bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV-Infected T Cells Induce Granzyme B-Secreting Regulatory B Cells in An Interleukin 21-Dependent Fashion,
Authors: Kaltenmeier Christof , Dahlke Karen , Gawanbacht Ali , Beyer Thamara , Hofmann Stefanie , Härter Georg , Grüner Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: October 2019

Abstract 3222

We and others have recently provided evidence that a series of lymphocyte subsets including, plasmacytoid dendritic cells, B cells and regulatory T cells are able to secrete the cytotoxic serine protease granzyme B (GrB) into the extracellular compartment, where it contributes to the suppression of T cell proliferation by a so far undefined GrB-dependent mechanism. For B cells, we found that viral antigens in the context of the acute phase cytokine interleukin (IL-) 21 can potently induce GrB. Here, we demonstrate that infection of CD4+ T cells with HIV-1 (NL4-3), but not mock infection, induces strong expression of IL-21 on both mRNA and protein levels in CD4+ T cells. Moreover, we found that HIV-infected CD4+ T cells are able to induce high levels of GrB in co-cultured B cells and that inhibition of IL-21 with specific antibodies abrogates T cell-mediated GrB induction in B cells. In support of these data, serum levels of both IL-21 and GrB are significantly higher in patients acutely infected with HIV as compared to healthy control subjects. Surprisingly, co-culture of CD4+ T cells with B cells during HIV-1 infection strongly suppressed both, proliferation of T cells as well as virus replication as indicated by significantly reduced p24 levels in culture supernatants. Notably, this effect was enhanced by external stimulation of B cells with IL-21, and was reduced by inhibition of GrB using specific GrB inhibitors. To further explore the underlying mechanisms of our findings, we performed confocal microscopy of T cell-B cell co-cultures and demonstrated that GrB-secreting B cells directly interact with CD4+ T cells, thereby transferring active GrB to them. Moreover, we found that GrB+ B cells decreased CD4+ T cell expression of the T cell receptor-zeta chain, a known GrB target, which is required for T cell proliferation, and known to be suppressed in HIV patients. In summary, we provide evidence that HIV induces the acute phase cytokine IL-21 in infected CD4+ T cells, thereby indirectly triggering the expression of GrB by B cells. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV, particularly in the early phase of acutely infected patients. Our study reveals a novel pathogenetic mechanism in HIV infection with potential relevance for the development of novel immunotherapeutic approaches.

Disclosures:

No relevant conflicts of interest to declare.
The Therapeutic Effect of Umbilical Mesenchymal Stem Cells Injected Directly Into Medullary Cavity of Ilium to Treat Refractory Aplastic Anemia and Bone Marrow Fibrosis,
Authors: Zheng Zhuanzhen , Qiao Zhenhua , Chen Wenliang , Gong Rong , Wang Yalin , Xu Lianrong , Ma Yanping , Hou Lihong , Zhang Li , Lu Yujin , Jiang Bo , Li Guoxia , Dong Chunxia
Published: October 2019

Abstract 3226

Objective

To evaluate mesenchymal stem cells(MSCs) combination with routine therapy to treat refractory aplastic anemia and bone marrow fibrosis, observe the course of the hematopoitic reconstitution and evaluate the security and curative effect. Methods 12 primary myelofibrosis and 19 chronic aplastic anemia patients who had all accepted routine therapy more than 6 months,because of pathogenetic condition, they needed infusing constituent blood such as at least condensed red blood cells 6 units and platelet 4 units per months, and despite this, whole blood cells counts were very lower. Those patients all had intermittent dizzy, fatigue, bad appetite and purpura. Because of age, complication, and psychological reasons, they refused Allogenic stem cell transplantation. So besides routine therapy, they received umbilical MSCs 1×107, every two weeks one time, those cells were injected directly into medullary cavity of ilium. Results 28 recipients showed hematopoietic reconstitution when PLT was up to 20×109/L and granulocytes was up to 2.0×109/L on day 28 post MSCs infusion. Bone marrow biopsy showed hyperplasia from hypoplasia to evident hyperplasia. On day 28, Th cells ascended from 23.0 to 36.2, Ts cells descended from 24.2 to 14.6, Th.. MTs from 0.95 to 2.47:1, IgG from 6.6 to 13.3, NK cells from 2.40 to 13.5, indicating earlier immune recorrect. At the last follow-up of 12 months, 17 patients showed normal hemoglobin and platelet counts, immune globulin was in the normal level, indicating the hematopoietisis and immune persistent reconstitution. 5 patients still needed infusing constituent blood cells, although, the interval of platelet and red blood cells infusion obviously prolonged, and the times to see doctor decreased and life quality elevated greatly. Conclusion This is a novel clinical research of successful treatment of refractory CAA and MF, it tell us infusion HLA-mismatched allogenic MSCs directly into medullary cavity of ilium is efficiency and security.

