bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial Microparticles Improve Primary and Secondary Hemostasis in Patients with Von-Willebrand Disease
Authors: Trummer Arne , Birschmann Ingvild , Werwitzke Sonja , Ganser Arnold , Tiede Andreas
Published: October 2019

Abstract 2287

The bleeding phenotype in patients with von Willebrand disease (vWD) type 1 and 3 can usually be correlated to the amount and function of von Willebrand factor (vWF:Ag, vWF:RCo) but also to the impairment of functional coagulation assays: the ristocetin-induced platelet aggregation (RIPA) for primary hemostasis and the parameters of thrombin generation in a calibrated automated thrombogram (CAT, Thrombinoscope®) for secondary hemostasis. We therefore evaluated whether addition and depletion of endothelial microparticles (EMP) might influence these assays for samples of vWD patients.

EMP were obtained from stimulated endothelial cell lines, isolated either by ultracentrifugation or magnetic beads selection for Annexin V and detected and quantified by flow cytometry after labelling for Annexin V and vWF. For CAT, EMP concentration was adjusted to 3×103/μl, for RIPA to 2×104/μl. vWD patient samples were diluted 3:1 or 2:1 with control buffer (HEPES/CaCl) or EMP solution.

For thrombin generation, there was a significant shortening of lag time (6.8min vs 16.5min, p=.029) and time-to-peak (13.6min vs. 23.0min, p=.049) in vWD type 3 plasma (n=4) after addition of EMP using no reagent or MP reagent while there was no significant effect on these parameters in normal plasma. After addition of a tissue factor blocking antibody, the EMP effect could be largely reversed. For RIPA, we found a median maximum aggregation of 4% in vWD patients (n=6, vWF-Ag <40%). After addition of EMP, aggregation levels increased to 17.3%, which was significantly higher compared to control buffer (9.8%, p=.028), but lower compared to 250 IU/ml of a plasma-derived vWF product (Humate P®, 88.7%). To evaluate if significant amounts of vWF are bound to circulating microparticles in human plasma, we used magnetic bead selection to reduce MP counts in plasma of DDAVP stimulated vWD patients (n=10), because depletion of microparticles by 0.2μm filtration showed significant disruption of large vWF multimers. After MP reduction (1464/μl vs. 1863/μl, p=.036), both vWF:Ag (134.7% vs 161.1%, p=.005) and vWF:RCo (131.8% vs. 167.2%, p=.017) were significantly decreased.

In summary, we could show that in vitro generated endothelial microparticles are able to improve thrombin generation and platelet aggregation in vWD patients and that significant amounts of vWF are bound to circulating microparticles in human plasma after DDAVP stimulation. Thus, in vitro generated EMP might have the potential to improve replacement therapy for vWD patients.

Disclosures:

No relevant conflicts of interest to declare.
Transfusion of Stored Autologous Blood Alters Reactive Hyperemia and Circulating Nitrite Levels in Healthy Volunteers
Authors: Berra Lorenzo , Dzik Walter Sunny , Coppadoro Andrea , Bloch Kenneth D. , Yu Binglan , Spagnolli Ester , Steinbicker Andrea U. , Feelisch Martin , Fernandez Bernadette , Stowell Christopher , Warren Shaw H , Lin Tian , Zapol Warren M.
Published: October 2019

Abstract 2327

Background:

Transfusion of human blood stored for over 2 weeks is associated with increased mortality and morbidity. During storage, packed red blood cells (PRBC) progressively release hemoglobin, which avidly binds nitric oxide (NO). We hypothesized that the NO mediated hyperemic response following ischemia would be reduced after transfusion of PRBC stored for 40 days.

Methods and results:

We conducted a cross-over randomized interventional study, enrolling ten healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced PRBC, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index (RHI) measurements were performed before and 10 minutes, 1 hour, 2 hours, and 4 hours after transfusion. The RHI was lower (13% mean post-transfusion difference) after transfusion of 40-day as compared to 3-day stored PRBC (p=0.01). Plasma cell-free hemoglobin and bilirubin levels were higher after transfusion of 40-day than after 3-day stored PRBC (p=0.02 and 0.001, respectively). Plasma levels of potassium, LDH, haptoglobin, cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored PRBC, but not after transfusion of 3-day stored PRBC (p=0.01).

Conclusions:

Transfusion of autologous PRBC stored for 40 days is associated with increased hemolysis and reduced RHI, possibly due to enhanced NO scavenging by cell-free oxyhemoglobin.

