bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacodynamics of CNTO 530 in a Murine Model of B-Thalassemia
Authors: Makropoulos Dorie A , Capocasale Renold J , Achuthanandam Ram , Wilson Krista , Volk Amy , Bugelski Peter 
Published: October 2019

Abstract
CNTO 530 is an erythropoietin (EPO) receptor agonist that links two EMP-1 peptides to a MIMETIBODYä scaffold. Here, we characterize the in vivo efficacy of CNTO 530 in a murine model of b-Thalassemia. Hematological analysis confirmed dysregulated erythropoiesis in these mice with decreased red blood cells (RBC), hemoglobin (Hgb), and hematocrit (Hct) indices compared to age matched C57BL/6 littermates. An increase in reticulocytes was also observed concurrent with decreased mean cell volume (MCV) and increased red cell distribution width (RDW) suggesting compensatory erythropoiesis. In addition, splenomegaly was observed consistent with increased hematopoiesis. To test the effectiveness of CNTO 530 in this model, a timecourse study was done comparing treatment with CNTO 530 to darbepoetin-a. A single subcutaneous (s.c.) dose of CNTO 530 at 10,000 U/kg dose stimulated a long-lived, approximately 4 g/dL increase in hemoglobin compared to the less than 1 g/dL, short lived increase in Hgb observed in response to darbepoetin-a at 10,000 U/kg. Mice dosed with CNTO 530 also showed an increase in RBC, Hct, and MCV where darbepoetin-a had no significant effect. Non-thalassemic littermates were also given a single s.c. dose of 10,000 U/kg of CNTO 530 or darbepoetin-a which showed these drugs indeed are efficacious in normal background animals. These results demonstrate that CNTO 530 caused a potent, reproducible and longer-lived response in Hgb than darbepoietin in a model of b-thalassemia.
A Novel Subset of CD8+, CD62L-High Tumor Infiltrating Lymphocytes (TIL) Exhibits Regulatory Function
Authors: Plautz Gregory , Wang Li-Xin 
Published: October 2019

Abstract
Tumor infiltrating lymphocytes (TIL) have demonstrated therapeutic effects in adoptive cell therapy of melanoma, and there is great interest in optimizing this response. However, the role of lymphocytes during tumor initiation and progression is complex and evolves over time. We have identified two distinct subsets of TIL that have divergent effector vs. regulatory function in murine tumor models. These subsets can be efficiently segregated in vitro based on differential expression of L-selectin (CD62L). Although initially present in small numbers, they can be activated in vitro with anti-CD3 mAb and expanded with a combination of IL-2 and IL-7 to provide sufficient numbers for adoptive transfer into secondary hosts with advanced tumors. Our initial studies demonstrated that the TIL CD62L-low subset is a mixture of CD4 and CD8 cells that individually or in combination mediate tumor-specific regression. By contrast, the CD62L-high subset, which is exclusively CD8+, does not have therapeutic efficacy. Moreover, when CD62Lhigh TIL are co-transferred with CD62L-low effector cells they abrogate their therapeutic efficacy, thus they have suppressor function. Because L-selectin is involved in lymphocyte homing to secondary lymphoid tissues, we hypothesized that the CD62L-high TIL cells might preferentially re-circulate into lymph nodes and inhibit primary sensitization of naïve T cells to tumor antigens. Tumor cells were inoculated subcutaneously, alone or with either CD62L-high TIL or CD62L-low TIL. The CD62L-high TIL actually enhanced the growth of subcutaneous tumors whereas the CD62L-low cells prevented tumor growth. More importantly, tumor-draining lymph nodes were harvested twelve days later and activated in vitro with anti-CD3 and IL-2/IL-7 for adoptive cell transfer to secondary tumor-bearing hosts. The presence of TIL suppressor cells during sensitization of tumor-draining lymph node cells partially inhibited the development of tumor-reactive effector cells. This inhibition was not tumor-antigen specific because TIL suppressor cells derived from the MCA205 fibrosarcoma were able to inhibit sensitization of B16/F10 draining lymph node effector cells. These data suggest that tumors acquire a population of CD8 CD62L-high T cells that can inhibit other effector T cells. The suppressor TIL cells also appear to modulate the tumor stroma to promote tumor growth and modulate antigen-presenting cells that migrate to draining lymph nodes thereby dampening the development of effector cells. Studies are ongoing to determine the mechanism of suppressor function.
Von Willebrand Factor-Glycoprotein Ib-Alpha Interactions Play a Role in Generating Platelet Microparticles: Data from Ex Vivo and in Vitro Studies
Authors: Artoni Andrea , Lecchi Anna , Mancuso MariaElisa , Santagostino Elena , Federici Augusto , Mannucci Pier M. 
Published: October 2019