Disclosures:

No relevant conflicts of interest to declare.
Immunosuppressive Drugs Upregulate the Immunoinhibitory Receptor Osteoactivin in Monocyte-Derived Dendritic Cells,
Authors: Schwarzbich Mark-Alexander , Gutknecht Michael , Guettler Lisa , Kanz Lothar , Salih Helmut R. , Gruenebach Frank , Rittig Susanne Malaika
Published: October 2019

Abstract 3232

Osteoactivin, also known as transmembrane glycoprotein NMB (GPNMB) and dendritic cell-associated transmembrane protein (DC-HIL), is a type I transmembrane glycoprotein that is detected abundantly in dendritic cells (DC), but not or only at low levels in monocytes. Its expression on antigen-presenting cells (APC) can inhibit T cell activation by binding the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells. We here studied the influence of different therapeutically used immunosuppressive agents such as the corticosteroid prednisolone and the calcineurin inhibitors tacrolimus or cyclosporine A on the expression of osteoactivin in human monocyte-derived DC. Therefore, DC were generated from blood monocytes isolated by plastic adherence and exposed to GM-CSF and IL-4. Prednisolone, tacrolimus or cyclosporine A were added to the culture medium at concentrations obtained upon clinical application in patients (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml) every second day starting from the first day of culture. Cells were harvested for immunophenotyping and osteoactivin expression analysis by immunostaining, western-blotting and real-time RT-PCR or for functional analyses in mixed lymphocyte reactions (MLR) on day 7 of culture. DC generated in the presence of therapeutic concentrations of the immunosuppressant drugs showed a reduced expression of CD1a. Furthermore, we observed a clear upregulation of osteoactivin on DC that had been treated with the immunosuppressive agents. In line with osteoactivin up-regulation, exposure to the immunosuppressive drugs resulted in reduced stimulatory capacity of the DC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. In summary, our results demonstrate that upregulation of osteoactivin is critically involved in the inhibition of DC function upon exposure to different classes of immunosuppressive agents. These findings suggest that upregulation of osteoactivin in DC constitutes a ubiquitous mechanism that may underlie inhibition of T cell activation by these drugs.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Generation Under Shear Force Is Modulated by Site-Specific Phosphorylation of Myosin-II Heavy Chain,
Authors: Spinler Kyle R , Shin Jae-Won , Discher Dennis E
Published: October 2019

Abstract 3262

Platelets are generated from megakaryocytes (MKs) under blood shear. When activated, platelet aggregration and contraction result in part from non-muscle myosin-IIA (NMM-IIA), which is the most abundant isoform of MKs. Defects in this motor protein are a common basis of May-Hegglin disease in which thrombocytopenia is a common symptom. Recently, we showed that reversible inhibition of NMM-IIA – rather than irreversible perturbations – together with soft 2D matrix increases polyploid MKs and also platelet generation (Shin et al., PNAS, 2011; 108:11458–63). While we identified specific phosphorylation sites in NMM-II heavy chain that can modulate MK polyploidization physiologically, it remained to be investigated whether the same sites can also influence platelet generation and activation. Since we showed previously that cytoskeletal proteins undergo forced disruption of specific domains and specific interactions within shear-stressed cells (Krieger et al., PNAS, 2011; 108:8269–74), it is possible that NMM-II heavy chain phosphorylation sites are also exposed under physical forces. Here, intravenous blood shear forces simulated by a cone and plate rheometer are shown to induce the de-activating and isoform-specific phosphorylation at Ser1943 (pS1943) of NMM-IIA in MKs. MEG01 cells show that the fraction of pS1943 NMM-IIA increases up to a low shear stress (0.5Pa), while further increases in shear stress lead to cell fragmentation and a decay in pS1943. Mutational analysis of this site in MEG01 cells together with knockdown and overexpression addresses whether pS1943 is both sufficient and necessary for cellular fragmentation under shear. The role of pS1943 in platelet activation will also be discussed in relation to May-Hegglin disease.

Disclosures:

No relevant conflicts of interest to declare.
CCR2 and CCR5 Co-Expressing CD11b+ Monocytes Affect Neovascularization in An Ischemic Hind Limb Mouse Model,
Authors: Kim Jeong-A , Noh Kyoungmi , Kim Hyunkyung , Broxmeyer Hal E.
Published: October 2019

