bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epigenetic Silencing of Mir-218 Regulates CDK6 Expression In Pre-B Acute Lymphoblastic Leukemia Cells
Authors: Li Xiaoqing , Liu Jun , Zhou Rui , Zheng Jine , Yao Junxia , He Yanli , Chen Xiangjun , Ding Jie , Huang Shiang , Chen Xian-Ming 
Published: October 2019

Abstract
Abstract 3621
Increasing evidence suggests that dysregulation of miRNAs plays an important pathological role in various malignant diseases including acute leukemia. To reveal the contributions of aberrant epigenetic modifications to the deregulated miRNA expression in precursor B-cell acute lymphoblastic leukemia, we examined the miRNA expression profile in NALM-6 cells after treatment with the combination of 5-AZA-2'-deoxycytidine (AZA) and trichostatin A (TSA). We found that the treatment significantly increased expression of 34 miRNAs and decreased the expression of 10 miRNAs. One of the most significantly upregulated miRNAs is miR-218, an intronic miRNA that can be transcribed from either pri-miR-218-1 or pri-miR-218-2, residing in the intron of the SLIT2 gene or SLIT3 gene respectively. Interestingly, we detected that pri-miR-218-1 and its host gene SLIT2, but not pri-miR-218-2 and SLIT3, were induced by AZA plus TSA treatment. Consistent with this observation, we showed that the CpG islands in SLIT2 promoter was highly methylated in NALM-6 cells and AZA plus TSA treatment significantly decreased DNA methylaiton in this region. We found that targeting of the 3'untranslated region of CDK6, a bona fide oncogenic factor, by miR-218 resulting in translational repression. Overexpression of miR-218 expression in NALM-6 cells by transfection of miR-218 precursor decreased cellular expression of CDK6 at the protein level, but not at the message level. AZA and TSA treatment decreased CDK6 expression in NALM-6 cells, presumably through upregulating miR-218. Our results indicate that epigenetic regulation plays an important role in controlling miRNA expression in human acute lymphoblastic leukemia cells. Epigenetic silencing of miR-218 may contribute to the overexpression of CDK6 in NALM-6 cells.

Disclosures:
No relevant conflicts of interest to declare.
High Throughput Sequencing of Acute Lymphoblastic Leukemia Small RNAs.
Authors: Wei Michael C , Osborn Elizabeth O , Cleary Michael 
Published: October 2019

Abstract
Abstract 3650
MicroRNAs are small, non-coding RNAs that regulate gene expression and play key roles in cancer by modulating oncogene and tumor suppressor pathways. We are investigating the clinical and prognostic roles of miRNA expression in pediatric leukemias using high-throughput sequencing as a profiling tool. To establish the methodology, we have utilized high-throughput sequencing to quantify small RNA expression from eight acute lymphoblastic leukemia cell lines and one MLL-rearranged infant ALL patient sample. We generated sequencing libraries from these cells, conducted high-throughput sequencing using the Illumina platform, and established a custom bioinformatics pipeline for data analysis. Over 50 million individual sequence reads were analyzed. These sequences were mapped against a database of human miRNAs, and the frequency of miRNA expression among samples was enumerated. Expression of hematopoietic-specific miR-142 and miR-181 cluster miRs was found in these leukemia samples, while the liver-specific miR-122 was not expressed. miR-196b, previously reported to be over-expressed in MLL-rearranged leukemias, was expressed in 3/3 MLL-rearranged leukemia cell lines and 1/5 non-MLL cell lines. Expression of individual miRNAs was validated by quantitative PCR. Additional analysis of MLL-associated miRNAs and novel small RNAs will be presented. Our results demonstrate the feasibility and potential of high-throughput sequencing to profile the expression of small RNAs from leukemia cells, and we plan to apply these methods to additional primary patient samples to examine prognostic and clinical correlations with small RNA expression patterns.

Disclosures:
No relevant conflicts of interest to declare.
Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation.
Authors: Shastri Aditi , Baker Kelty R. , Thiagarajan Perumal 
Published: October 2019

Abstract
Abstract 3652
A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin.
The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site.
We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin.
Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms.

