 children with SCD who were measured before enrollment (HUG-KIDS Pre) and during treatment (HUG-KIDS HU) in a phase I-II trial of hydroxyurea for children with symptomatic SCD. The third comparison group included 1887 and 1889 children with height and weight data from the Cooperative Study of Sickle Cell Disease (CSSCD). Fifty-three of 55 surviving patients (20 F, 33M) had growth monitored after HCT. There was no significant difference in the distribution of repeated measurements between males and females, and 2 or more measurements of height and weight were performed in 89% and 92% of patients, respectively. The mean period of follow-up in the HCT and comparison groups was 3.3 ± 2.4 years after HCT, 5.5 ±3.3 years in the CSSCD, 4.0 ± 2.7 years in HUG-KIDS (pre) and 1.8±0.5 years in HUG-KIDS (HU). To accommodate the non-identical distribution of the ages among children treated by HCT, the comparison age range in the CSSCD and HUG-KIDS (HU) cohorts was 5 to 15 years for males, and 6 to 15 years in females, and was 3 to 13 years for males, and 4 to 13 years for females in the HUG-KIDS (pre) cohort. A Hierarchical Linear Model was used to model height and weight over the period of follow-up. Its design allowed for group comparisons of height and weight at baseline and linear growth velocity during the follow-up period. The height and weight data were modeled separately for males and females. At the baseline, there were no significant height or weight differences between the HCT and comparison groups among females, however, males in the HUG-KIDS (pre) group were approximately 4 cm taller than males in the HCT group. In all comparisons except that of males in the HCT and CSSCD groups, there were no strong differences in the linear growth velocity. However, males treated by HCT grew on average 0.67 cm/year more than males in the CSSCD group, and gained on average 1.0 kg/year more weight than males in the CSSCD, and these differences reached statistical significance (p=0.03, p=0.002, respectively). These data support the conclusion that growth after HCT for SCD is not impaired compared to children who receive standard therapy or HU. Moreover, in males, growth was more rapid after HCT than among males enrolled in the CSSCD. This analysis supports the notion that HCT for SCD offers a significant therapeutic benefit without a negative impact on growth.
Progression of Avascular Necrosis of the Hip in Sickle Cell Disease: 2 Year Follow-Up of Randomized Trial of Aggressive Physical Therapy and Hip Coring Decompression.
Authors: Vichinsky Elliott P. , Neumayr Lynne , Earles Ann , Aguilar Christine , Robertson Shanda , Orringer Eugene , Al-Lamki Zakia , Casella James , Wang Winfred , Ramirez Gloria , Daeschner Charles , Olivieri Nancy , McClain Kenneth , Grossi Mauro , Pegelow Charles , Toledano Stuart , Files Bea , Krisnamurti Lakshmanan , Kutlar Abdullah , Abboud Miguel
Published: October 2019

Avascular necrosis (AVN) of the hips affects half of sickle cell anemia (SCA) patients and can rapidly lead to total collapse of the hip. Treatment with supportive therapy alone is unsuccessful and results in half of all patients requiring hip surgery within 2 years of diagnosis. Prognosis in advanced cases is dismal, with surgery eventually required in all patients. Alternative therapies including aggressive physical therapy (PT) and hip coring decompression (HCD) may improve the natural history of AVN but neither have been evaluated prospectively in SCD. The goal of the prospective randomized National AVN Trial in SCA is to evaluate the safety of HCD and PT for AVN. 46 patients were randomized to have either HCD of their study hip followed by 6 weeks of an aggressive PT protocol, or to undergo only the PT protocol. Both treatment arms are balanced for Ficat stage and patient age. Outcome measures include clinical improvement as measured by the Children’s Hospital Oakland Hip Evaluation Scale (CHOHES), a 100 point validated scale that assesses hip function and pain associated with AVN in SCA. Additional measures include X-ray progression and the need for surgical intervention. This report presents hip survival rates in 39 study patients: 17 patients underwent HCD and 22 were treated in the PT arm. Average patient age was 26 (range 10 to 50). 67% had bilateral disease. The average follow-up is currently 40 months. In the survival graph, treatment failure was defined as additional hip surgery. 4 of the 39 study hips (10%) were treatment failures: 1/9 (11%) of Stage I, 3/19 (16%) of Stage II, and 0/11 (0%) of Stage III hips.

Figure Figure

In addition to a low treatment failure rate, hips in each Ficat stage show evidence of clinical improvement. CHOHES scores improved 17 points at 1 year and 22 points at 2 years; even Stage III shows a 20 point improvement at 1 year and 21 points at 2 years. Our data indicates that both HCD and aggressive PT are beneficial in the treatment of AVN in SCA. PT may be effective in the treatment of even fairly advanced Stage III AVN. The AVN Trial in SCA is ongoing; patients will be followed for 3 to 5 years, and a comparison of HCD to aggressive PT will be made upon completion.
Growth Suppressors IFI-204 and IFI-205 Inhibit Primitive Hematopoietic Cell Proliferation In Vitro and in Vivo.
Authors: Klarmann Kimberly , Gough Daniel , Asefa Benyam , Clarke Chris , Renn Katie , Johnstone Ricky , Keller Jonathan
Published: October 2019