Abstract 3270

Monocyte/myeloid -lineage cells are known to be involved in neovascularization. However, the specific monocyte /myeloid population that participates in neovascularization has not been clearly identified. Several subsets of monocyte/myeloid cells have been proposed to be important for this process. For example, CD11b+CXCR1hiCCR5+ resident monocytes regulate tissue healing by increasing angiogenesis, and CD11b+CXCR1loCCR2+ inflammatory monocytes are mobilized to the inflammation site. Interestingly, we found that CD11b+ myeloid cells isolated from ischemic muscle of post-femoral artery dissected C57BL/6 mice are positive for both CCR2 and CCR5. To study the relationship between CD11b+CCR2+CCR5+ monocytes and neovascularization in an ischemic hind-limb model of C57BL/6 mice, we addressed the following questions:(1) Are the number of tissue-resident CD11b+CCR2+CCR5+ monocytes increased in ischemic muscle, post-ischemic injury?; (2) Are CD11b+CCR2+CCR5+ monocytes recruited to the ischemic muscle and do they incorporate into the blood vessels in the ischemic site?; (3) Do CD11b+CCR2+CCR5+ monocytes modulate neovascularization in ischemic hind-limbs of C57BL/6 mice upon direct injection of these cells into ischemic muscle. Results demonstrate that numbers of CD11b+CCR2+CCR5+ monocytes markedly increase in ischemic muscle by day 4 after the surgery (n=5 each; 0.8±0.4×105 cells/g tissue in non-surgically treated mice vs 9.5±1.7×105 cells/g tissue in the ischemic muscle, p<0.05) then return to basal levels at day 21. Following immunostaining of ischemic muscles, CD11b+CCR2+CCR5+ monocytes localize in perivascular areas of non-surgically treated C57BL/6 mice. At day 4 after the surgery, these cells infiltrate into ischemic muscles. Infiltrated CD11b+CCR2+CCR5+ cells make numerous vessel-like structures 21 days after the surgery. Following direct injection of purified CD11b+CCR2+CCR5+ monocytes from ischemic muscle at day 4, an increase in blood flow was manifest by 28 days in the CD11b+CCR2+CCR5+ monocyte-injected group (n=7 each, 67.6±5.7% in the CD11b+CCR2+CCR5+ monocyte-injected group vs 39.9±6.7% in the control group, p<0.05). Thus, we found that CD11b+CCR2+CCR5+ monocytes infiltrated and formed numerous vessel-like structures in the ischemic muscle, and additional injection of CD11b+CCR2+CCR5+ monocytes increased the blood flow. These findings strongly suggest that CD11b+CCR2+CCR5+ monocytes play an important role in neovascularization.

Disclosures:

No relevant conflicts of interest to declare.
Splenectomy and the Incidence of Venous Thromboembolism and Sepsis In Patients with Immune Thrombocytopenia,
Authors: Wun Ted , Boyle Soames F. , Brunson Ann , White Richard H.
Published: October 2019

Abstract 3284

Introduction:

Splenectomy is efficacious for many patients with chronic immune thrombocytopenia (ITP) refractory to initial corticosteroid therapy. However, there are concerns about short and long term complications, especially venous thromboembolism (VTE) and sepsis. Information regarding the incidence and risk factors for these complications might inform therapeutic decisions.

Methods:

Using the California Office of Statewide Health Planning and Development Patient Discharge Dataset, we identified all the records of Californians discharged with a diagnosis of ITP from 1991–2009. Similarly, all records with a procedure code for splenectomy, or principal or secondary diagnosis of VTE or sepsis were identified. The resultant datasets were then merged with the ITP dataset so that incident splenectomy, VTE, and sepsis could be determined for the ITP cohort.

Results:

A total of 37,780 unique patients were initially identified with an ITP code, of which 13,798 were in the principal position. Records where the first ITP code was on the same date as a splenectomy were then excluded as we wished to study a cohort of ITP cases that warranted admission but may or may not have undergone splenectomy later. This resulted in 10,752 cases that constitute the ITP analysis cohort. This cohort was 60% female, 57% White, 23% Hispanic, 9% Asian, and 8% African-American with a median age of 54 years. One thousand nine hundred fifty-five ITP patients underwent splenectomy subsequent to their first ITP hospitalization; 8,797 did not. The cumulative incidences of VTE were 4.4% and 1.8%, and for sepsis were 7.9% and 6.7%, for patients who did and did not undergo splenectomy, respectively. Cox proportional hazard models for VTE and sepsis are given in the tables.

Discussion:

In this large cohort of cases with ITP, splenectomy was associated with hazard ratios (HR) of 2.6 to 4.8 for VTE and a HR of 8.2 in older patients for sepsis, confirming the results of smaller studies. Age and diagnosis of lupus also increased the risk of VTE. Asians had a lower risk of VTE, consistent with previous findings. The absolute risk of VTE was < 5% and would not seem to warrant long-term primary prophylaxis. Age, lupus, and co-morbidities also increased the risk of sepsis in patients with ITP. The increased risk of sepsis in Asians is unexplained. Of note, the cumulative incidence of sepsis in patients that did not undergo splenectomy was still considerable and may have reflected the use of immunosuppressive therapy.

Disclosures:

Wun: Glycomimetics, Inc.: Research Funding; Eli Lilly, Inc.: Research Funding.
A Novel Missense Mutation Asp69Gly in the Platelet Glycoprotein Ib(beta) Gene in Two Families with Bernard-Soulier Syndrome,
Authors: Hassenpflug Wolf A , Weber Belinda , Obser Tobias , Budde Ulrich , Schneppenheim Reinhard
Published: October 2019

Abstract 3300

Bernard-Soulier Syndrome (BSS) is characterized by macrothrombocytopenia and impaired platelet function due to defects of the GpIb-IX-V complex. Patients with Bernard-Soulier Syndrome are rare and only few disease causing mutations have been identified in the platelet gylcoprotein Ib(alpha), Ib(beta) or glycoprotein IX genes, respectively.