Disclosures:
No relevant conflicts of interest to declare.
Intracranial Hemorrhage In Patients with Inherited Bleeding Disorder In Children.
Authors: Park Ji Kyoung , Lee Soon Yong , Park Young Sil 
Published: October 2019

Abstract
Abstract 3657
Intracranial hemorrhage (ICH) is known to be a severe although uncommon complication of inherited bleeding disorder. A 12-year retrospective study was conducted to investigated the episodes of ICH in children with an inherited bleeding disorder who where followed by a regional hemophilia comprehensive center in South Korea. We review cases of ICH events in patients with inherited bleeding disorders in children and investigate characteristics, treatment, and prognosis. Between January 1998 and August 2010, a total of 13 ICHs in 9 patients were identified.
ICHs were occurred for 7 patients with hemophilia A, one patient with hemophilia B, and one patient with factor VII deficiency. All patients were male and severe hemophilia A(2 patients); moderate hemophilia A(5 patients); moderate hemophilia B(1 patients). One of the patients with severe hemophilia A had inhibitors. The median age of patients was 6years 11months old. (range, 7 days≂f15 years old). Types of ICHs were 8 SDH, 2 EDH, 1 intracerebral with IVH, 1 intracerebral with SDH and 1 SDH with SAH. 8 cases were occurred spontaneously and 5 cases were occurred after traumatic events. Presenting symptoms were headache (6 events), seizure (2 events), vomiting (1 event), stuporous mentality (1 event), lethargy (1 event), irritability (1 event) but one patient with traumatic SDH had no symptom. Initial diagnostic modality was CT scan. One patient with severe hemophilia A who had inhibitors was initially treated with FEIBA® and controlled well. The remaining patients with hemophilia were treated with factor eight/nine concentrate. The patients with factor VII deficiency was treated with recombinant factor VIIa (Novoseven®). The patient with moderate hemophilia B required surgical evacuation. The mean duration of hospitalization was 12.8 days (range, 3–25 days). ICH was the first significant bleeding event in 3 patients – one with moderate hemophilia A, aged 7 days, and one with moderate hemophilia A, aged 5months, and one with factor VII deficiency, aged 1months. The median follow-up duration was 73 months (range, 4–149 months). Two younger patients had complication with long term sequale like hydrocephalus, cerebral palsy, and epilepsy. For one patient, antibody to factor VIII was developed due to factor administration.
ICH can occur in patient with a severe inherited bleeding disorder and also in patients with moderate hemophilia. Younger patients with ICH may have poor prognosis and neurologic sequale more frequently. Symptoms of younger patients was atypical so their parental recognition and medical diagnosis delayed. The median duration between onset of symptom and administration of factor is 2.6 days(range, 0–8 days). Delayed administration of factor caused the patients to have long term sequale. Rapid and appropriate factor administration is important to treatment course and prognosis.

Disclosures:
No relevant conflicts of interest to declare.
Suicide Gene (HSVtk) Ablation of Hematopoietic Stem Cells and Their Progeny In the Rhesus Macaque Model: An Approach to Deletion of Dangerous Clones.
Authors: Barese Cecilia , King Connor , Sellers Stephanie , Krouse Allen E , Metzger Mark E , Traversari Catia , Bordignon Claudio , Kornblau Steven M. , Marini Frank , Donahue Robert E , Dunbar Cynthia E. 
Published: October 2019

Abstract
Abstract 3759
For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy.

Disclosures:
No relevant conflicts of interest to declare.
Feline Leukocyte Adhesion Deficiency.
Authors: Giger Urs , Pratt Suzanne M. , Palena Christina , Kennedy Doug R , Bauer Thomas R. 
Published: October 2019

Abstract
Abstract 3779
Leukocyte adhesion deficiency (LAD) caused by CD18 deficiency is an autosomal recessive immunodeficiency which has been described in humans, Irish Setter dogs, Holstein cattle, and transgenic mice. We report here on the characterization of the clinical features and leukocyte function abnormalities of a novel feline model of leukocyte adhesion deficiency.
The proband was a 5 year old male domestic longhair cat with a life-long history of infections, inflammation and granulomas. Severe gingivitis was recognized at 8 weeks of age which progressed to peridontitis and loss of all deciduous and adult teeth before 1 year of age. Over the next years this cat experienced recurrent severe upper respiratory infections, gastroenteritis, anterior uveitis, various abscesses, and epidermal sloughing with sepsis secondary to small grooming lacerations. The various illnesses seemed to be responsive to various long-term antibiotic as well as glucocorticoid treatments. Complete blood cell counts revealed a persistent severe leukocytosis (50-91,000/μl) due to mature neutrophilia with few toxic changes, and mild lymphocytosis, monocytosis and eosinophilia. Serum immunoglobulin concentrations were normal.
Compared to feline control cells, neutrophils of the proband did not express any CD18 on the cell surface as determined with two monoclonal anti-CD18 antibodies but normal CD45 expression. Adhesion of affected neutrophils with and without PMA activation was severally impaired; the adhesion of normal feline neutrophils could be completely blocked by the anti-CD18 antibody. In a proliferation assay the proband's T-cells responded weakly at 1 pg SEA but near normally at 100 pg SEA, suggesting a CD18-independent T-cell response in cats. Sequencing of the feline CD18 β-integrin gene is being completed.
In conclusion, this naturally-occurring feline model of CD18 deficiency exhibits similar features to LAD in other species. However, feline LAD seems clinically milder allowing for a longer life expectancy possibly due to T-cell independent activities. This LAD model may be helpful in developing and assessing novel therapies.

