bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.