We characterized 2 independent families with Bernard-Soulier Syndrome. Affected members of both families presented with a bleeding history including gross hematoma after minimal trauma, recurrent epistaxis, menorrhagia and prolonged bleeding after surgery. Routine laboratory tests showed reduced platelet numbers and increased platelet volume. Consistent with the diagnosis of Bernard-Soulier syndrome FACS analysis demonstrated reduced GpIb-IX expression on the platelet surface. Platelet agglutination in the presence of ristocetin (RIPA) was impaired.

Using standard techniques we sequenced the GpIb(alpha), GpIb(beta) and GpIX genes. We identified a novel mutation Asp69Gly in the GpIb(beta) gene in both families. The mutation was homozygous in two children of the first family and one child of the second family. As expected, the parents of the affected index patients were heterozygous mutation carriers. To further investigate the impact of the Asp69Gly mutation on GpIb expression we used structure prediction software and homology-based protein modeling. Asp69Gly lies within an extracellular region flanking a leucin-rich element and cysteine-cluster. It affects the highly conserved center aspartic acid of a CDC motif that is thought to form one of two cystein-loops close to the transmembrane domain. We propose that this mutation disrupts the regular conformation of the cystein loop, potentially leading to abnormal protein folding and protein decay in the endoplasmic reticulum.

In addition to a mere defect of biosynthesis our data hint at additional functional consequences of the Asp69Gly mutation. The father and the mother of the index patient in the first family, who are heterzygous for the Asp69Gly mutation, express only slightly reduced levels of platelet GpIb-IX based on FACS analysis. Nevertheless both of them suffer from epistaxis and menorrhagia, respectively. Expression studies are underway to clarify the influence of the Asp69Gly mutation on GpIb expression and function. Preliminary data show reduced cell surface expression of the GPIb-IX complex and reduced binding to von Willebrand-factor (VWF).

While the functional and structural effects of mutations in the GpIb(alpha) and GpIX genes have been thoroughly studied, the impact of mutations in the GpIb(beta) gene is less clear. Our results could help to elucidate how the GpIb(beta) protein contributes to the assembly and surface expression of the GpIb-IX-V complex.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Mediators and Markers in Sepsis Associated Coagulapathy,
Authors: Hoppensteadt Debra , Cunanan Josephine , Khan Hussein , Litinas Evangelos , Sharma Gautam , Kaul Inder , Fareed Jawed
Published: October 2019

Abstract 3341

Thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), fibrinopeptide A (FPA) and thrombin antithrombin complex (TAT), along with tissue factor (TF), microparticles (MP), D-dimer (DD) and antithrombin (AT). Baseline plasma samples from one hundred patients diagnosed with sepsis associated DIC were analyzed for various parameters by using ELISA methods. The MP levels were measured by using a functional method utilizing annexing trapping whereas the antithrombin was measured using both the functional and immunologic methods. Plasma samples from 50 normal male and female individuals comprised the control group. The results are tabulated in the following table. Both TF and MP levels were markedly elevated in comparison to the control group. All of the other markers of thrombin generation showed elevated levels F1.2 (3.4 fold), FPA (4.3 fold) and TAT (4.0 fold). Marked elevation of D-dimer was also noted (5.0 fold). Both the immunologic and functional levels of AT were also decreased. While the data was widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes such as the antithrombin, thrombomodulin and TFPI may play an important regulatory role in the pathogenesis of this syndrome.MarkerNormal ControlsDIC BaselineF 1.2 (pM)108.6+12.3473.7+320FPA (ng/ml)1.6+0.86.8+3.1MP (nM)8.47+1.124.56+2.1AT (% NHP)94.2+2.780.4+3.1DD (ng/ml)415.4+65.23685+112TAT (ng/ml)3.4+0.317.0+3.1

Disclosures:

No relevant conflicts of interest to declare.
Non-Muscle Myosin II Mediates Microparticle Release From Endothelial Cells Activated by Antiphospholipid Antibodies,
Authors: Betapudi Venkaiah , Hsi Linda , Allen Kristi L , Willard Belinda , McCrae Keith R.
Published: October 2019