Disclosures:
No relevant conflicts of interest to declare.
Human Interleukin-3/Granulocyte Macrophage-Colony Stimulating Factor Knock-In Mice Support Human Myeloid Cell Reconstitution and Human Immune Responses In the Lung.
Authors: Willinger Tim , Rongvaux Anthony , Takizawa Hitoshi , Eynon Elizabeth E. , Stevens Sean , Manz Markus G , Flavell Richard A. 
Published: October 2019

Abstract
Abstract 3789
Humanized mice, i.e. mice with a functional human immune system, have great potential to study human immunology in vivo and to allow vaccine testing. To this end, mice need to fully support engraftment with human immune cells, allow infection with human pathogens, and mount effective human immune responses to pathogens. A major limitation of current humanized mice is the poor development and function of human myeloid cells. Here we report a novel strategy to overcome this limitation by generating human interleukin-3/granulocyte macrophage colony stimulating factor knock-in (hIL-3/GM-CSF KI) mice to create a better environment for human myeloid cells. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis (PAP) due to elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the PAP syndrome. In addition, these mice showed an enhanced systemic inflammatory response to LPS. Finally, humanization of IL-3 and GM-CSF lead to a stronger innate immune response against influenza virus infection. In summary, hIL-3/GM-CSF KI mice represent a new mouse model to study human immune responses in the lung and against pathogens such as influenza.

Disclosures:
Stevens: Regeneron Pharmaceuticals: Employment; AnaptysBio Inc: Employment.
Toll Receptors and Langerhans Cell Histiocytosis.
Authors: Very Gayane badalian , Vergilio Jo-Anne , Calicchio Monica , Fleming Mark D. , Rollins Barrett 
Published: October 2019

Abstract
Abstract 3793

Introduction:
Langerhans cell histiocytosis is a clonal proliferative disease of antigen presenting cells. Clinically, it is associated with aberrant secretion of cytokines from both Langerhans cells (LCs) themselves and immune cells associated with the disease. Toll-like receptors (TLRs) are membranous and cytoplasmic proteins involved in pattern-based recognition of bacterial and viral products. Activation of TLRs results in production of pro-inflammatory cytokines, modulation of the immune response, and maturation of LCs. TLRs activate signal transduction pathways involving IRFs (interferon regulatory factors), NFkB (Nuclear Factor-k-B) and MAPK (mitogen activated protein kinases).
In this study, we have examined LCH samples for their expression of TLRs and downstream effector proteins.

Methods:
We examined archived samples from 42 LCH patients. Paraffin embedded samples were sectioned and stained for immunohistochemistry (IHC) using antibodies directed against TLR1-9; IRF3, 7 and 8; MEK1/2 and ERK1/2. RNA was isolated from the same samples using laser-capture microdissection, and the expression of TLR receptors, effectors and downstream targets was analyzed using qRT-PCR. These data were compared with gene expression data published in Gene Expression Omnibus using data set GSE16395.

Results:
IHC results showed upregulation in pathologic LCs compared to normal LCs of TLR 1, 3, 5, 7, 8 and 9, but not TLR 4 and 6. Moreover, upregulation of downstream pathways involving MEK1/2 and ERK1/2 and IRF8 in was also observed in pathologic LCs. Gene expression analysis showed up-regulation of the effectors of TLRs including CASP8, MAP3K7, and PPARA as well as genes in the NFKB pathway (CCL2, NFKb1, NFKB2, NFKBIL1, NFRKB, and REL), the JNK/p-38 pathway (ELK1, c-FOS, MAP3K1 and MAP3K8), and the interferon regulatory factor pathway (CXCL10, IRF7, IRF8 and TBK1). Comparison with the gene expression data base confirmed the results obtained from qRT-PCR for the gene expression of TLRs, their effectors, and downstream.

Conclusion:
Upregulation of TLRs and the pathways they control occurs commonly in LCH. Therapies directed toward inhibition of TLRs and their downstream targets may provide a means of modulating the adverse inflammatory aspects of LCH.