Disclosures:

Bloch: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.
“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis
Authors: Komeno Yukiko , Yan Ming , Matsuura Shinobu , Lo Miao-Chia , Downing James R. , Zhang Dong-Er
Published: October 2019

Abstract 2329

Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications.

Disclosures:

No relevant conflicts of interest to declare.
C/EBPα-Induced MicroRNA 30c Inactivates Notch1 During Granulopoiesis and Is Downregulated In Acute Myeloid Leukemia
Authors: Katzerke Christiane , Madan Vikas , Gerloff Dennis , Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Mueller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Abstract 2368

The transcription factor CCAAT Enhancer Binding Protein alpha (C/EBPα) is crucial for normal granulopoiesis and frequently disrupted in acute myeloid leukemia (AML). Loss of expression or function of C/EBPα leads to a block of myeloid differentiation. MicroRNAs inhibiting translation of mRNA into protein were identified as critical players in stem cell development. We and others have already shown that C/EBPα exerts its effects by regulating microRNAs such as miR-223 and miR-34a. In a global microRNA-array screen we found miR-30c as a novel target of C/EBPα during granulocytic differentiation. Wild-type C/EBPα-p42 upregulates miR-30c expression, whereas the C/EBPα-p30 mutant, found in AML, does not. Furthermore, G-CSF upregulates miR-30c expression during granulocytic differentiation of primary human CD34-positive progenitor cells. C/EBPα induces miR-30c and downregulates Notch1, a putative target of miR-30c, on protein, but not mRNA level. A block of miR-30c by LNAs prevents C/EBPα–induced downregulation of Notch1 protein expression. miR-30c is a tumor suppressor and downregulated in various subtypes of AML. In mice, miR-30c shows a high expression in LSK (including hematopoietic stem cells), GMP (granulocytic monocytic precursors) and granulocytes. An induced knock-out of C/EBPα in mice leads to a significantly downregulation of miR-30c expression in bone marrow cells. Our data indicates that C/EBPα-induced miR-30c inactivates Notch1 during granulopoiesis and is downregulated in AML. These data reveal the importance of deregulated microRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Key Autophagy Genes in Primary AML Patients and Their Functional Role in Neutrophil Differentiation of APL Cells
Authors: Tschan Mario P. , Brigger Daniel , Schläfli Anna M. , Fey Martin F.
Published: October 2019

Abstract 2456

There is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. Autophagy may support the remodelling processes that are taking place during differentiation and maturation of different cell types. Generally, autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Furthermore, autophagy-inducing drugs support degradation of aggregated PML-RARA protein in acute promyelocytic leukemia (APL). We used customized 384-well micro fluidic cards to determine the expression of key autophagy genes (ATGs) in a large cohort of primary acute myeloid leukemia (AML) samples (n=122) and normal neutrophils (n=14). In general, we found significantly lower ATG mRNA levels in AML as compared to neutrophils for 21/38 autophagy genes investigated. Among the most significantly downregulated genes in AML were ATG1, ATG4D, ATG14, ATG16L2, WDFY3, BNIP3L, FIP200, GATE-16 and GABARAPL1. Given the low expression of these ATGs in AML, a disease mainly characterized by a block in differentiation, we hypothesized that these genes are implicated in myeloid differentiation. We therefore analyzed expression of selected ATGs in two APL cell line models for neutrophil differentiation. We found a significant 2- to 40-fold increase of ATG1, ATG7, WDFY3, GATE16 and GABARAPL1 in NB4 and HT93 APL cell lines upon all-trans retinoic (ATRA)-induced neutrophil differentiation. The expression of these genes did not change in ATRA-resistant NB4-R2 cells upon neutrophil differentiation indicating that their increased expression is functional in neutrophil differentiation. Next, we knocked down ATG1, WDFY3, GATE16 and GAPARAPL1 in NB4 cells using lentivirally delivered shRNAs. Inhibition of ATG1, WDFY3 and GATE16 but not GABARAPL1 significantly attenuated ATRA-induced neutrophil differentiation as measured by reduced levels of the myeloid markers CD11b, CEBPE and G-CSF-R. Analysis of the autophagic markers p62/SQSTM1 and LC3B revealed that inhibiting ATG1, WDFY3 and GATE16 not only diminished neutrophil differentiation of APL cells but also myeloid differentiation-associated autophagy (MDAA). In summary, low ATG expression is generally associated with an immature AML phenotype. Furthermore, knocking down key autophagy genes attenuates ATRA-induced neutrophil differentiation and MDAA of APL cells. Our studies indicate that autophagy is a putative tumor suppressor mechanism in AML/APL and already available autophagy-inducing drugs such as lithium chloride may be used in combination with differentiation-inducing drugs.