Abstract
Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins such as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb might be critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in relationship with VWF levels and multimeric composition as well as shear stress exploiting ex vivo and in vitro natural models. PMP and TFMP were analyzed by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. In the first set of experiments, levels of PMP and TFMP were measured before and after DDAVP administration in 9 patients with mild hemophilia A. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma at baseline and 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed after DDAVP; peak levels were achieved after 2–8 h. Correlation was found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). We then studied 27 patients with different types of von Willebrand disease (VWD) and 15 normal controls: citrated platelet rich plasma was exposed to increasing shear stress (from 0 to 90 dyne) for 45 seconds in a cone and plate viscometer and generated PMP were then enumerated by flow cytometry. In normal controls the number of MP at 25 dyne was significantly higher than the number at 0 dyne (p<0.05). In patients with type 1 VWD and type 2B PMP significantly increased as in normal controls (p<0.05 at 25 dyne), while in type 3 patients no PMP increase was observed (p n.s. till 90 dyne). Interestingly, in type 2M patients PMP started to increased significantly at 50 dyne (p<0.05), while type 2A patients had the same behaviour of type 3 patients. This study support the hypothesis that ultralarge VWF multimers released by the endothelium interact with platelets and trigger MP generation, both in vivo and in vitro.
A Chemokine Receptor CXCR4 Plays a Crucial Role for Repairing Ischemic Tissue through Regulation of HIF-2a
Authors: Nagano Masumi , Kimura Ken-ichi , Yamashita Toshiharu , Hamada Hiromi , Ohneda Kinuko , Yoshikawa Hiroyuki , Ohneda Osamu 
Published: October 2019

Abstract
Endothelial progenitor cells (EPCs) are potential therapeutic tools for treatment of ischemic tissues. We previously divided EPCs into two populations on the basis of aldehyde dehydrogenase (ALDH) activities and demonstrated that EPCs with low ALDH activity (Alde-Low EPCs) possess a greater ability for repairing ischemic tissue than those with high ALDH activity by using mouse dorsal skin flap model (Nagano et al., Blood, 2007). To explore molecular basis of the function of Alde-Low EPCs, we transduced small interfering RNAs (siRNAs) into Alde-Low EPCs to reduce the expression of hypoxia inducible transcription factors (HIF-1a or HIF-2a) and examined the ability to repair ischemic tissues by using mouse skin flap model. Injections of Alde-Low EPCs or EPCs with HIF-1a siRNAs via tail vein significantly reduced the necrotic area (4.4±4.5 and 13.2±2.8% of total ischemic area, respectively, n=3 in each group) compared to control PBS injection (30.5±3.2%, n=4). In contrast, EPCs with HIF-2a siRNA showed no positive effect on reducing necrotic area (34.1± 5.2%, n=4). These data suggest that HIF- 2a plays a major role for repairing ischemic tissues in Alde-Low EPCs. We then analyzed the expression of hypoxia-inducible genes in these cells. While the VEGF mRNA level was 30% reduced in both HIF-1a and HIF-2a siRNA treated cells, the expression of a chemokine receptor CXCR4 was reduced in HIF-2a, but not HIF-1a siRNA transduced cells. Moreover, transduction of CXCR4 siRNA into Alde-Low EPCs significantly lost the ability to repair ischemic regions in a mouse flap model (35.5± 3.5%, n=3). Collectively, these results indicate that CXCR4 plays an essential role for repairing ischemic tissue through regulation of HIF-2a.
B Cells at the Interface of Innate and Adoptive Immunity: Identification of IRF8 Target Genes in Germinal Center B Cells
Authors: Shin Dong-Mi , Morse Chang-Hoon Leeand Herbert 
Published: October 2019

