bluuebunny/crossref_metadata_2025_split huggingface dataset:
Incidence of Thrombosis in Children with Tunneled Central Venous Access Devices Versus Peripherally Inserted Central Catheters (PICC lines)
Authors: Kanin Maralee , Young Guy 
Published: October 2019

Abstract
Abstract 1146

Background:
The recent proliferation in deep vein thrombosis (DVT) in pediatric patients has been attributed to the improved care of children with serious and life-threatening disorders as well as the prolonged survival of children with chronic disease. Often such treatment involves the insertion of central venous catheters. Yet the incidence of DVT associated with peripherally-inserted central catheters (PICC) and tunneled Lines (TL) has not been investigated in depth. Thus we report a retrospective database study to determine the incidence of DVT in pediatric patients with PICC and TL lines.

Methods:
Children <18 years of age who were admitted to Children's Hospital Los Angeles from January 1, 2003- December 31, 2009 were eligible for inclusion. Data were extracted from the CHLA hospital discharge database which includes data on all procedures and up to 20 diagnoses per admission. Diagnoses and procedures were identified by International Classification of Disease, Ninth Revision (ICD-9) coding in the hospital discharge database. PICC codes used were: 365.69 and TL codes used were: 365.57, 365.58, 365.60, 365.61, 365.63, 365.65, 365.66. DVT codes were: 415.11, 415.12, 415.19, 452, 453.0, 453.1, 453.3, 453.41, 453.42, 453.8, 453.9. Patient diagnoses other than DVT were grouped according to ICD. 9 coding into categories termed Complex Chronic Conditions (CCC) as previously described. To ensure that the DVT event was related to the individual line insertion, patients were excluded if their lines were placed after three days of hospital admissions and if they received more than one line or more than one type of line. A limitation of the data is the lack of information regarding the length of time the catheters remained in place.

Results:
Over the 6 year period of this study, 1449 eligible subjects were identified of whom 29 had DVTs (2%). There were 947 PICC insertions and 502 TL insertions. The mean age of PICC patients was 12±4 years and 56% were male. The mean age of TL patients was 8±6 years and 54% were male. There were 9 (0.95%) DVTs in PICC patients, and 20 (3.9%) DVTs in TL patients. The odds ratio for developing a DVT with a TL was 3.6 (p-value.002; confidence interval 1.6–8.1) which remained significant at 3.8 (p=.001; confidence value 1.7–8.55) when controlling for the presence of CCCs.

Conclusions:
Despite the proliferation in the utilization of PICC in recent years, it appears that they are not associated with a higher risk for DVT than TL, and in fact, the data from this study suggests that TL were associated with more DVT than PICC. Although it is possible that the results could be explained by the fact that TL in general remain in place for a longer period of time than PICC, this study nonetheless provides important reassurance that PICC appear to have relatively low thrombotic potential when compared to TL.

Disclosures:
No relevant conflicts of interest to declare.
The Balance of Gfi-1 and Gfi-1b Maintains the Undifferentiated, Multipotent State of Hematopoietic Stem Cells and Regulates Lineage-Commitment
Authors: Foudi Adlen , Hock Hanno 
Published: October 2019

Abstract
Abstract 1210
Gfi-1 and Gfi-1b are homologous transcriptional repressors that are expressed in hematopoietic stem cells (HSCs). Gfi-1 is crucial for the terminal maturation of neutrophils, and Gfi-1b is critical for erythropoiesis and thrombopoiesis. HSCs give rise to all mature blood lineages through a tightly regulated multistep differentiation process, but the mechanism of their early lineage specification remains largely elusive. Here, we have dissected the role of the Gfi-family factors in HSC maintenance and early lineage-commitment. To this end, we generated conditional targeted alleles for Gfi-1 and Gfi-1b that allowed for time controlled induced disruption of their genes. Acute disruption of Gfi-1 resulted in a rapid, severe decrease of HSCs numbers in the bone marrow and ablated their function in competitive repopulation assays. Surprisingly, and sharply contradicting recent claims to the opposite, acute disruption of Gfi-1b also led to decreased numbers of long-term repopulating HSCs in the bone marrow and decreased fitness in competitive transplantation. After induced, combined disruption of both factors, no HSC and progenitor cells were maintained in the bone marrow for more than 2 weeks. To elucidate the molecular mechanisms of the Gfi-family mediated HSC maintenance we performed global gene expression profiling of Gfi-1−/− and Gfi-1b−/− HSCs. Unexpectedly, both factors regulate highly distinct gene sets involved in differentiation of alternative lineages. Thus, surprisingly, their action in HSCs is not redundant but synergistic. Consistent with this, disruption of individual Gfi-family factors renders HSCs prone to differentiation to specific alternative lineages, while combined disruption is entirely incompatible with HSCs maintenance, in large part due to unchecked differentiation. Together, our data reveal that balanced expression of Gfi-1 and Gfi-1b is required for maintaining the undifferentiated, multipotent state of HSCs, while altering the balance is sufficient for inducing commitment to specific lineages.