Members of the interferon inducible-200 (IFI-200) family of proteins inhibit cell growth and may be important mediators of differentiation. We examined IFI-204 and IFI-205 mRNA expression in purified populations of hematopoietic stem and progenitor cells at different stages of maturation using quantitative RT-PCR and found that their expression markedly increased during myeloid maturation. To evaluate the effect of IFI-205 and IFI-204 on hematopoietic stem cell (HSC) growth, we transduced these genes into mouse bone marrow cells (BMC) using retroviral vectors. The presence IFI-204 or IFI-205 resulted in a decrease in cell growth in response to hematopoietic growth factors. Further analysis revealed the infected cells were 98% c-Kit+ Sca-1+, indicative of the stem cell surface phenotype, suggesting they may be blocked in a primitive stage of maturation. When transplanted, BMC transduced with IFI-204 or IFI-205 failed to engraft lymphoid, myeloid, or erythroid lineages in both short and long term reconstitution assays, suggesting that constitutive expression of IFI-204 and IFI-205 inhibited HSC development both in vitro and in vivo. However, based on the quantitative RT-PCR results, which show that IFI-205 increased during myeloid differentiation, we know its endogenous, regulated expression must permit the cells to mature. Therefore, to study of the effects of these genes on differentiation we transduced the mulitpotential EML (erythroid, myeloid, lymphoid) cell line with IFI-204 and IFI-205 to circumvent severe growth inhibition caused by expression of IFI-204 and Ifi-205 in normal cells. Single cell analysis of EMLs transduced with IFI-205 demonstrated that expression of IFI-205 in this cell line did not significantly inhibit cell growth. We have isolated EML clones from the transduced cells and verified IFI-205 expression. In addition, we generated transgenic mice that express IFI-205 under control of the Vav and MRP8 promoters, and we identified transgenic lines that express IFI-205 at higher levels compared to wild type controls. Analysis of hematopoiesis in these animals is currently in progress. Altogether, our data demonstrate 3 findings: 1) IFI-204 and IFI-205 expression increases during myeloid development based on quantitative RT-PCR analysis, 2) constitutive expression of IFI-204 and -205 results in potent inhibition of growth and maturation of normal hematopoietic stem and progenitor cells in vivo and in vitro and 3) these genes did not significantly inhibit the proliferation of the EML cell line, which provides us with a means to study the mechanism by which these molecules regulate myeloid maturation. Finally, the considerable inhibitory effects of these family members on normal hematopoietic cell growth suggest their potential as therapeutic modalities for treatment of leukemia.
The HGFIN Gene Mediates Cell Cycle Quiescence of CD34+/CD38-: Implications for Hematopoietic Stem Cell Expansion and Gene Therapy.
Authors: Ahmeti Era , Sadoshima Junichi , Harrison Jonathan S. , Rameshwar Pranela
Published: October 2019

Hematopoietic stem cells (HSC) are difficult to expand in vitro since expansion media mostly include exogenous factor, e.g., cytokines. Growth factors cause lineage commitment with the HSC exhibiting short-term immune reconstitution. The recently cloned HGFIN (or nmb) gene has been detected in CD34+/CD38− and differentiated immune cells. HGFIN is a single transmembrane protein with multiple consensus regions for p53. In malignant cells, HGFIN mediate tumor progression and in normal cells, HGFIN maintain cell cycle quiescence. We hypothesize that HGFIN negatively regulates proliferation of HSC by prolonging the G0 phase. Short-term knockdown of HGFIN in HSC could cause the cells to exit G0/G1 phase so as to accommodate the insertion of a replacement gene, without changing the pluripotent property of HSC. RT-PCR detected HGFIN mRNA in CD34+/CD38− cells, but not in CD34+/CD38+ cells from bone marrow (BM) aspirates. Treatment of CD34+/CD38− cells with double stranded HGFIN siRNA oligos for 2 h led to their exit from G1 to G2/S (flow cytometry). During this time, adenovirus-LacZ (ß-galactosidase assay) was incorporated in >80% CD34+/CD38− cells, compared to 10–20% exposed to mutant siRNA, or untreated. After 24 h exposure to siRNA oligos, the CD34+/CD38− cells reverted to G0/1 phase. The latter cells (with adenovirus-LacZ) were studied in 12-wk long-term culture initiating assay. The results (ß-gal positive progenies) from the LTC-IC assay indicate that despite siRNA treatment, the CD34+/CD38− cells retained multipotential properties. Future investigations with specific HGFIN antibody will map pathways among HGFIN and other molecules that regulate cell cycle progression in CD34+/CD38− cells. In summary, the results show that HGFIN regulate cell cycle quiescence of CD34+/CD38− cells. Transient knock down of HGFIN in these cells allow adenovirus to be inserted without loss of multipotential properties. Understanding the biology of HSC at the molecular level will be relevant to HSC expansion and restoration of immune competence via gene therapy.
Notch Signaling in Hematopoietic Precursor Cells Maintains the Expression of Genes Required for Stem Cell Self-Renewal and Promotes the Expression of Genes Associated with T cell Differentiation.
Authors: Kato Keizo , Varnum-Finney Barbara , Bernstein Irwin D.
Published: October 2019

Notch Signaling in Hematopoietic Precursor Cells Maintains the Expression of Genes Required for Stem Cell Self-renewal and Promotes the Expression of Genes Associated with T cell Differentiation.