Disclosures:

No relevant conflicts of interest to declare.
Activated K-RAS Down-Regulated Small GTPases and Cell Motility of Myeloid Sarcoma-Inducing Leukemia Cells Through Erk Signaling Cascade
Authors: Fu Jen-Fen , Shih Lee-Yung
Published: October 2019

Abstract 2459

The molecular mechanism of myeloid sarcoma (MS) formation in the patients with acute myeloid leukemia (AML) is not clear. By analyzing the genetic mutations, we found that AML patients with combined MLL/AF10 and activated N-/K-RAS mutation had a high occurrence of association with MS. We had previously performed a retroviral transduction/transplantation assay and demonstrated that the activated KRAS mutation could not immortalize murine bone marrow (BM) cells, whereas MLL/AF10 alone induced myeloproliferative disease-like myeloid leukemia (MPD) in the transplanted mice. Combination ofMLL/AF10 and activated K-RAS mutation induced MPD in a shorter latency, in addition, multiple MS were detected in the abdominal-, subcutaneous-, or intramuscular fat of the transplanted mice. To understand the role of activated KRAS mutation in cooperation with MLL/AF10 in the MS formation, we performed an in vivo imaging analysis to compare cell migration between cells carrying single and combined mutations. Our results showed that the cells carrying combined mutations were slowly migrating to the omental and gonadal adipose tissues of the transplanted mice after intra-peritoneal injection. On the contrary, the cells carrying only MLL/AF10 were rapidly condensed and entered into blood stream. An in vitro transwell migration assay confirmed that the cells carrying combined mutations exhibited a reduced cell motility in response to SDF-1 treatment. Analysis of the Cxcr4 gene expression did not show significant difference between cells carrying single and combined mutations, suggesting that the slow migration was not attributed to lacking receptor for SDF-1. Further determination of the cell migration-related small GTPases revealed that the amount of total and GTP–bound Rac, Cdc42 and Rho was reduced in the cells carrying combined mutations. Moreover, treatment of SDF-1 further down-regulated protein expression and activity of these small GTPases. To understand which signaling pathway(s) linked the KRAS mutation to the down-regulated small GTPases, we analyzed the amount of total and phosphorylated signaling molecules and found that Erk, but not Jnk1 and p38 Mapk, had a similar protein expression and activation pattern as those of the GTPases, suggesting that the reduction in cell motility of the MS-inducing leukemia cells might be mediated via KRAS-Mek-Erk axis.

Disclosures:

No relevant conflicts of interest to declare.
Source and Purity of Factor VIII Products As Risk Factors for Inhibitor Development In Previously Untreated Patients with Severe Hemophilia A
Authors: Mancuso Maria Elisa , Mannucci Pier Mannuccio , Rocino Angiola , Garagiola Isabella , Tagliaferri Annarita , Santagostino Elena
Published: October 2019

Abstract 25

Background:

Inhibitor development is influenced by several genetic and environmental factors and the type of factor VIII (FVIII) products may play a role. Methods: We designed a cohort study whose novelty resides in the classification of products not only according to the plasmatic (pdFVIII) or recombinant (rFVIII) source of FVIII but also to the degree of purity expressed as FVIII specific activity per mg of protein. The role of FVIII product as risk factor for inhibitor development was evaluated in a multivariate model adjusting for potential confounders (i.e. age at first FVIII exposure, intensive treatment and prophylaxis). Cumulative incidences of all and high-responding inhibitors were calculated for the whole cohort of 721 patients with severe and moderate hemophilia A followed-up in 3 Italian Hemophilia Centers. Detailed treatment data from the first FVIII infusion up to inhibitor development or 150 exposure days were available for 377 patients and in this group of patients risk factors for inhibitor development including the type of FVIII product and its degree of purity (i.e. low/intermediate-, high-purity pdFVIII and rFVIII) were analysed. Results: The overall cumulative incidence of inhibitors was 22% (n=160; 130 high-responders, 18%) and it was lower in patients first treated with pdFVIII (107/586, 18%) than in those treated with rFVIII (53/135, 39%). Similar results were obtained by evaluating only high-responding inhibitors and patients with severe hemophilia. The adjusted hazard ratio of inhibitor development was 4.9 with rFVIII and 2.0 with high-purity pdFVIII (95%CI: 2.9–8.3 and 1.1–4.0), taking as reference low/intermediate-purity pdFVIII. There was no difference in the frequency of inhibitor testing between treatment groups. Sensitivity analyses - in patients who never switched product type, previously untreated patients, those treated on-demand and those with high-risk F8 mutations - confirmed an increased inhibitor risk in patients first treated with rFVIII or high-purity pdFVIII than in those treated with low/intermediate-purity pdFVIII. In fact, in all the aforementioned subgroups by multivariate analysis the risk of inhibitor development was invariably 3- to 6-fold higher in patients first treated with rFVIII than in those first treated with pdFVIII, and similar results were obtained for both all inhibitors and high-responding inhibitors. Conclusions: This study shows that the degree of purity of FVIII products influences inhibitor development independently from other risk factors, and emphasizes that differences exist also within pdFVIII products.