Abstract
IRF8 is a transcription factor widely expressed in hematopoietic cells and best known for its effects on myeloid and dendritic cell (DC) differentiation and function. Recent studies showed that IRF8 is expressed at low levels in naïve B cells, at significantly increased levels in germinal center (GC) B cells but little if at all in plasma cells. In GC, IRF8 was shown to contribute to the regulation of AID and BCL6. To better understand IRF8 contributions to the biology of mature B cells and possibly to transformation, we studied three mouse and three human lymphoma cell lines of GC origin by ChIP-Chip (C-C) analyses for IRF8 and POLII targets. We identified 87 IRF8 targets that were common to mouse and human B cells and were also positive for POLII. 17/18 of these putative IRF8 targets tested by PCR ChIP analyses were validated. IRF8 is transcriptionally activated by IFNg, and many of the newly identified targets were previously known to be IFN-responsive. Analyses of the targets identified 14 that contribute to various aspects of innate immunity at the cell surface (CD37, HSP90B1, Ly86), via signal transduction (AZI2, NFKBIZ, STAT2, IRF9), or with direct anti-viral activity (ADAR, PML, S100, ZC3HAV1 and others). Another 19 are active contributors to acquired immune functions of B cells with many involved in peptide processing and antigen presentation by both MHC class I and class II molecules (B2M, CD74, TAP1, TAP2, CTSS and 20S proteosomal components) and some in signal transduction (BLK, BLNK). Still others are active in B cell differentiation and function (MS4A1 [CD20], ELF1 and GNA13). CYLD plays in both arenas as a negative regulator of RIG-I-mediated antiviral responses and by regulating proper B cell activation after BCR ligation. The regulatory contributions of IRF8 to innate immunity in DC, macrophages, granulocytes and now B cells as well as elements of acquired immunity in B cells suggest that it contributes to an evolutionary bridge that positions a single cell type to integrate critical defense mechanisms from the innate and acquired immune repertoires.
Largazole, a Novel Cyclic Peptide Histone Deacetylase Inhibitor, Demonstrates Anti-MM Effects Alone and Increases the Anti-MM Effects of Bortezomib.
Authors: Li Zhi-Wei , Hu Mengyin , Leung Crystal , Steinberg Jeffrey A , Shen Jing , Li Mingjie , Wang Cathy S , Sanchez Eric , Bowers Albert , Greshock Thomas , Miller Kenneth , Newkirk Tenaya , West Nathan , Bradner James E. , Williams Robert M , Bonavida Benjamin , Chen Haiming , Berenson James R. 
Published: October 2019