Keizo Kato, Barbara Varnum-Finney, Irwin D. Bernstein

Clinical Research Division, Fred Hutchinson Cancer Research Center,

Seattle, WA, USA

We have previously shown that Notch signaling promotes the self-renewal of hematopoietic precursors, including short-term repopulating cells, and induces early T-cell differentiation. Here we evaluate gene expression in murine Lin-Sca-1+c-kit+ Hoechst side population (SKSP) bone marrow cells during culture with the immobilized Notch ligand, Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human-IgG1 for 28 days with SCF, IL-6, IL-11 and Flt-3-ligand. We analyzed hematopoietic stem cell (HSC) associated genes, including polycomb genes, Bmi-1 and Rae28, required for HSC self-renewal, and Rex-1 required for embryonic stem cell self-renewal, together with early T-lymphoid differentiation associated genes such as GATA-3, pre-Ta and CD3e, by semi-quantitative or real-time RT-PCR. After culture for 7 or 14 days with Delta1ext-IgG, the expression of Bmi-1, Rae28 and Rex-1 was greater in cultures containing Delta1ext-IgG compared to those without. Bmi-1, Rae28 and Rex-1 were likely not direct targets of Notch signaling since the expression in SKSP cells was equivalent after 3 hours culture in the presence or absence of Notch ligand, whereas rapid up-regulation of the direct Notch target Hes1 was observed 3 hours after incubation with Delta1ext-IgG. Expression of GATA-3, pre-Ta and CD3e was induced by Notch signaling since their expression was seen by 7-14 days with but not without Delta1ext-IgG. Furthermore, the expression of these genes was dependent on Notch signaling since removal of cells from Notch ligand after culture for 28 days led to a rapid reduction of Hes1 expression within 3 hours, and a slower reduction in genes associated with self-renewal observed after 2 days. Our results suggest that Notch signaling regulates the self-renewal of hematopoietic precursors by maintaining the expression of genes known to be required for stem cell self-renewal, while also promoting the expression of T-cell differentiation-associated genes.
Proaerolysin Multi Color Flow-Fish Reveals Shortened Telomere Length in GPI− as Compared to GPI+ Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).
Authors: Beier Fabian , Balabanov Stefan , Buckley Tom , Rojewski M. , Becker T. , Kanz Lothar , Schrezenmeier Hubert , Brummendorf Tim H.
Published: October 2019

Objective: Replication-dependent telomere shortening can result in cellular senescence or genetic instability. Telomere length both reflects and limits the replicative potential of normal somatic cells. Shortened telomeres have been linked to disease stage, degree of cytopenia as well as to response to treatment in patients with bone marrow failure syndromes. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Pancytopenia in PNH is thought to result from an auto-immune mediated depletion of the GPI+ stem cell compartment. In the current study, we aimed to develop a methodology that allows the selective analysis of telomere length in granulocytes from patients with PNH dependent on GPI expression. The aim was to find out whether replicative stress posed on the residual GPI− HSC is reflected in shortened telomere length compared to their GPI+ counterparts.

Methods: The telomere length of GPI+ and GPI− granulocytes of 12 patients with PNH and 22 healthy individuals was analysed. For this purpose, we developed Proearolysine Multi-color flow FISH which is based on the crosslinking of a fluorescence-labeled non-toxic derivative of the bacterial toxine Aerolysine (that selectively binds to the GPI anchor of cells) in combination with fluorescence in situ hybridization and flow cytometry.

Results: We found significantly (p<0.05) shortened telomeres in GPI− granulocytes (6.68±0.3 telomere fluorescence unit (TFU), n=12) compared to age-matched healthy individuals (mean±S.E.: 7.73±0.8 TFU, n=22), but no significant shortening in GPI+ (7.06±0.3 TFU, n=12) granulocytes from PNH patients. Telomere length in GPI− granulocytes was found to be significantly shorter as compared to their GPI+ counterparts (deltaTEL: 0.38±0.1, p=0.002). These results were confirmed by Multi-color flow FISH using an anti-CD59 antibodies.

Conclusion: Autoimmune-mediated damage to the GPI+ HSC compartment leads to compensatory hyperproliferation of predominantly residual GPI− HSC. In line with this hypothesis, coexisting GPI-negative HSC clones showed even more accelerated telomere shortening as compared to their GPI+ counterparts. Potentially, replicative exhaustion of individual HSC clones could contribute to disease progression in PNH. Whether acquired genetic instability due to progressive telomere shortening also accounts for an increased incidence of secondary clonal disorders will need to be evaluated in future prospective studies.
His2315/Gln2316 of the Factor VIII C2 Domain Interact with Phospholipid Membranes and Influence Activity of the Factor Xase Complex.
Authors: Gilbert Gary E. , Kaufman Randall J. , Price Patricia , Mao Hongzhi , Pipe Steven W.
Published: October 2019