Disclosures:

No relevant conflicts of interest to declare.
The TOR Kinase Inhibitor INK128 Is Effective in Pre-B Acute Lymphoblastic Leukemia Models
Authors: Fruman David A , Janes Matthew Robert , Mallya Sharmila , Vu Collin C , Shieh Marie P. , Lilly Michael B. , Sender Leonard S , Martin Michael B , Ren Pingda , Liu Yi , Rommel Christian
Published: October 2019

Abstract 2585

Pre-B Acute Lymphoblastic Leukemia (B-ALL) remains a significant cause of morbidity and mortality in both adults and children. Targeting signaling pathways that sustain B-ALL cell survival is a promising strategy to improve treatment outcomes. The target of rapamycin (TOR) is a serine/threonine kinase that integrates signals from oncoproteins and the microenvironment to support ALL cell proliferation and survival. A single TOR enzyme in cells forms two multiprotein complexes, TORC1 and TORC2, with distinct regulation and substrates. The classical TOR inhibitor rapamycin inhibits only some functions of TORC1 and does not acutely inhibit TORC2. A new generation of TOR inhibitors has been developed that inhibit all outputs of both TORC1 and TORC2. These ATP-competitive, TOR kinase inhibitors (TOR-KIs) have improved anti-cancer activity in models of both solid tumors and hematological malignancies. Previously we reported that the TOR-KI compound PP242 is effective in mouse models of Philadelphia Chromosome-positive (Ph+) B-ALL, with minimal toxicity to normal hematopoietic cells or immune function (MR Janes et al., Nat. Med. 2010; 16:205). Here we present promising results on the efficacy of a second generation TOR-KI, INK128, in both Ph+ and non-Ph B-ALL. INK128 is an orally active, highly selective TOR-KI that is currently in phase I clinical trials for solid tumors, multiple myeloma and Waldenstrom's Macroglobulinemia. We find that INK128 has similar biological effects as PP242 on Ph+ B-ALL cells, but these effects are apparent at 5-fold lower concentrations in vitro and 80-fold lower doses in mice. In methylcellulose assays using Ph+ and non-Ph B-ALL specimens, INK128 greatly reduces colony numbers and enhances the efficacy of standard-of-care drugs. Both adult and pediatric non-Ph B-ALL specimens are sensitive to growth suppression by INK128. INK128 also causes apoptosis in B-ALL cells cultured on stromal cells. Importantly, INK128 suppresses human Ph+ B-ALL xenograft expansion in mice at doses that do not impair normal hematopoietic cell proliferation. INK128 is also less immunosuppressive than rapamycin. Together these results provide rationale for testing INK128 and other TOR-KIs in clinical trials of B-ALL therapy.

Disclosures:

Fruman: Intellikine: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janes:Intellikine: Employment. Martin:Intellikine: Employment. Ren:Intellikine: Employment. Liu:Intellikine: Employment. Rommel:Intellikine: Employment.
ALL Progression Induces PD1 on T Cells and Blockade of PD1 Enhances Adoptive Immunotherapy
Authors: Burk Chad R , Fix William , Qin Haiying , Fry Terry J
Published: October 2019

Abstract 2587

Background:

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and, despite tremendous success in therapy over the past 3 decades, remains a primary cause of cancer-related mortality in children. Enthusiasm for the use cellular immunotherapy for ALL has been tempered by the poor response to donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation. However, ALL blasts are susceptible to T cell and NK cell mediated lysis in vitro suggesting that poor response to in vivo immune interventions may be due to events occurring during the priming of the immune response. Using a murine model of precursor B cell ALL we examined the impact of leukemia progression on T cells in vivo. Methods: We developed a transplantable syngeneic model of pediatric ALL derived from transgeneic mice expressing human E2aPBX1, a recurring translocation present in 5% of pediatric leukemia (Bijl et al, Genes and Development, 2005). This murine line displays a precursor B cell phenotype and results in 100% lethality following injection of 100,000 cells (Qin et al, ASH, 2010). Using congenic (CD45.1) B6 recipients, we tracked the early progression of ALL in vivo and examined the T cells in the leukemia-containing compartments by flow cytometry and PCR. Results: Using congenic markers, ALL cells can be detected in bone marrow as early as 3 days following intravenous injection of 1,000,000 cells with a sensitivity of 0.01%. Spleen and lymph node involvement was seen later (10 days) followed by the detection of circulating blasts by 2 weeks. E2aPBX1 cells express variable levels of costimulatory molecules in vitro with no change in expression during in vivo progression. Notably, PDL1 and PDL2 are expressed both in vitro and in vivo at higher levels than on non-malignant precursor B cells in leukemia-bearing mice. Remarkably, although PD1+ T cells are not seen in the bone marrow of non-leukemia-bearing mice, PD1 expression on bone marrow T cells was markedly increased during progression such that 60–80% of all bone marrow CD4 and CD8 T cells were positive by 2 weeks following leukemia injection (figure). In addition to expression of PD1, these T cells also co-expressed Tim3, a phenotype associated with T cell exhaustion. Blockade of PD1 or PDL1 starting 3 days following leukemia injection had no impact on leukemia progression. However, combining PD1 blockade with the adoptive transfer of T cells from leukemia-primed donors resulted in improved survival compared to primed T cells alone (p=0.0004). Conclusions: Early progression of ALL results in the induction of PD1 and Tim3 on T cells in vivo. Combination of PD1 blockade plus adoptive T cell therapy results in therapeutic benefit suggesting that this axis may be an attractive target in ALL.

Disclosures:

No relevant conflicts of interest to declare.
CXCR3 Is Required for the Efficient Recruitment of Bone Marrow-Resident Memory T Cells to Inflamed Tissues,
Authors: van der Voort Robbert , Brandao Claudia , Volman Thomas J. , Verweij Viviènne , van Gisbergen Klaas , Lu Bao , Gerard Craig , Nolte Martijn A. , Dolstra Harry
Published: October 2019

Abstract 3242

Although the importance of the bone marrow (BM) in hematopoiesis is well known, its function in adaptive immune responses has only recently been acknowledged. Currently it is known that the BM contains fully functional CD4+ and CD8+ T cells that can engage in both primary and secondary immune responses. Interestingly, most of these T cells belong to the memory T cell lineage, identifying the BM as one of the largest memory T cell reservoirs in the body. Since not much is known about the trafficking of BM T cells, we compared the homing phenotype and function of T cell subsets in the BM, blood, spleen and peripheral lymph nodes (pLN). In addition, we determined the expression of chemokine mRNA and protein levels in the BM and other lymphoid organs. We confirmed that at least 80% of the CD4+ and 60% of the CD8+ BM T cells have a memory phenotype, and that most CD4+ T cells belong to the effector memory lineage, while the CD8+ population predominantly consists of central memory T cells. Most BM T cells expressed the chemokine receptor CXCR3, the adhesion molecules P-selectin glycoprotein ligand 1 and VLA-4, and increased levels of CD44 and LFA-1, as compared to T cells from the spleen. In addition, L-selectin was absent from most CD4+ BM T cells, but present on virtually all CD8+ T cells. Notably, the percentage of CXCR3+ T cells within the effector memory and central memory subsets from BM was higher than within the same subsets from pLN. Furthermore, BM contained significant mRNA levels of the CXCR3 ligands CXCL9, CXCL10 and CXCL11. An in vivo migration assay using a mixture of fluorescent-labeled T cells from CXCR3-deficient mice and control mice indicated however that during homeostasis CXCR3 does not play a major role in BM entry or retention. These data suggest that CXCR3 expressed by memory T cells is rather involved in BM exit, than in BM entry. Indeed, we observed that, as compared to control mice, CXCR3−/− mice contained significantly more CD4+ and CD8+ T cells in their BM. Additional in vitro assays demonstrated that CD4+ and CD8+ BM T cells migrated vigorously in response to CXCL9 and CXCL10, generally released in high concentrations during inflammation. Finally, we demonstrate that CXCR3−/− effector/effector memory T cells, but not wild type T cells, accumulate in the BM of mice infected with lymphocytic choriomeningitis virus. Altogether, these data demonstrate that the BM is a major reservoir of memory T cells that employ CXCR3 to quickly respond to chemotactic signals from inflamed tissues.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Platelet Receptor Expression in ITP,
Authors: Qiao Jianlin , Tran Huy , Mu Fi-Tjen , Andrews Robert K , Gardiner Elizabeth E
Published: October 2019