Abstract
Abstract 4925
Multiple myeloma (MM) remains an incurable malignancy. Therefore, there is a need for the development of new agents to improve the survival for these patients. Histone acetylation, which is controlled by the balanced activities between histone acetyltransferase (HAT) and histone deacetylase (HDAC), is a major epigenetic modification that contributes to tumorigenesis. Through inhibition of HDAC activity, HDAC inhibitors (HDACis) increase acetylation levels of histones as well as other tumor suppressor gene products; and, therefore, are potential anti-cancer agents. Based on the structures, currently available HDAC inhibitors can be divided into four groups, including hydroxamates, cyclic peptides, aliphatic acids, and benzamides. Largazole is a novel member of the cyclic peptide family of HDACis some of which have shown anti-cancer effects in preclinical studies and early clinical trials including for patients with MM especially when used in combination with the proteasome inhibitor bortezomib. In this study, we have explored the potential anti-MM activity of largazole alone and in combination with bortezomib in preclinical in vitro studies. As demonstrated using the MTS assay, this HDACi inhibits the growth of cells from the MM cell lines RPMI8226, U266 and MM1S with an IC50 of approximately 0.2 μM in all three cell lines. In addition, largazole also induces apoptotic death of MM cells as determined with Annexin V staining followed by flow cytometric analysis. Largazole was also shown to induce histone acetylation in MM cells as determined with Western blot analysis using an antibody against acetyl-histone H4. Furthermore, the combination of this HDACi and bortezomib demonstrated synergistic anti-MM effects in these cell lines. Importantly, treatment of mice with largazole shows excellent tolerability at doses that produce concentrations in vivo that are higher than those shown to produce anti-MM effects in our in vitro studies. Thus, largazole may be a potential new agent for the treatment of MM alone and in combination with bortezomib. Currently, we are evaluating the cytotoxic effects of largazole on normal peripheral blood and bone marrow mononuclear cells in vitro and in vivo using our severe combined immunodeficiency mouse models of human myeloma alone as well as in combination with several other drugs including bortezomib used in the treatment of MM. Updated results from these ongoing studies will be presented at the meeting.

Disclosures
Berenson: Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Incidence and Risk Factors for Intracranial Hemorrhage in Californians with Immune Thrombocytopenia
Authors: Brunson Ann , White Richard H. , Wun Ted 
Published: October 2019

Abstract
Abstract 1161

Introduction:
Intracranial hemorrhage (ICH) is thought to be an uncommon event in patients with immune thrombocytopenia (ITP), with reported cumulative incidences of approximately 1%. Most studies have drawn from single institution experiences. We determined the incidence using a large population-based administrative database.

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. We then limited analysis to cases with ITP as the principal diagnosis. Similarly, all cases with a diagnosis of ICH (see types below) in any of the first 5 diagnosis fields were identified and merged with the ITP cohort. Univariate analysis and Cox proportional hazard modeling were then done to determine associations with ICH.

Results:
A total of 37,780 unique patients were initially identified with an ITP code, of which 13,651 were in the principal position and comprise the analysis cohort. This cohort was 60% female, 60% White, 22% Hispanic, 8% Asian, and 7% African-American with a median age of 53 years. Three hundred sixty-four cases had an ICH (cumulative incidence 2.67 %) at a median of 844 days after the first admission for ITP; 109 occurred with the first hospitalization for ITP. The median age of patients with ICH was 67 years. One hundred thirty-five cases with ICH had a splenectomy a median of 296 days prior to their hemorrhage. Of the ICH cases, 65 were known to have died (death statistics available only through 2007); 41 of these deaths were during the admission for the intracranial hemorrhage.

Discussion:
The cumulative incidence of ICH in this large cohort of Californians with ITP was 2.67%, higher than that reported from another population-based study with a smaller number of patients, and from single institution studies. The median age in this study is older than in other reports. The death rate for ICH hospitalization was 11.3% (41/364). Increasing age and certain medical co-morbidities were associated with increased hazard of ICH, as was diagnosis of lupus. Splenectomy was not associated with either increased or decreased hazard, but as this procedure was likely performed only in more severely affected but otherwise healthier patients this result is difficult to interpret. The finding of increased hazard of ICH in Hispanics and Asians with ITP is novel and unexplained. Intracranial hemorrhage may be a more common complication of ITP than previously appreciated.