Abstract 3343

The antiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLA). The majority of pathogenic APLA are actually directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. Microparticles are small phospholipid-rich vesicles that may express tissue factor and are thought to play an important role in thrombosis. Increased release of microparticles may be stimulated by cellular activation or apoptosis, and previous work from our laboratory and others has demonstrated increased levels of circulating microparticles in patients with APS, even far removed from thrombotic events. Though several mechanisms have been proposed for mediating release of microparticles from cells, this process is poorly understood and there is little information available concerning the mechanisms of microparticle release from endothelial cells stimulated by APLA/anti-β2GPI antibodies. To address this issue we assessed the role of non-muscle myosin II motor protein in mediating the release of microparticles from endothelial cells activated by anti-β2GPI antibodies. We observed that incubation of endothelial cells with β2GPI and anti-β2GPI antibodies caused significant increases in the release of microparticles compared to cells treated with β2GPI and control antibodies. Proteomic and biochemical studies demonstrated that anti-β2GPI antibodies caused increased phosphorylation of endothelial cell Myosin II light chain (MLC), an essential step in the activation of Myosin II motor protein. Moreover, examination of endothelial cells stimulated by anti-β2GPI antibodies using confocal microscopy revealed a dramatic increase in the formation of actin-myosin II filaments. To assess the effects of these changes on microparticle release, we inhibited MLC phosphorylation in anti-β2GPI-treated endothelial cells using ML-7, a highly specific inhibitor of MLC. ML-7 caused a significant decrease in the release of microparticles from anti-β2GPI-treated cells. In conclusion, our results demonstrate that non-muscle myosin II motor protein activation by anti-β2GPI antibodies is necessary for the increased release of microparticles from activated cells. These microparticles may play important roles in the propagation of thrombosis and other clinical sequelae observed in patients with APS.

Disclosures:

No relevant conflicts of interest to declare.
Zebrafish Growth Factor Independence Transcription Factors Establish a New Paradigm for Regulation of Primitive and Definitive Hematopoietic Lineages,
Authors: Cooney Jeffrey D. , Shafizadeh Ebrahim , McBride Paul F. , Carroll Kelli J. , Anderson Heidi , Ganis Jared J. , North Trista E. , Paw Barry H.
Published: October 2019

Abstract 3379

The Growth Factor Independence (Gfi) zinc finger transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic lineage specification. In mammals, Gfi1 regulates hematopoietic stem cell (HSC) and lymphoid populations, while Gfi1b is required for megakaryocyte and erythroid development (van der Meer, et al. 2010 Leukemia 11:1834–43). In zebrafish, gfi1.1 plays an essential role in primitive hematopoiesis, preserving primitive HSC populations and regulating the erythroid-myeloid balance (Wei, et al. 2008 Cell Res. 6:677–85). However, little is known about the role of gfi1.1 in definitive hematopoiesis or about the role of additional hematopoietic gfi family members in zebrafish. Here, we report the isolation and characterization of an additional zebrafish gfi family transcription factor, gfi1.2b. We compare and contrast gfi1.1 and gfi1.2b, showing that they are highly expressed in the intermediate cell mass (ICM) and aorta-gonad-mesonephros (AGM), the respective sites of primitive and definitive hematopoiesis in zebrafish. Using antisense morpholino oligos (MO), whole mount in situ hybridization (WISH) and fluorescent activated cell sorting (FACS) of transgenic reporter fish, we demonstrate that gfi1.1 and gfi1.2b have distinct, essential roles in preserving primitive and definitive HSC populations, respectively. Loss of gfi1.1 specifically silences expression of scl and gata-1, markers of primitive HSC and erythroid progenitors. Conversely, loss of gfi1.2b silences expression of Tg(cd41:eGFPlo) cells, indicating an essential role for gfi1.2b in preserving definitive hematopoietic progenitors (Ma, et al. 2011 Blood 118:289–297). Consistent with the discrete roles of gfi1.1 and gfi1.2b in primitive and definitive lineages, knockdown of gfi1.2b silences lymphocyte rag-1 expression in the developing thymus, while knockdown of gfi1.1 has no effect on the thymic lymphocyte population. gfi1.1 and gfi1.2b have overlapping roles in erythropoiesis, as loss of either gfi factor reduces erythrocyte populations, while loss of both gfi paralogs results in a more profound silencing of erythrocytes. We further demonstrate that loss of gata-1 reduces gfi1.1 expression and silences gfi1.2b, suggesting that gata-1 plays an essential role in regulating the transcription of both genes. Together, these studies demonstrate that gfi1.1 and gfi1.2b have distinct and overlapping roles in zebrafish hematopoiesis and establish a new paradigm for the regulation of primitive and definitive hematopoietic lineages by gfi transcription factors.

Disclosures:

No relevant conflicts of interest to declare.
Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,
Authors: Zhao Xinyang , Vu Ly P. , Perna Fabiana , Liu Fan , Xu Hao , Nimer Stephen
Published: October 2019

Abstract 3403

RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases.

Disclosures:

No relevant conflicts of interest to declare.
Membrane-Bound Human Stem Cell Factor Promotes Differentiation of Human Granulocytes and Mast Cells In Vivo,
Authors: Takagi Shinsuke , Saito Yoriko , Hijikata Atsushi , Tanaka Satoshi , Watanabe Takashi , Koseki Haruhiko , Ohara Osamu , Saito Takashi , Taniguchi Shuichi , Shultz Leonard , Ishikawa Fumihiko
Published: October 2019