A major phospholipid-binding motif of factor VIII is localized in the C2 domain. Two pairs of hydrophobic amino acids in the factor VIII C2 domain (Met2199/Phe2200, Leu2251/Leu2252) have previously been shown to interact with phospholipid membranes. However mutations of hydrophilic amino acids (Gln2213, Arg2215, Asn2217, Arg2220, Lys2249) that were predicted to interact with the membrane surface, based upon the crystallographic structure, have not altered phospholipid binding activity. The established hydrophobic interactions do not account for the demonstrated hydrophilic interactions of factor VIII with the phospholipid bilayer. We hypothesized that His2315 and/or Gln2316, interact with phospholipid membranes, based upon their contribution to the epitope for phospholipid-blocking mAb B02C11 and based upon phosphatidylserine binding by C2 domain peptides that include these residues. Factor VIII His2315Ala/Gln2316Ala (fVIII 2315/16) was prepared by PCR mutagenesis and expressed from COS-1 cells. Secreted factor VIII was purified by immunoaffinity chromatography and evaluated in ELISA assays, aPTT assays with factor VIII deficient plasma, and in defined assays with varying phospholipids. The specific activity of fVIII 2315/16 was 67 ± 9% of wild type factor in a commercial aPTT assay with a large excess of phospholipid. The apparent affinity for extruded phospholipid vesicles (0.1 μm diameter) of composition phosphatidylserine:phosphatidylethanolamine: phosphatidylcholine 4:20:76 was 32 ± 10% of wild type factor VIII in a factor Xase assay with varying phospholipid. When the phospholipid concentration was saturating, the Vmax was 55% of factor Xase with wild type factor VIII. We conclude that His2315 and/or Gln2316 constitute the first identified hydrophilic amino acids of factor VIII that interact with a phospholipid membrane. His2315/Gln2316, combined with the two pairs of phospholipid-interactive hydrophobic amino acids constitute three spatially separated phospholipid-interactive points that define a phospholipid-interactive plane on the factor VIII C2 domain. This is consistent with the hypothesis that the phospholipid-binding plane of the C2 domain overlaps substantially with the epitope for BO2C11 and is nearly coincident with the phospholipid-binding surface predicted from the crystallographic structure.
Mutation of the Hydrophobic Residues in Factor Va2 C1 Domain Affects the Phosphatidylserine Mediated Prothrombin Activation.
Authors: Majumder Rinku , Quinn-Allen Mary Ann , Kane William H. , Lentz Barry R.
Published: October 2019

Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. The binding of factor Va to PS membranes regulates assembly of the prothrombinase complex. The C-terminal domain (C2) of factor Va (residues 2037–2196 in human factor Va) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp2063/Trp2064 (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent results have shown that mutating these Trp residues abolishes FVa2-membrane binding, but does not affect the assembly and activity of the prothrombinase in the presence of 25% PS membranes or soluble C6PS. Our data also indicates that there is another site on factor Va2 that might be specific for PS or C6PS and might serve as a regulatory site for assembly or activity of the FVa2-FXa complex. A pair of solvent exposed amino acids, Tyr1956/Leu1957 in the C1 domain is located analogously to the critical Trp residues in C2. Recently, we showed that prothrombin activation in the presence of the factor Va mutant (Y1956, L1957) A was markedly impaired on phospholipid vesicles containing 10% or less PS but was essentially normal on vesicles containing 25% PS (Saleh, M., Peng, W., Quinn-Allen, MA., Macedo-Ribeiro, S., Fuentes-Prior, P., Bode,W. and Kane, WH. Thromb. Haemost. 2004,91:16–27). Our work aims to test the hypothesis that the PS regulatory site in Va2 is located analogously to the C6PS binding site in the C2 domain. We have used factor Va mutants with mutations in either the C1 domain, (Y1956, L1957) A or in both the C1 and C2 domains, (Y1956, L1957, W2063, W2064) A. We determine the rate of thrombin formation in the presence of 400 mM C6PS and wild type, C1 and C1C2 mutated factor Va2 to be 170, 12 and 11 nM/S−1/M−1, respectively. Mutations in the C1 and C1C2 domains of factor Va2 reduced the rate of activation of prothrombin to thrombin by 14–15 fold. We have also determined the effect of these mutations on the assembly of factors Xa–Va2 complex by monitoring the change in fluorescence of dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK)-Xa with the addition of wild type, C1 and C1C2 mutated factor Va2 in the presence of 400 mM C6PS. Our data shows that the Kd’s of factor Xa with factor Va2 (wild type, C1 mutant and C1C2 mutant) are 3, 564, 624 nM respectively. Our results support the hypothesis that a PS regulatory site is located in the C1 domain of factor Va and includes residues Tyr1956 and Leu1957.
NMR Structural Analysis of Factor XI Apple 4 Domain.
Authors: Samuel Dharmaraj , Cheng Hong , Riley Paul W. , Walsh Peter N. , Roder Heinrich
Published: October 2019

Blood coagulation is achieved by two closely coordinated mechanisms: i) the contact factor, or intrinsic, pathway initiated by assembly of coagulation proteins on negatively charged surfaces, and ii) the extrinsic pathway initiated by exposure of tissue factor at the site of vascular injury. Although factor XI (FXI), a 160 KDa homodimeric plasma coagulation protein, can be activated either by FXIIa (generated by the contact factor pathway) or by thrombin (generated by the extrinsic or tissue factor pathway), recent evidence suggests that its unique dimeric structure is required for FXI-activation by thrombin on the platelet surface, leading to the initiation of the intrinsic pathway that is required for normal hemostasis. Each FXI monomer consists of an N-terminal heavy chain and a C-terminal trypsin-like catalytic light chain. The heavy chain consists of four homologous subunits called apple domains (designated A1 to A4). The A2 and A3 domains of one monomeric subunit bind to FIX, whereas the A3 domain of the other monomeric subunit binds to platelets. The A4 domain, which shares high (25–38%) sequence identity with other Apple domains, facilitates FXI dimer formation through an intermolecular disulfide bond at Cys-321. In the present study the rA4 domain was cloned and purified to determine its three-dimensional structure. Multidimensional heteronuclear NMR experiments were carried out using C13, N15, H2 labeled samples. Chemical shifts of the C13, N15 and H1 resonance of all the residues were assigned. Assignment of NOE cross peaks between inter- and intra-subunit amino acids is in progress. Preliminary results indicate that the monomeric structure of the A4 domain consists of six anti-parallel β-strands and an α-helix, stabilized by three cystine cross links. The orientations of charged residues and hydrophobic patches on different sides of the molecule may play important roles in the dimerization process of FXI.
Ximelagatran Versus Warfarin: A Comparison of Bleeding Outcomes in Patients with Nonvalvular Atrial Fibrillation.
Authors: Douketis James D. , Arneklev Karin , Goldhaber Samuel , Spandorfer John , Halperin Frank , Horrow Jay
Published: October 2019