Disclosures:
No relevant conflicts of interest to declare.
Contrasting Effects of Enhanced Sirt1 Activity in Normal and BCR-ABL1-Depedent Haematopoiesis
Authors: Hu Ming , Khorashad Jamshid S , Chaidos Aristeidis , Nteliopoulos Georgios , Khongkow Mattaka , Roberts Irene , Goldman John , Reid Alistair , Lam Eric W-.F. , Apperley Jane , Karadimitris Anastasios 
Published: October 2019

Abstract
Abstract 1215
Sirt1 is a NAD+-dependent class III histone deacetylase that regulates various cellular processes including cell growth, apoptosis, senescence and stress responses. Variations in Sirt1 expression and activity are associated with pleiotropic and profound biological effects that are often cell context-dependent. Here we tested the impact of enhanced Sirt1 activity in normal and BCR-ABL1-dependent leukemic haematopoiesis in vitro.
Using 5-FU enriched murine primary hematopoietic stem and progenitor cells (HSPC) as a model, we report that: 1. Sirt1 over-expression induces 2–3 fold increased, both in frequency and absolute number, of HSPC cells (Lin-Sca1+ckit+) in ‘stem cell-like’ culture condition and enhanced clonogenicity in serial transplantation assay. qRT-PCR analysis showed significantly higher expression of GATA-2 gene in HSPC cells suggesting that Sirt1 promotes GATA-2 mediated HSPC cell proliferation. 2. When HSPC cells were induced to differentiation in the presence of lineage specific growth factor EPO, G-CSF and IL-7, respectively, Sirt1 expression significantly increased the frequency and absolute number of terminal differentiated erythroid (ter119+, 2–3 folds), granulocytic (Gr-1+Cd11b+, 2–3 folds) and B (B220+CD19+, 3–4 folds) cells. Thus, enhanced Sirt1 activity promotes HSPC self-renewal and multiple lineage differentiation.
We next tested the effects of Sirt1 activity in Chronic Myeloid Leukaemia (CML). By transducing Sirt1 retrovirus into IL3-dependent BaF3, IL3-independent BCR-ABLp210-expressing BaF3 and primary HSPC cell, we found that Sirt1 over-expression significantly inhibited BCR-ABL1-driven cell proliferation and clonogenicity in cytokine depleted media. In addition, Sirt1 over-expression further reduced the growth rate of BCR-ABLp210 expressing BaF3 cells when treated with TGF-b1, suggesting that Sirt1 might play as a downstream effector of TGF-b signal. Finally, Resveratrol (RSV), a Sirt1 activator, alone and in co-operation with Imatinib, induced cell death and inhibited clonogenicity of CD34+ cells from CML patients with chronic phase.
Taken together, our data demonstrate that Sirt1 exhibits contrasting effects in normal and BCR-ABL1-dependent haematopoiesis and suggest that manipulating Sirt1 activity may be a useful strategy to differentially promote normal and inhibit leukemic haematopoiesis in CML.