Abstract 3419

Recently, advances in xenograft models for human hemamtopoietic stem cells (HSCs), or the humanized mice, have begun to allow investigators to examine the differentiation of human hematopoietic and immune cells in vivo. However, lymphoid-skewed human hematopoietic development in the mouse bone marrow is one of the remaining limitations in the humanized mouse models. The inefficient human myeloid development could at least partly be attributed to the mouse microenvironment not fully supporting differentiation and maturation of human myeloid lineage. To overcome this problem, we focused on the role of membrane-bound human stem cell factor in supporting the maintenance of human HSCs and inducing the development of human myeloid cells and created human stem cell factor transgenic NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. Transplantation of 5000–50000 cord blood-derived Lin-CD34+CD38- cells resulted in significantly higher engraftment of human CD45+ leukocytes at 3–6 months post-transplantation in the bone marrow, spleen, and peripheral blood of hSCF Tg NSG recipients compared with those of non-transgenic NSG recipients. The enhanced human CD45+ engraftment was most prominent in the bone marrow (hSCF Tg recipients: 98.0 +/− 1.3%, n= 15, non-Tg NSG controls: 75.3 +/− 7.3%, n=7). In the bone marrow, the frequency of human CD33+ myeloid cells within the total human CD45+ population was significantly higher in the hSCF Tg NSG recipients than in the non-Tg NSG recipients and constituted the majority of human hematopoietic cells (hSCF Tg recipients: 54.6 +/− 4.5%, n=15 and non-Tg NSG controls: 29.3 +/− 4.0%, n=7). Flow cytometric analysis demonstrated that the majority of engrafted human myeloid cells in the hSCF Tg recipient bone marrow were side-scatter high, HLA-DR negative granulocytes. Reflecting the effect of human SCF on the development of human mast cells, human c-Kit+CD203c+ mast cells were identified in the bone marrow, spleen, and gastrointestinal tracts of the hSCF Tg NSG recipients. Altogether, the in vivo humanized mouse model demonstrates the essential role of membrane-bound SCF in human myeloid development. The hSCF Tg NSG humanized mice may facilitate the in vivo investigation of human HSCs, myeloid progenitors and mature myeloid lineage.

Disclosures:

No relevant conflicts of interest to declare.
Outcomes in Children with Severe Aplastic Anemia Receiving Bone Marrow Transplantation from an HLA-Matched Family Donor or Intensive Immunosuppressive Therapy As First-Line Treatment,
Authors: Yoshida Nao , Kikuchi Akira , Kobayashi Ryoji , Yabe Hiromasa , Kosaka Yoshiyuki , Muramatsu Hideki , Takahashi Yoshiyuki , Kato Koji , Suzuki Ritsuro , Ohara Akira , Kojima Seiji
Published: October 2019

Abstract 3421

Bone marrow transplantation (BMT) from an HLA-matched family donor (MFD) is the treatment of choice for severe aplastic anemia (SAA) in children. For children without an MFD, immunosuppressive therapy (IST) with a combination of antithymocyte globulin and cyclosporine has been successful. However, this treatment approach is based on the results of comparative studies between these therapies conducted in the 1980s, and the outcomes of both BMT and IST have improved markedly over the past three decades. Therefore, updated evidence for treatment decisions in pediatric SAA is required. In the present study, we compared the outcomes of children with SAA who received IST (subjects enrolled in the prospective multicenter trials of IST conducted by the Japan Childhood Aplastic Anemia Study Group) or BMT from an MFD (subjects registered in the Transplant Registry Unified Management Program conducted by the Japan Society for Hematopoietic Cell Transplantation). The influence of potential risk factors on overall survival (OS) and failure-free survival (FFS) was assessed according to first-line treatment, time period of treatment (1992–1999 and 2000–2009), age and other variables related to each treatment. FFS was defined as survival with treatment response. Death, primary or secondary graft failure, relapse and secondary malignancy were considered treatment failures in patients who received BMT. Death, relapse, disease progression requiring stem cell transplantation from an alternative donor or 2nd IST, clonal evolution and evolution to paroxysmal nocturnal hemoglobinuria were considered treatment failures in patients who received IST. Between 1992 and 2009, 599 children with SAA younger than 17 years received BMT from an MFD (n=213) or IST (n=386) as first-line treatment. While the OS did not differ between patients receiving IST and BMT (88±2% vs. 90±2% at 15 years), FFS was significantly inferior in patients receiving IST as compared to those receiving BMT (54±3% vs. 84±3% at 15 years, P<0.0001). There was no significant improvement in outcomes over the two time periods; OS and FFS at 10 years in 1992–1999 vs. 2000–2009 were 87±2% vs. 93±2% and 66±3% vs. 67±3%, respectively. On multivariate analysis, age <10 years was identified as a favorable factor for OS (P=0.007) and choice of first-line IST was the only unfavorable factor for FFS (P<0.0001). These updated data support the current algorithm for treatment decisions, which recommends BMT when an MFD is available.