Disclosures:
Wun: Daiichi Sankyo Company, Ltd. and Eli Lilly and Company: Research Funding.
Limited Effect of TET2 Mutations on Promoter DNA Methylation in Chronic Myelomonocytic Leukemia
Authors: Yamazaki Jumpei , Taby Rodolphe F , Vasanthakumar Aparna , Macrae Trisha , Ostler Kelly R , Shen Lanlan , Kantarjian Hagop M. , Estecio Marcos R , Jelinek Jaroslav , Godley Lucy , Issa Jean-Pierre 
Published: October 2019

Abstract
Abstract 1365
TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30 patients). By contrast, only 1/30 patients had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A. By bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutant and wild-type cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We confirmed only two non-CpG island promoters, AIM2 and SP140, as hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14 475 genes) previously found to be hypermethylated in TET2 mutant cases. This finding shows that hypermethylation of both AIM2 and SP140 are bona fide markers of TET2 mutant cases in CMML. On the other hand, total 5-methylcytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases. Thus, TET2 mutations have a limited impact on promoter DNA methylation in CMML. To confirm this, we performed genome-wide analysis using a next-generation sequencing method for DNA methylation levels in three TET2 mutant cases. TET2 mutant CMMLs had an average of 230 (1.9%) promoter CpG island sites hypermethylated compared to normal blood, which is close to what is generally observed when one compares cancer to normal. By contrast, all three cases had near normal to increased levels of methylation outside CpG islands. The median methylation levels in non-promoter, non-CpG island sites was 88.7% in normal blood compared to 91.7%, 92.1% and 94.6% in the three TET2 mutant cases. Thus, TET2 mutant CMMLs escape the general hypomethylation phenomenon seen in many cancers. All together, our data suggest that TET2 mutant CMML cases may have distinct DNA methylation patterns primarily outside gene promoters.

Disclosures:
No relevant conflicts of interest to declare.
A Subnuclear Targeting Defective Mutant of Runx1 Uncouples the Roles of Runx1 In Embryonic Development and Adult Definitive Hematopoiesis
Authors: Dowdy Christopher R , Frederick Dana , Zaidi Sayyed K. , Lian Jane B. , van Wijnen J. , Stein Janet L. , Stein Gary S. 
Published: October 2019

Abstract
Abstract 1387
As shown by gene ablation or replacement studies, Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC). Runx1 is also the most common target for point mutations and chromosomal translocations in human leukemia. Localization of Runx1 to distinct foci within the nucleus through a unique subnuclear targeting signal is linked to physiological roles of the protein. Many leukemia-related chromosomal translocations (e.g., 8;21 translocation that encodes for AML1-ETO protein) disrupt the Runx1 C-terminus and modify its biological activity as well as alter subnuclear localization. We hypothesize that altered subnuclear routing of leukemic proteins plays a key role and provides a common mechanism in human leukemias. We have previously developed a knock-in mouse model with a Runx1 C-terminal truncation (Runx1 Q307X), which similar to a complete ablation of Runx1 or a genetic knock-in of the leukemic AML1-ETO, causes mid gestation lethality. The Runx1 Q307X mutation also abolishes subnuclear targeting and alters Runx1-dependent myeloid gene expression. To further dissect the specific functional contributions of altered Runx1 subnuclear targeting, we developed a knock-in mouse model in which endogenous Runx1 is replaced with a triple point mutation (Runx1 HTY350-352AAA) that modifies the subnuclear targeting. In contrast to Runx1 Q307X or AML1-ETO knock-in mice, animals homozygous for Runx1 HTY350-352AAA bypass embryonic lethality. However, expression of key Runx1 target genes that regulate hematopoiesis is altered. In addition, bone marrow from Runx1 HTY350-352AAA animals shows increased proliferation ex vivo, resulting in an increase of myeloid and a decrease of B-lymphoid lineage cells. Importantly, red blood cell development, previously thought unrelated to Runx1 expression, is also modified in the Runx1 HTY350-352AAA homozygous animals, as shown by decreased mean corpuscle volume and selective effects on globin gene expression. Together, these findings show that altered subnuclear targeting uncouples the critical role of Runx1 during hematopoietic stem cell emergence and differentiation into various lineages in the adult.

Disclosures:
No relevant conflicts of interest to declare.