Disclosures:
No relevant conflicts of interest to declare.
Mesenchymal Stromal Cells Accelerate Hematopoietic Reconstitution and Mediate An IL6-Dependent Regeneration of the Intestinal Epithelium of Lethally Irradiated Mice.
Authors: Francois Moira , Birman Elena , Coutu Daniel L. , Forner Kathy-Ann , Galipeau Jacques 
Published: October 2019

Abstract
Abstract 3846
Apoptosis of radiation-sensitive stem cells in the bone marrow and the gut are the main complications arising from radiation exposure, whether therapeutic or accidental. Bone marrow-derived mesenchymal stromal cells (MSC) were shown to exert regenerative properties through the secretion of factors which act in a paracrine manner. In this study, we demonstrated that a single intra-peritoneal injection of C57Bl/6 MSC rescued lethally irradiated Balb/c mice. Histological analysis and Ki67 immunostaining of jejunum sections collected 48 hours and 5 days post MSC injection indicated that MSC protected the gastro-intestinal epithelium from severe radiation-induced damages and prompted the regeneration of the gut by stimulating proliferation of the crypt stem cell pool. Using interleukin-6 null MSC, we demonstrated that IL-6 derived from the MSC played a role accelerating gastro-intestinal epithelium regeneration. We also observed that MSC injection prevented severe anemia and restored granulocyte and platelet counts to normal values faster than the control group. Accelerated regeneration of the bone marrow and extra-medullar hematopoiesis in the spleen of MSC transplanted mice corroborated this observation. Our results suggest that IL-6 derived from MSC or used on its own may be exploited to alleviate radiation induced injuries to patients undergoing radiotherapy or subject to accidental radiation exposure

Disclosures:
No relevant conflicts of interest to declare.
Microenvironment-Mediated Regulation of Micrornas In B-Lymphocytes as a Novel Mechanism for Terminal B-Cell Differentiation and Lymphomagenesis.
Authors: Lin Jianhong , Lwin Tint , Tam Wayne , Zhao Jian-Jun , Crespo Luis , Choi Yong Sung , Zhang Xinwei , Tannenbaum Victoria , Sotomayor Eduardo M. , Moscinski Lynn C. , Dalton William S. , Wright Ken , Tao Jianguo 
Published: October 2019

Abstract
Abstract 3853
B-cell differentiation is tightly regulated by synchronized suppression and/or induction of specific transcription factors. Among them, B-cell lymphoma 6 (BCL6) and PRDM1 are considered to be master regulators for germinal center formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 also have been associated with lymphomagenesis. Their regulation still need further study especially at the posttranscriptional level. Here, by using co-culture system and whole genomic microRNA microarray profiling, we show for the first time that direct B lymphoma cell-stroma cell contact between follicular dendritic cells and B-lymphocytes could induce upregulation of miR-30 family and downregulation of miR-9 and let-7 family. In silico analysis showed that miR-30s can target genes BCL6 and miR-9/let7 can target PRDM1 with direct binding sites in 3`UTR region of their mRNAs. The microarray data can be proved by microRNA specific Q-RT-PCR. Specifically, by both gain of function and loss of function studies, we functionally verified that FDCs Regulate Expression of BCL6 and PRDM1 via Cell-Cell Direct Contact induced correlated microRNA dysregulation. To further validate the direct interaction between BCL6 and miR-30, we constructed luciferase reporters containing the BCL6 3`-UTR that included miR-30 binding sites and a mutant 3`-UTR harboring mutations in the “seed pairing” sequences of the miR-30 binding site. Co-transfection of miR-30 and reporter construct into cells significantly decreased luciferase activity in wild-type but not in mutant BCL6-3`-UTR transfected cells, supporting the role of miR-30 family in the regulation of BCL6 expression. BCL6 and PRDM1 and their regulation miRNAs, let-7 and miR-30, also can be validated in primary normal B-lymphocytes and lymphoma cells by using our co-culture system. Dysregulation of BCL6 and PRDM1 is often associated with lymphomagenesis. We firstly identified that BCL6 is the direct target of miR-30 family and also verified PRDM1 is the target of miR-9, and let-7 in our system. Our studies provide a novel mechanism of post-transcriptional regulation of BCL6 and PRDM1 by several microRNAs. In the context of micro-environment, it provides a clue for germinal center B-cell differentiation as well as B-cell lymphomas progression regulated by lymphocyte cell-stroma cell contact through microRNAs.

Disclosures:
No relevant conflicts of interest to declare.