Disclosures:

No relevant conflicts of interest to declare.
Beneficial Effects of Nucleoside Treatment in Zebrafish Models of Diamond Blackfan Anemia Deficient in Ribosomal Proteins L11 or S19,
Authors: Danilova Nadia , Sakamoto Kathleen M , Lin Shuo
Published: October 2019

Abstract 3439

Deficiency of ribosomal protein (RPs) is associated with Diamond Blackfan Anemia, (DBA) a congenital syndrome with bone marrow failure and variable malformations. Recent studies of ours and other labs established p53 network activation as a major contributor to DBA. Therefore, modulation of p53-dependent pathways emerges as a most promising approach to DBA treatment. Direct suppression of 53 may, however, lead to increased risk of malignant transformation. Search for more physiological modulators of p53 pathways is therefore warranted. p53 has been shown both to respond to metabolic alterations and to change metabolism. Specifically, nutrients can influence p53 status through mTOR and other pathways. Recently, Ortega et al reported that treatment of Caco-2 cells with exogenous nucleosides modulated expression and activity of many transcription factors including p53 (J Nutr Biochem, 22:595, 2011). We applied treatment with exogenous nucleosides to RP-deficient zebrafish and found positive effects. As a DBA model, we use zebrafish mutant for ribosomal protein RPL11; we also employ morpholino nucleotides to create deficiency of various ribosomal proteins including RPS19. Treatment of both models resulted in inhibition of p53-dependent pathways. Specifically, expression of genes associated with cell cycle arrest such as p21 and p53 targets inducing apoptosis such as puma, was decreased by nucleoside treatment. We hypothesize that nucleoside treatment affects p53 by modulating the activity of mTOR and MEK/Erk pathways. Previously, Gu et al. showed that nucleotide depletion strongly inhibited mTOR and activated MEK/Erk. We also study how nucleoside treatment influences other stress-response pathways and affects interaction of p53 and mTOR pathways. Our data indicate that nucleosides may be effective in modulation of p53-dependent signal transduction pathways and suggest their usefulness for treatment of p53-associated disorders.

Disclosures:

Sakamoto: Abbott: Research Funding; Genentech: Research Funding.
Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation
Authors: Santos Daniel Garcia , Schranzhofer Matthias , Chies José Artur Bogo , Ponka Prem
Published: October 2019

Abstract 344

Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Disclosures:

No relevant conflicts of interest to declare.
Haploinsufficiency of Dnmt1 Impairs Leukemia Stem Cell Function Through Derepression of Bivalent Chromatin Domains,
Authors: Trowbridge Jennifer J. , Sinha Amit U. , Armstrong Scott A. , Orkin Stuart H.
Published: October 2019

Abstract 3459

Leukemia stem cells (LSCs) are an attractive target in treatment of many types of blood cancers. There remains an incomplete understanding of the epigenetic mechanisms driving LSC formation and maintenance, and how this compares to the epigenetic regulation of normal hematopoietic stem cells (HSCs). One of the major epigenetic modifications, DNA methylation, is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed decreased expression of Dnmt3a and Dnmt3b in LSCs isolated from a model of MLL-AF9-induced acute myeloid leukemia (AML) compared to normal HSCs. In contrast, expression of Dnmt1 was maintained in LSCs compared to HSCs, suggesting that Dnmt1 may have a critical function in the formation and maintenance of LSCs. Supporting this hypothesis, we found that conditional knockout of Dnmt1 fully ablates the development of AML. Furthermore, haploinsufficiency of Dnmt1 (Dnmt1fl/+ Mx-Cre) was sufficient to delay progression of leukemogenesis and impair LSC self-renewal. Strikingly, haploinsufficiency of Dnmt1 did not functionally alter normal hematopoiesis or HSCs, suggesting an enhanced dependence of LSCs on DNA methylation. Mechanistically, we observed that haploinsufficiency of Dnmt1 in LSCs resulted in derepression of genes that had been silenced by MLL-AF9-mediated transformation and marked by bivalent H3K27me3/H3K4me3 chromatin domains. These results suggest that the formation and maintenance of LSCs depends not only upon activation of a leukemogenic program, but also upon silencing of a specific gene signature that is active in HSCs through crosstalk between two epigenetic mechanisms, polycomb-mediated repression and DNA methylation-mediated repression. This silenced gene signature includes known and candidate tumor suppressor genes as well as genes involved in lineage restriction. These studies present evidence that distinct epigenetic regulatory mechanisms are dominant in LSCs compared to HSCs and provide novel gene candidates for targeted reactivation in AML therapy.

Disclosures:

Armstrong: Epizyme: Consultancy.
Predictors of Outcome In Adult Patients with Acute Myeloid Leukemia In First Relapse,
Authors: Ravandi Farhad , Faderl Stefan , Pierce Sherry , Garcia-Manero Guillermo , Brandt Mark , Shan Jianqin , Kadia Tapan , Borthakur Gautam , Estrov Zeev , o'Brien Susan , Cortes Jorge E. , Kantarjian Hagop M.
Published: October 2019