Abstract 3289

In this study we assess platelet receptor expression and shedding in patients with immune thrombocytopenia (ITP) before and during treatment. The aim is to evaluate the potential value of quantitative measurement of platelet receptors for diagnosis and/or monitoring treatment in thrombocytopenia due to immune or other causes. The platelet-specific collagen receptor, glycoprotein (GP)VI, is associated with the Fc receptor γ-chain (FcRγ). GPVI/FcRγ is coassociated on platelet surface with the GPIb-IX-V complex; GPIbα of GPIb-IX-V binds von Willebrand factor and other ligands. Our previous studies showed engagement of platelet FcγRIIa by antiplatelet antibodies induced ectodomain shedding of GPVI, generating soluble ectodomain (sGPVI) in plasma. However, apart from one individual with an anti-GPVI antibody, whether anti-platelet antibodies associated with ITP affect GPVI/GPIb expression/shedding has not been addressed. In this study we used flow cytometry and a sGPVI ELISA to assess 1) whether patients with ITP had dysregulated expression/shedding of GPVI or GPIbα, and 2) whether platelet receptor expression changes prior to recovery of platelet count in individuals undergoing treatment for ITP. In 9 ITP patients (mean age=48.6, range 29–79; 6 female) with platelet count 61±9 × 109/L (range, 33–122 × 109/L), GPVI surface expression (GeoMean±SE, 137±17) was lower than healthy controls (274±26; n=17; platelet count 247±13), and sGPVI in patient plasma was significantly higher (39±4 ng/mL) compared to 17 healthy donors (19±3 ng/mL) (P=0.0006). In longitudinal samples analysed at weekly intervals during 2-month treatment with steroids, decreased GPVI surface expression and increased sGPVI in plasma remained essentially unchanged as the platelet count normalized, consistent with persistent anti-platelet antibody. However, while levels of intact platelet GPIbα were significantly reduced in ITP compared to healthy donors (P=0.0053), they approached healthy levels within 1 week of treatment, preceding improvement in platelet count or other measures. GPIbα expression/cleavage has been previously implicated in platelet clearance in experimental models, and our analysis suggests the proteolytic status of human GPIbα may be a novel early marker for evaluating response to treatment in ITP.

Disclosures:

No relevant conflicts of interest to declare.
Activated Protein C and Septic Shock: A Propensity-Matched Cohort Study,
Authors: Rimmer Emily K. , Kumar Anand , Doucette Steve , Marshall John , Dial Sandra , Gurka David , Dellinger Phillip , Sharma Sat , Penner Charles , Kramer Andreas , Wood Kenneth , Ronald John , Zarychanski Ryan
Published: October 2019

Abstract 3307

Background:

Septic shock is a highly inflammatory and pro-coagulant state associated with high mortality. In a single randomized controlled clinical trial, recombinant human activated protein C (rhAPC, drotrecogin alfa) reduced mortality in patients with severe sepsis at high risk of death. Further clinical trials in patients with a lower severity of illness and in children have failed to reproduce these results, and the effectiveness of rhAPC remains the subject of ongoing debate. Insufficient data exists regarding the use of rhAPC outside of a clinical trial.

Objective:

To evaluate the effectiveness of rhAPC on mortality in a cohort of patients with septic shock.

Methods:

We conducted a retrospective, propensity-matched, multi-center cohort study in 28 academic and community intensive care units in 3 countries. Propensity scores were based more 33 variables that were either known or suspected to be associated with rhAPC use. Our primary outcomes were mortality over 30 days and 30-day mortality stratified by APACHE II quartile. Secondary outcomes were hospital and ICU mortality and length of stay, ventilator free and vasopressor free days, and the use of acute dialysis.

Results:

Using a propensity matched Cox proportional hazard model we observed a 5.9% absolute reduction in 30-day mortality associated with activated protein C use [115/342 (33.6%) vs. 135/342 (39.5%), HR 0.83, 95% CI 0.65–1.07, p=0.15]; however this reduction failed to reach statistical significance. Similarly, we observed non-significant reductions in mortality among the 3 lowest APACHE II quartiles (APACHE II <20: 12.5% vs. 18.2%, HR 0.66, 95%CI 0.29 – 1.49, p=0.32; APACHE II 21–25: 20% vs 33.3%, HR 0.55, 95% CI 0.30–1.02, p=0.06; and APACHE II 26–30: 36.7% vs. 48.7%, HR 0.73, 95% CI 0.45 – 1.19, p=0.21). Hospital mortality (40.6% vs. 46.8%, HR 0.83, 95%CI 0.66–1.04, p=0.10) and ICU mortality (31.0 vs. 35.1%, HR 0.87, 95%CI 0.67–1.13, p=0.28) were not significantly reduced among patients who received rhAPC compared to matched controls. The use of rhAPC was not associated with significant differences in ICU or hospital length of stay, ventilator free days, vasopressor free days or the use of acute dialysis.