Disclosures:
No relevant conflicts of interest to declare.
Mesenchymal Stem Cells Engineered to Inhibit Complement-Mediated Damage
Authors: Soland Melisa , Bego Mariana , Colletti Evan , Porada Christopher D , Zanjani Esmail , St. Jeor Stephen , Almeida-Porada Graca 
Published: October 2019

Abstract
Abstract 1253
Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express low levels of molecules, such as CD59, which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. It has been demonstrated that transduction of MSC with a retrovirus encoding HCMV US proteins resulted in higher levels of MSC engraftment and diminished recognition by the immune system, due to a decrease in HLA-I expression. Here we investigate whether engineering MSC to express US2, US3, US6, or US11 HCMV proteins can alter complement recognition, and thereby protect MSC from complement attack and lysis. US HCMV proteins increased MSC CD59 expression to differing degrees, as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI) (n=3). CD59 MFI on untransduced MSC was 128±33, and this value remained largely unchanged on MSC transduced with an empty retroviral vector (MSC-E) and on MSC transduced with US11 (MSC-11). In contrast, a significant increase in CD59 MFI was seen in MSC transduced with US2 (MSC-2), US3 (MSC-3), and US6 (MSC-6), with MFIs of 273±35 (p<0.05), 319±64 (p<0.05), and 265±16 (p<0.05), respectively. Although overexpression of HCMV proteins on MSC did not change the MFI for membrane cofactor protein (CD46) and complement decay accelerating factor (CD55), it significantly altered the percentage of MSC that expressed these two complement-protective proteins (n=3). Specifically, while no statistically significant difference was seen in the percentage of MSC-E, MSC-6, or MSC-11 expressing CD46 (17.9±1%, 19±0.1%, 21.3±1.4%, respectively), 27.7±0.7% of MSC-3 (p<0.05) and 27.7±1.6% of MSC-2 (p<0.05) expressed CD46. Similarly, while comparable percentages of MSC, MSC-E, and MSC-3 expressed CD55 (37.8±3%, 42.8±1%, and 45.2±1, respectively), the overexpression of HCMV US2, US6, and US11 each led to a significant increase in the percentage of MSC expressing CD55 (MSC-2: 49±1% (p<0.05); MSC-6: 56±0.6% (p<0.05); MSC-11: 60±1.3% (p<0.05)). Because the HCMV US2 protein was the most efficient at up-regulating all three complement regulatory proteins, we used two different complement-mediated cytotoxicity assays to investigate whether MSC-2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis of MSC-2 by 59.10±12.89 % when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV US proteins in complement evasion, and our results demonstrate that over-expression of HCMV US proteins on MSC could serve as a strategy to generate cells protected from complement lysis.

Disclosures:
No relevant conflicts of interest to declare.
Reactivation of Telomerase in Late Generation Tert−/− Mice Results in Reversal of Anemia
Authors: Raval Aparna , Kusler Brenda , Garbuzov Alina , Artandi Steven , Mitchell Beverly S. 
Published: October 2019

Abstract
Abstract 1258
We previously reported that telomere shortening in a late generation telomerase K/O mouse model results in significantly reduced RBC and erythroblast (CD71+Ter119+) numbers in the peripheral blood and bone marrow, respectively. We also observed significant decreases in erythroid progenitor (Lin-cKit+Sca1-CD34-CD16/CD32-) and HSC (Lin-cKit+Sca1+ CD34-CD150+) numbers in G5 Tert−/− mice. We now report the mechanism underlying the specific defect in the erythroid lineage in these mice. First, there is a higher number of CD34-KLS CD150lo and CD150neg cells in the mutant mice relative to age matched G0 +/− mice. Methyl cellulose colony formation assay showed that CD34-KLS CD150hi cells give rise to a greater number of CFU-GEMM colonies while CD34-KLS CD150lo and CD34-KLS CD150neg cells both had a reduction in the numbers of GEMM colonies, suggesting that the loss of erythroid differentiation is associated with loss of CD150hi expression within the CD34-KLS population. Thus, a significant reduction in CD150hi HSC (p value < 0.001) and an increase in CD150lo (p value < 0.001) and CD150neg (p value < 0.04) HSC in G5 Tert−/− mice underlies the decreased number of erythroblasts in these mice. Secondly, we found that the reduction in telomere length as measured by quantitative PCR is greater in sorted erythroblasts (CD71+Ter119+) than in similarly purified myeloid progenitors (Gr1+Mac1+) in G5 Tert −/− mice. This finding correlates with the presence of increased DNA damage and apoptosis in erythroid progenitors and erythroblasts as compared to myeloid progenitors. In order to establish a cause and effect relationship between telomere shortening and defective erythropoiesis, we reactivated telomerase activity using a tamoxifen-inducible Cre recombinase to excise a Lox-Stop-Lox cassette located in intron 2 of telomerase gene in G5 Tert−/− mice. Reactivation of telomerase activity resulted in increased CD150hi HSC, erythroid progenitors and erythroblasts numbers in the bone marrow of these mice 3 months after tamoxifen treatment. In addition, RBC numbers and Hb levels in the peripheral blood improved in the treated mice, whereas neither improved in control G5 Tert−/− mice treated with sunflower oil. These data show for the first time a direct link between the loss of telomerase function, telomere shortening and a selective defect in erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments underway to define the intracellular signaling pathways that are affected in the HSC and erythroid precursors of G5 Tert−/− mice, demonstrate the lineage-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes.