Background: The conventional anticoagulant management of patients with nonvalvular atrial fibrillation (AF) involves warfarin, administered to achieve a target international normalized ratio (INR) of 2.0–3.0. Ximelagatran is a novel oral direct thrombin inhibitor that, unlike warfarin, has a predictable anticoagulant effect and does not require dose adjustments based on the INR. In the SPORTIF III and V trials, ximelagatran was as effective as warfarin in preventing stroke and other thromboembolic complications in patients with nonvalvular AF. However, these studies were not designed to determine if ximelagatran was associated with a lower risk of major bleeding. Furthermore, these studies did not compare the case-fatality rate, time course, and anatomic sites of bleeding in ximelagatran- and warfarin-treated patients.

Methods: We undertook a pooled analysis of the SPORTIF III and V trials that involved 7329 patients with AF who received oral ximelagatran, 36 mg twice daily, or warfarin, administered to achieve a target INR of 2.0–3.0. Patients had nonvalvular AF and 1 or more risk factors for stroke: hypertension; age ≥75 yrs; previous stroke, transient ischemic attack or systemic embolism; left ventricular dysfunction; age ≥65 yrs and coronary artery disease; or age ≥65 yrs and diabetes. Major exclusion criteria were: mitral stenosis; previous heart valve surgery; transient AF; and increased risk for bleeding. Bleeding event rates were compared using Fisher’s exact test and the log-rank test and were expressed as the number of bleeds per year. The case-fatality rate of major bleeding was defined as the number of fatal bleeds divided by fatal plus nonfatal bleeds.

Results: The annual incidence of major bleeding was 2.0% in ximelagatran-treated patients and 2.7% in warfarin-treated patients (P = 0.029). The annual incidence of any (major or minor) bleeding was 31.7% in ximelagatran-treated patients and 38.8% in warfarin-treated patients (P < 0.0001). If episodes of intracranial hemorrhage were excluded, the annual incidence of major bleeding was 1.9% in ximelagatran-treated patients and 2.4% in warfarin-treated patients (P = 0.054). The case-fatality rate of major bleeding was 8.2% in ximelagatran-treated and 8.1% in warfarin-treated patients (P = 0.98). The time course and anatomic sites of major bleeding were not significantly different in ximelagatran-treated and warfarin-treated patients. Most bleeds involved the gastrointestinal tract, urinary tract or soft tissues.

Conclusion: In patients with nonvalvular AF who require long-term anticoagulation, treatment with oral ximelagatran, 36 mg twice daily, is associated with a lower risk of bleeding complications than warfarin.
Venous Thromboembolism Treatment with Low-Molecular-Weight Heparin during Pregnancy: A Systematic Review.
Authors: Greer Ian A. , Nelson-Piercy Catherine
Published: October 2019

Introduction Low-molecular-weight heparin (LMWH) has become the standard therapy for managing acute venous thromboembolism (VTE) during pregnancy. In the UK, Europe and Australasia, LMWH is widely used for the treatment of both deep-vein thrombosis and pulmonary embolism occurring during pregnancy, and this practice is endorsed by a peer-reviewed Royal College of Obstetricians and Gynaecologists (RCOG) guideline. However, there are currently no LMWHs licensed for use during pregnancy, and efficacy and safety data are derived mostly from small case series. Our aim was to evaluate the efficacy and safety of LMWH for VTE treatment during pregnancy by performing a systematic review of data from published literature.

Methods Data from published studies included in this systematic review were identified by searching MEDLINE and EMBASE up to the end of 2003. The reference lists from identified articles were also hand searched. Data on the LMWH treatment regime, VTE recurrence, and side effects of VTE treatment were extracted and entered into pre-piloted forms.

Results Sixteen studies (including six case reports) reporting 177 patients were included. One hundred and eight (61%) women were treated with enoxaparin, 49 (28%) with dalteparin and 20 (11%) with nadroparin. In 31 (18%) cases, VTE was initially treated with unfractionated heparin for two days to two weeks after diagnosis. LMWH was administered twice daily in 154 (87%) cases. Recurrent VTE was reported in three (1.7%) women: one receiving dalteparin 10,000 IU od, one receiving enoxaparin 40 mg bd, and one receiving enoxaparin 1mg/kg bd. There were no maternal deaths. Severe maternal bleeding occurred in three (1.7%) women. In two cases, the LMWH could have contributed to the extent of bleeding originating primarily from obstetric causes, and in the other from epistaxis. Minor allergic reactions occurred in two (1.1%) women, thrombocytopenia (unrelated to LMWH) in one (0.6%) woman, and osteoporosis in none.