Abstract 3569

Treatment of patients with acute myeloid leukemia (AML) in first relapse has remained challenging and although relatively high response rates have been reported using selected combination chemotherapy regimens, long term leukemia-free survival has been less than 20% in most studies. A number of predictors of outcome have been reported of which the most significant are duration of first CR (CR1), cytogenetics at diagnosis, age at relapse, and whether allogeneic stem cell transplant (SCT) was performed in CR1 (Breems, DA, JCO, 2005). We sought to examine predictors of outcome after first relapse of AML incorporating some of the more recent relevant covariates such as molecular data, when available. From January 2000 to March 2010, 447 patients with AML (excluding acute promyelocytic leukemia) in first relapse were seen at the University of Texas – MD Anderson Cancer Center (MDACC) and have available survival data; 275 received therapy at MDACC and 66 (24%) achieved a second CR (CR2), with 14 (5%) patients achieving CR without platelet recovery (CRp2). For the overall population, the median survival from relapse is 5 months (range, 1 to 103+) and the median follow-up in surviving patients is 34.3 months (range, 5 to 103+). On univariate analysis, the following factors predicted survival in first relapse: the duration of CR1 (p<0.001), unfavorable cytogenetics at diagnosis (p<0.001) and having NPM1+/FLT3- status at diagnosis (p=0.001), as well as the following variables at relapse: age (p< 0.001), WBC (p<0.001), platelets (p=0.005), peripheral blood blast % (p<0.001), bone marrow blast % (p<0.001), performance status (p<0.001), creatinine (p<0.001), LDH (p<0.001) and albumin (p<0.001). Having FLT3-ITD at diagnosis and undergoing allogeneic stem cell transplant in CR1 were of borderline significance (p value for both =0.08). On multivariate analysis, taking into account only variables with a p<0.1, duration of CR1, age at relapse, unfavorable cytogenetics at diagnosis, and LDH as well as bone marrow blast % at relapse were significant (p<0.001). Positive FLT3-ITD at diagnosis was also significant (p=0.04). When multivariate analysis was conducted in the subset of 121 patients with complete FLT3 and NPM1 mutational status at diagnosis, neither the NPM1+/FLT3- status, nor having FLT3-ITD at diagnosis remained significant. In conclusion, we confirm that the most important predictors for survival in first relapse are duration of CR1, age at relapse, unfavorable cytogenetics, and possibly FLT3-ITD at diagnosis, as well as percentage of bone marrow blasts and LDH at relapse.

Disclosures:

No relevant conflicts of interest to declare.
A Meta-Analysis of CAG (cytarabine, aclarubicin, G-CSF) Regimen for the Treatment of 1045 Patients with Leukemia in China and Japan,
Authors: Wei Guoqing , Ni Wanmao , Chiao Dicky , Huang He , Cai Zhen , Liu Delong
Published: October 2019

Abstract 3638

Background:

CAG regimen (cytarabine, aclarubicin, G-CSF) has been commonly used in China and Japan for the treatment of AML and MDS. This study is to summarize the data and to analyze the efficacy as well as the toxic effects of CAG regimen in acute leukemia (AL) and MDS pts.

Methods:

The databases of PubMed, Wanfang Data, as well as American Society of Hematology (ASH) annual meeting abstracts were searched for articles published in English, Chinese and Japanese languages between January 1995 and December 2010. Eligible studies were relevant clinical trials on AL and MDS pts treated with CAG regimen. Complete remission (CR) rates and odds ratio (OR) were compared through a meta-analysis using a random-effects or fixed-effects model.

Results:

37 trials with a total of 1045 AL and MDS pts were included for analysis. Among the 1045 pts treated with CAG, 819 pts were AML, 215 pts were de novo MDS or transformed AML (MDS/tAML), 6 pts were ALL, and 5 pts were biphenotypic acute leukemia (BAL). The AML CR rate of CAG from 29 studies was 58.0% (95% CI, 53.1%-62.7%). The MDS/t-AML CR rate from 12 studies was 45.7% (95% CI, 39.0%-52.4%). The AML CR rate was significantly better than that of MDS /tAML (Q=8.072, p<0.01). Among 819 AML pts, 327 pts were newly diagnosed, 370 pts were relapsed/refractory (R/R) AML. The AML status was not specified in the rest 122 pts. Interestingly, no significant difference in CR rates was noted between the newly diagnosed (57.0%, 95% CI 51.5%-62.3%) and R/R AML pts (60.1%, 95% CI 50.5%-68.9%) (Q=0.312, p>0.05). The CR rate for the 367 elderly AML pts was 52% (95% CI 51.5%-62.3%). The CR rate was also significantly higher in pts with favorable (64.5%, 95% CI 38.8%-83.9%) and intermediate (69.6%, 95% CI 60.4%-77.5%) cytogenetics than those with unfavorable one (29.5%, 95% CI 19.7%-41.8%) (p<0.05). There were 7 trials that compared the CR rates of CAG regimen with those of other induction regimens in AML pts. Surprisingly, the CR rate of CAG was significantly higher than those of other induction regimens (OR 2.425, 95% CI, 1.515–3.880). CAG regimens were well tolerated with cardiotoxicity in 0.42% cases (4/954) and early death occurred in 4.40% cases (44/1000).

Conclusions:

CAG regimen induced significantly higher CR rates in AML than in MDS pts. The CR rates of CAG regimen was significantly better than those of other induction regimens in AML pts. This regimen was well tolerated with low cardiotoxicity and early death rate.

Disclosures:

No relevant conflicts of interest to declare.