Disclosures:
No relevant conflicts of interest to declare.
Suppression of the Hematopoietic Defect in TF-1 Cells Depleted of Shwachman-Diamond Syndrome Protein: Correlation with Decreased eIF6 Levels
Authors: Singh Sharon , Blanc Lionel , Henson Adrianna , Sezgin Gulay , Ellis Steven R , Liu Johnson M. 
Published: October 2019

Abstract
Abstract 1270
Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6.
Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p<0.01 135+/−6.8 p=0.64 Erythroid 89+/−8.4 48+/−4 p<0.001 89+/− 4.7 p=0.94

Disclosures:
No relevant conflicts of interest to declare.
A Homozygous Telomerase Reverse Transcriptase T Motif Variant Resulting in Markedly Reduced Telomerase Repeat Addition Processivity in Siblings with Hoyeraal Hreidarsson Syndrome
Authors: Gramatges M. Monica , Qi Xiaodong , Sasa Ghadir S. , Chen Julian J-L , Bertuch Alison A. 
Published: October 2019

Abstract
Abstract 1272
Hoyeraal Hreidarsson syndrome (HHS) is a severe form of dyskeratosis congenita (DC) characterized by bone marrow failure, intrauterine growth retardation, developmental delay, microcephaly, cerebellar hypoplasia, immunodeficiency, and extremely short telomeres. As with DC, mutations in genes that encode factors required for telomere maintenance, such as the telomerase reverse transcriptase (TERT), have been found in patients with HHS. We describe two sibling cases of HHS caused by a homozygous mutation (p.T567M) within the TERT T motif, which is a highly conserved motif specific to the telomerase-class of reverse transcriptases. This mutation resulted in a marked reduction in the capacity of telomerase to processively synthesize telomeric repeats, indicating for the first time a role for the T motif in this unique aspect of telomerase function. The consanguineous parents, heterozygous for this mutation, exhibited telomere lengths around the first percentile and no evidence of a DC phenotype. Although heterozygous processivity defects have been associated with familial adult-onset pulmonary fibrosis, these cases demonstrate the severe clinical and functional impact of biallelic processivity mutations. Thus, despite retaining the capacity to catalyze the addition of short stretches of telomeric repeats onto the shortest telomeres, the sole expression of telomerase repeat addition processivity mutants leads to a profound failure of telomere maintenance and early onset disease. In the course of our investigation of this mutant, several commonly used algorithms predicted no effect of TERT p.567M upon protein function. To examine this further, we compiled missense changes that are associated with a clinical phenotype and correlated each with its reported telomerase activity and presence of the phenotype in more than one family member. We then summarized the predicted functional impact for each of these mutations based upon four commonly used algorithms. Our results demonstrate these algorithms to be limited in their capacity for predicting the effect of missense changes upon telomerase function, emphasizing the importance of in vitro functional analyses including processivity assays for TERT variants of unknown significance.

Disclosures:
No relevant conflicts of interest to declare.
Adaptor Protein Lnk Binds to and Inhibits Normal and Leukemic FLT3
Authors: Lin Dechen , Yin Tong , koren-Michowitz Maya , Ding Ling-Wen , Gueller Saskia , Gery Sigal , Tabayashi Takayuki , Bergholz Ulla , Kazi Julhash , Rönnstrand Lars , Stocking Carol , Koeffler Phillip 
Published: October 2019

Abstract
Abstract 1312

Background
The production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML.
FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3.

Methods and Results
Co-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL.

Conclusions
This study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases.

Disclosures:
No relevant conflicts of interest to declare.