Conclusion Our findings are consistent with LMWH being both effective and safe in the treatment of acute VTE during pregnancy.
CHIR258: A Multitargeted Growth Factor Receptor Tyrosine Kinase (RTK) Inhibitor Induces Significant Apoptosis of Human FLT3 ITD Acute Myelogenous Leukemia Cells In Vitro and in Vivo.
Authors: Menezes Daniel L. , Wiesmann Marion , Lee Sang Hoon , Lee Isabelle , Peng Jing , Tang Yan , Ye Helen , Oei Yoko , Goldbeck Cheryl , Garrett Evelyn N. , Heise Carla
Published: October 2019

Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. CHIR258, a novel, orally active, small molecule exhibits potent activity against FLT3 kinase and other RTKs involved in myeloid leukemias as well as endothelial and solid tumor cell proliferation (biochemical IC50 for FLT3 = 1 nM; cKIT = 2 nM; VEGFR1/2/3 ~ 8 nM, FGFR1/3 ~ 10nM, PDGFRβ = 27 nM, CSF-R1 = 36 nM). CHIR258 was tested in two human leukemic cell lines with differing FLT3 mutational status (MV4;11, FLT3 ITD vs. RS4;11, FLT3 WT). Cell cycle analysis of leukemic cells incubated with CHIR258 (0.1 μM, 48 h) showed a significant increase in the sub-Go/G1 cell population in MV4;11cells compared to the RS4;11 cells. The pro-apoptotic activity of CHIR258 against MV4;11 cells in culture was confirmed by AnnexinV staining, and appearance of cleaved poly ADP-ribose polymerase (PARP) after 24 h. Interestingly, cleaved PARP fragments were detected with either pulsed (1 h) or continuous incubations of MV4;11 cells with CHIR258. In addition, cell cycle arrest and apoptosis was consistent with degradation of survivin and loss of phosphorylation of Retinoblastoma (Rb) protein observed at CHIR258 concentrations ≥ 0.1 μM. The mechanism of action of CHIR258 was examined in a subcutaneous (s.c.) xenograft model of leukemia in SCID-NOD mice. In s.c. xenografts, daily oral doses of CHIR258 resulted in dose-dependent increased efficacy against FLT3-ITD MV4;11 tumors compared to RS4;11 WT tumors. Modulation of FLT3 downstream signaling molecules (p-FLT3, p-ERK) was achieved in MV4;11 tumors at biologically active doses in vivo, confirming molecular mechanism of action. Target modulation (p-FLT3) was maintained for up to 24 h post-dose. MV4;11 tumor responses to CHIR258 were evidenced within 1–2 days of initiating drug treatment. Immunohistochemical analyses of MV4;11 tumors revealed that early responses to CHIR258 correlated well with decreased tumor cellularity, and proliferation. Tumors stained positive for active caspase-3 and cleaved PARP suggesting cell death was mainly mediated via apoptosis. Efficacy was also demonstrated in an intravenous MV4;11 bone marrow (BM) engraftment model, wherein daily CHIR258 treatments substantially increased the life span of mice, with 33% long term survivors or “cures”. Strikingly, CHIR258-treated mice were devoid of MV4;11 tumor cells in BM as confirmed by flow cytometry and immunohistochemical analyses. In conclusion, our data indicates that CHIR258 might become a highly effective therapy in AML associated with FLT3 expression and warrants clinical trials with AML patients.
Variant Forms of Human Interleukin-3 (IL-3) Linked to Truncated Diphtheria Toxin (DT388), Have Enhanced Cytotoxicity Against Acute Myeloid Leukemia (AML) Progenitors.
Authors: Wong Siaw Hui , Frankel Arthur E. , Hogge Donna E.
Published: October 2019

To develop a cytotoxic drug which targets the IL-3 receptor (IL-3R) on human AML cells we previously developed a fusion protein containing a truncated form of diphtheria toxin which lacks the native binding site (DT388) fused to human IL-3. This molecule kills leukemic progenitors from many AML patients while showing little toxicity to normal hematopoietic progenitors. However, some AML samples showed little or no cell kill after exposure to this molecule. To attempt to improve the cytotoxicity of DT388IL-3 two variants of the toxin were constructed which contain alterations in the IL-3 residues that are designed to enhance binding affinity to the IL-3R. The two variants, DT388IL3[K116W] and DT388IL3[D125-133], have substitution of a hydrophobic tryptophan group at the 116 position and an eight amino acid deletion from the C-terminus of the IL-3 molecule, respectively, while the catalytic and translocation domains of DT388 remain unchanged. These variant DT388IL3 molecules and the unmodified ‘native’ fusion toxin were compared for their ability to kill AML colony forming cells (AML-CFC) from the peripheral blood of 13 newly-diagnosed AML patients and myeloid CFC from 3 normal bone marrows (NBM). AML and NBM cells were cultured for 24h with or without fusion toxin at concentrations varying between 1 and 250 ng/ml prior to plating in CFC assays. Little or no AML-CFC kill was observed for 3/13 samples. The mean % AML-CFC kill for the remaining 10 AML samples ranged from 21 – 61% for the lowest and highest concentration of native DT388IL3 tested and was significantly higher (P<0.05, paired T test) for both variants at all concentrations tested (mean 36 – 72% kill and 44 – 91% kill for the D125-133 and K116W variants, respectively, at 1-250 ng/ml). There was no significant increased kill of NBM CFC with either variant (mean % kill ranging from 4 – 44%, 17 – 58% and 19 – 54% for the native toxin, D125-133 and K116W variants, respectively, at 1-250 ng/ml). The K116W variant achieved the greatest AML-CFC kill for 7 of the 10 responding AML samples, with >90% kill achieved for 6 of these at concentrations as low as 1 ng/ml. The concentration of DT388IL3[Kll6W] required to achieve ≥50% kill of AML-CFC was on average ≥5-fold lower than the concentration of native toxin required to achieve the same effect. Thus, the variant DT388IL3 molecules tested show enhanced cytotoxic activity against AML progenitors with little change in the toxicity profile against normal hematopoietic precursors. In particular, the DT388IL3[K116W] variant warrants further testing against more primitive normal and leukemic progenitors as a potentially promising new therapeutic agent for AML. Such studies are underway.
Phase I Study of Clofarabine Plus Idarubicin and Clofarabine Plus Idarubicin Plus Cytarabine (ARA-C) in Patients (PTS) with Relapsed and Primary Refractory Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), and Myeloid Blast Phase of Chronic Myeloid Leukemia (CML).
Authors: Faderl Stefan , Ferrajolil Alessandra , Wierda William , Verstovsek Srdan , Ravandi-Kashani Farhad , Garcia-Manero Guillermo , Estey Elihu , Thomas Deborah A. , Kornblau Steve , Kwari Monica , Gandhi Varsha , Kantarjian Hagop M.
Published: October 2019