Conclusion:

In this retrospective, multi-center propensity matched cohort study, the use of rhAPC in patients with septic shock was not associated with statistically significant reductions in 30-day mortality. However, the absolute reduction in 30-day mortality was in similar to that observed in previously published clinical trials. Prospective clinical trials evaluating the efficacy of rhAPC are currently ongoing and will serve to define the role of rhAPC in patients with septic shock.

Disclosures:

Kumar: Pfizer: Research Funding; Astellas: Research Funding; GSK: Research Funding; Roche: Research Funding.
Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,
Authors: Zhang Wei , Dang Suying , Wisniewski Thomas
Published: October 2019

Abstract 3350

HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Mir-130a-Mediated Downregulation of SMAD4 Contributes to Reduced Sensitivity to TGFβ Stimulation in Promyelocytic Cells,
Authors: Hager Mattias , Pedersen Corinna Cavan , Larsen Maria Torp , Andersen Mette Klarskov , Hother Christoffer , Grønbæk Kirsten , Jarmer Hanne , Borregaard Niels , Cowland Jack B
Published: October 2019

Abstract 3383

MicroRNAs (miRNA) are noncoding RNA molecules that regulate the synthesis of proteins and, if dysregulated, can result in development of various form of cancers. We have found that miR-130a is highly expressed in immature proliferating granulocytic precursors. In more mature granulocytes the miR-130a expression is significant lower. In acute myeloid leukemia the granulocyte precursors have lost the ability to undergo terminal maturation, leading to accumulation of non-functional, immature granulocytes (myeloblasts). We hypothesize that a sustained high expression of miR-130a during granulopoiesis may sustain continuous cell proliferation. TGF-β is a strong inhibitor of cell proliferation and lack of TGF-β expression is associated with various form of cancer. Smad4 is an essential compound in the TGF-β signaling pathway. Using microRNA target-prediction software, we identified Smad4 as a putative target for miR-130a. This was confirmed experimentally by demonstrating that transient overexpression of miR-130a results in reduction in the amount of Smad4 protein. Luciferase reporter constructs with the 3`-UTR of Smad4 also respond to miR-130a – an effect that is abolished by point mutations in the miRNA–binding site. In agreement, we observed that stable overexpression of miR-130a in a granulocytic cell line reduces the level of Smad4 protein, and render the cells less sensitive to TGF-β-induced growth inhibition. This was also confirmed with cell cycles analysis. Furthermore, the effect was diminished when transfecting the same clones with SMAD4 lacking the 3-‘UTR. In line with our hypothesis, the most immature granulocyte precursors demonstrate the highest expression of miR-130a is highest, and the lowest expression of Smad4 protein. As the granulocyte precursors mature, the expression of miR-130a decreases dramatically whereas the level of Smad4 protein expression increases demonstrating inverse correlation between miR-130a and Smad4 protein. The level of Smad4 mRNA is comparable at all stages of granulopoiesis. High miR-130a levels and low or no expression of Smad4 was found in primary cells from patients with acute myeloid leukemia and in a cell line (Kasumi-1) with the t(8;21)(q22;q22) chromosomal translocation. The level of Smad4 increased in Kasumi-1 cells when the endogenous level of miR-130a was inhibited by anti-miR-130a LNA. Our data indicate that miR-130a is involved in cell cycle regulation of normal and malignant granulocytic cells through engagement of Smad4 in the TGF-β-pathway. Grant acknowledgment: Lundbeck foundation, Carlsberg foundation, Swedish Research Council.

Disclosures:

No relevant conflicts of interest to declare.
Two Distinct Transactivation Domains of GATA1 Contribute Megakaryopoiesis,
Authors: Kaneko Hiroshi , Kobayashi Eri , Shimizu Ritsuko , Yamamoto Masayuki
Published: October 2019

Abstract 3384

Transcription factor GATA1 orchestrates erythroid/megakaryocytic cell differentiation by coordinately regulating the expression of multiple genes encoding proteins responsible for the proliferation, differentiation and survival of progenitors. Mice lacking GATA1 expression die in utero due to insufficient primitive erythropoiesis. GATA1 retains three functional domains; N-terminus (NT) transactivation domain (TAD) and N-terminus and C-terminus zinc finger domains that are both critical for DNA-binding and interaction with co-factors. We previously showed that each domain of GATA1 confers distinguishable properties to GATA1 during primitive and definitive hematopoiesis in vivo. Especially, GATA1 mutant lacking NT domain (GATA1s) is sufficient to rescue the GATA1-deficient mice from embryonic lethality, but the rescued mice suffered from anemia and thrombocytopenia. Showing very good agreement with this finding, inherited mutation yielding GATA1s was found in a family with dyserythropoiesis and dysmegakaryopoiesis. Whereas previous biochemical studies argued that NT domain was a unique TAD in GATA1, these findings suggest that alternative TAD domain is vital for the GATA1 activity in vivo. In this study, we focus on the C-terminus (CT) region of GATA1. The transactivation activity of GATA1 lacking the CT region (G1-DCT) was significantly reduced to approximately 30% of the wild-type GATA1, which was similar to the GATA1s or NT deletion level. Practically no transactivation activity was found in the GATA1 lacking both NT and CT domains. We also examined transactivation potential of the NT and CT fused with GAL4-DBD. CT-GAL4-DBD fusion protein showed Luciferase reporter activity over 20-fold higher than the GAL4-DBD. Furthermore, retrovirally transduced G1-DCT failed to control the hyperproliferation of megakaryocyte progenitors lacking endogenous GATA1, similar to the salient feature reported for GATA1s. These results thus indicate that the coordinated function of CT- and NT-TADs of GATA1 is required in the megakaryopoiesis. GATA1s mutation was found in majority cases of Down syndrome patients with transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Since CT domain is rich in Ser, Thr and Pro residues, while the NT domain is an acidic transactivation domain rich in Glu, we surmise that GATA1 regulates target gene expression by utilizing two distinct transactivation domains. The imbalance in gene expression in megakaryocytic progenitors, which is caused by GATA1s loosing the function of NT-TAD, may be the key aspect for the understanding of the onset of TMD and AMKL.

Disclosures:

No relevant conflicts of interest to declare.
The Tibetan PHD2 Polymorphism Asp4Glu Is Associated with Hypersensitivity of Erythroid Progenitors to EPO and Upregulation of HIF-1 Regulated Genes Hexokinase (HK1) and Glucose Transporter 1 (SLC2A/GLUT1),
Authors: Lorenzo Felipe R , Swierczek Sabina , Huff Chad Daniel , Prchal Josef T.
Published: October 2019

Abstract 3388

The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. As a high-altitude population, many Tibetans have developed a remarkable ability to protect against several hypoxic complications, including polycythemia and other harmful responses exhibited by non-adapted populations upon exposure to severe hypoxia. We have identified 10 genes involved in high-altitude adaptation in Tibetans, including a principal negative regulator of HIF-1a and HIF-2 a peptides, i.e. PHD2 (EGLN1), as well as HIF2A (EPAS1) (Simonson, Science 2010). At this meeting last year (Lorenzo, Abstract# 2602 ASH 2010), we reported a novel PHD2 Asp4Glu mutation that we found in 57 of 94 Tibetan, 2 of 88 Asian and 0 of 38 Caucasian chromosomes. In most Tibetan samples, this variant is associated with a previously reported, unvalidated PHD2 polymorphism, Cys127Ser (found in 70 of 94 Tibetan, 27 of 88 Asian and 4 of 38 Caucasian chromosomes).

To study the functional consequences of this PHD2 Asp4Glu mutation, we recruited five Tibetan volunteers living in Utah, four of whom were homozygous and 1 heterozygous for PHD2 Asp4Glu and Cys127Ser. We unexpectedly found that homozygotes for the exon 1 PHD2 mutation had markedly hypersensitive erythroid BFU-E (Fig.1) compared to the range of normal controls we have standardized over several decades. Interestingly, erythroid progenitors from individuals with Chuvash polycythemia or a HIF-2a gain-of-function mutation also have hypersensitive BFU-E. To determine if the Tibetan erythroid hypersensitivity data may be explained by increased HIF activity, we have quantified HIF target gene expression in subject granulocytes and found a significant increase in hexokinase (HK1) and glucose transporter (GLUT1/SLC2A) mRNA levels.

These data report the first molecular defect with functional consequences that is associated with the complex Tibetan adaptation to extreme hypoxia.

Disclosures:

No relevant conflicts of interest to declare.
The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,
Authors: He Zhixu , Shu Liping , Yang Xiaoyan , Deng Min , Liu Tingxi
Published: October 2019

Abstract 3413

Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown.

Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish.

Disclosures:

No relevant conflicts of interest to declare.
MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,
Authors: Zhou Beiyan
Published: October 2019

Abstract 3461

The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein.

We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells.

Disclosures:

No relevant conflicts of interest to declare.