Phase I and II clinical studies demonstrated activity of Clofarabine in acute leukemias. In previous studies we have investigated clofarabine, plus ara-C combinations and reported a CR rate of 24% in relapsed AML and 52% in previously untreated AML ≥ 50 years (yrs) with acceptable toxicity profile. Anthracyclines are active in AML. To explore clofarabine further in AML combinations we conducted a phase I study of clofarabine with idarubicin with or without ara-C in pts with relapsed AML, MDS, and CML. Considered as dose-limiting toxicities (DLT) are ≥ grade 3 drug-related toxicities. Maximum tolerated dose (MTD) will be determined by “3+3” dose escalation scheme. On the clofarabine (C)/idarubicin (I) combination (CI), 9 AML pts are enrolled (2 primary refractory, 7 first relapse). Median age: 58 yrs (range 24–71). Median first remission duration (CRD1): 3.1 mos. (0–7.6). For the first dose level, C was given at 22.5mg/m2 i.v. daily x 5d and I at 12mg/m2 i.v. daily x 3d. Among the first 6 pts, 2 ≥ gr. 3 toxicities (diarrhea, rash, ↑ bili) occurred necessitating dose de-escalation of C to 15mg/m2 i.v. daily x 5 and I 8mg/m2 i.v. daily x 3. Among 3 pts, 1 ≥ gr.3 toxicity (↑ bili) was observed. No responses occurred. On the CI + ara-C arm (CIA), 7 AML pts are enrolled (1 primary refractory, 6 first relapse). Median age: 58 yrs. (24–78). Median CRD1: 11.2 mos. (0–13.1). First dose level: C 22.5mg/m2 i.v. daily x 5d, I 8mg/m2 i.v. daily x 3d, A 1g/m2 i.v. daily x 5d. Of 3 pts, 2 developed ≥ gr.3 toxicities (↑ bili, diarrhea) leading to the following de-escalation: C 15mg/m2 i.v. daily x 5d, I 6mg/m2 i.v. daily x 3d, A 0.75g/m2 i.v. daily x 5d. Of 4 pts (1 ≥ gr. 3 rash, ↑ bili), 3 pts achieved CR. The phase I study is ongoing until determination of DLT and MTD for each arm. Our preliminary results indicate clinical activity of CIA even at the low dose level.
Interleukin-12 Gene Expression into Acute Myeloid Leukemia-Derived Dendritic Cells Overcomes T-Cell Functional Impairment Induced by Leukemic Microenvironment.
Authors: Curti Antonio , Pandolfi Simona , Aluigi Michela , Isidori Alessandro , Alessandrini Isabella , Chiodoni Claudia , Testoni Nicoletta , Colombo Mario P. , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

Acute myeloid leukemia (AML) cells are poorly immunogenic and release soluble factors inhibiting T-cell function. AML-derived dendritic cells (AML-DCs) have better antigen presentation capacity than leukemic blasts but share with AML cells some immunosuppressive features. In this study, we show that AML-DCs generated from CD14− AML samples (which represent 80% of total AML patients) are defective in IL-12 production. We, then, transfected CD14−-derived AML-DCs with IL-12 gene through the novel non-viral method nucleofection. IL-12 gene-nucleofected AML-DCs produce significant amount of IL-12 while maintain leukemia-specific karyotype, DC-like phenotype and function. In presence of the supernatant from the human leukemic cell line K562, allogeneic T-cell proliferation and interferon (IFN)-γ production induced by mock-transduced AML-DCs are significantly reduced. This effect is mainly directed on T cells, since AML-DC phenotype and cytokine production are not affected by leukemic supernatant. However, when stimulated by IL-12-producing AML-DCs, T cells produce higher concentrations of IFN-γ, thus maintaining a Th1 cytokine profile. In conclusion, IL-12 gene can be expressed into AML-DCs defective in endogenous IL-12 production by using a novel non-viral method which does not modify their phenotypical, cytogenetic and functional features. IL-12 gene expression into AML-DC counteracts the inhibitory effect of leukemic microenvironment on T lymphocytes
Reduced Intensity Compared to High Dose Conditioning for Allotransplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome: A Comparative Clinical Analysis.
Authors: Flynn Catherine , Barker Juliet N. , Miller Jeff S. , Wagner John E. , DeFor Todd , Blazar Bruce R. , Burns Linda J. , MacMillan Margaret L. , Arora Mukta , Weisdorf Daniel
Published: October 2019

We evaluated the comparative safety and efficacy of stem cell transplantation using reduced intensity (RI) vs myeloablative (MA) conditioning for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) who underwent allogeneic hematopoietic stem cell transplantation (HSCT). Thirty two patients [18 males, median age 54 (range 19–69)] underwent a RI HSCT from January 2000- September 2003, and these were compared to 187 patients [93 males, median age 39 (range 18.4–60.3)] who received MA transplants between January 1990 – September 2003. Disease status in the 2 groups was similar, (AML in CR1: RI 38% vs MA 38%; CR2: 22% vs 15%; AML relapse: 13% vs 20% and MDS 28% vs 27%). Patients were eligible for RI if age >55 with HLA matched sibling donor (Sib) or >45 with unrelated bone marrow (BM) or umbilical cord blood (UCB) donor; extensive previous therapy including previous transplant or major co-morbidity.

RI donors were Sib (n=17, 53%), unrelated UCB (n=14, 44%), or unrelated BM (n=1). MA donors were Sib (n=176, 94%) and UCB (n=11, 6 %), p<0.01. RI conditioning included an alkylator (busulfan (Bu) or cyclophosphamide (Cy), a nucleoside analogue (fludarabine or cladribine) and 200 cGy total body irradiation (TBI), while MA conditioning was Cy/TBI (1320 cGy) (n=177) or Bu/Cy (n=10). Median follow up is 1.7 years in RI recipients and 4.2 years in MA. Neutrophil engraftment was more rapid in the RI group (median =11.5 days vs 21 days) with 3% and 6% graft failure respectively in the RI and MA groups. The cumulative incidence of Grade III–IV acute graft versus host disease (GVHD) was 22%±14% in RI vs 12±4% after MA HSCT. The cumulative incidence of relapse or disease progression at 1 year was 31% after RI vs 22% after MA HSCT (p=0.06). Disease free survival (DFS) at 1 year was 31± 16% in RI and 37%± 7% in the MA group (p=0.14). Multivariate regression analysis showed that neither conditioning intensity (NST RR=1.4, p=0.14) nor donor choice (UCB RR 0.9, p=0.63) significantly altered DFS. More failures (relapse or death) followed transplant for AML in relapse (RR=1.5, p=0.05), recipient seropositive for CMV (RR1.4, p=0.03) and development of Grade III/IV GVHD (RR 1.9, p<0.01), with MDS having an outcome similar to AML in remission (RR 1.2,p=0.28). These data suggest that allografts for AML or MDS using RI conditioning, even for older patients or using unrelated UCB can yield satisfactory outcomes and extended DFS. Advances in GVHD management and new approaches for patients in relapse are needed to improve these outcomes.
Total Body Irradiation (TBI), Fludarabine (F), Melphalan (M) and Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Advanced Pediatric Hematologic Malignancies.
Authors: Petropoulos Demetrios , Worth Laura L. , Mullen Craig A. , Mahajan Anita , Choroszy Mary S. , Chan Ka Wah
Published: October 2019

The FM reduced-intensity preparative regimen has been used successfully in adults for allogeneic HSCT. Its role in pediatric hematologic malignancies is unclear. We studied the engraftment efficacy and the antineoplastic effect of adding TBI to the FM combination. Between July 1998 and January 2004 a total of 29 pediatric (19 males) patients were treated. Twenty-two had acute lymphoblastic leukemia (ALL), 6 had acute myeloid leukemia (AML), and 1 anaplastic large cell lymphoma. Disease status at the time of SCT was: CR2 (19 patients), CR3 (5), CR1 (2, with Ph+ leukemia), and induction failure/relapse (3). Six patients received this regimen for a second HSCT. The donor was unrelated in 21 cases: 1–2 antigen mismatched cord blood (CB) in 20, and peripheral blood stem cells (PBSC) in one. Of the 8 related-donor HSCT, 2 were not genotypically identical. The conditioning regimen was TBI 9Gy (3 fractions on day -7, -6, and -5), F 30 mg/m2 IV daily (days -4 to -1) and M 140 mg/m2 IV on day -1. GVHD prophylaxis consisted of tacrolimus and mini-methotrexate. No anti-thymocyte globulin was used. The regimen was well-tolerated, with grade II-III oral mucositis and diarrhea the most common side effects seen. Twenty-seven patients achieved ANC engraftment after a median time of 16 days (range 11–35), and 23 also had platelet engraftment at a median of 42 days (range 14–200). There was one case of primary graft failure and one early death. Six of 27 evaluable patients developed grade III-IV acute GVHD and three chronic GVHD. There were seven deaths in the first 100 days (3 from interstitial pneumonitis, 1 GVHD, 1 renal failure, 1 VOD, 1 graft failure), all in patients with ALL. Nine patients (7 with ALL) relapsed at a median of 8 months post-transplant (range 2–54). With a median follow-up of 54 months (range 7–79), 7 of 22 ALL, 5 of 6 AML, 1 of 1 lymphoma patients are alive and in remission (12 are over two years post HSCT). In conclusion, the addition of TBI to FM leads to successful engraftment of allogeneic HSC (including 1–2 antigen mismatched unrelated cord blood units) in heavily pretreated pediatric patients with hematologic malignancies, without the inclusion of ATG. The regimen was well tolerated and its activity in AML deserves further evaluation.