bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mesenchymal Stem Cells Convert Human Macrophages to a Novel Type of Alternatively Activated Macrophages.
Authors: Kim Jaehyup , Hematti Peiman 
Published: October 2019

Abstract
Abstract 3632
Poster Board III-568
Mesenchymal stem cells (MSCs) are capable of modulating the immune system through interaction with a wide range of immune cells. This study investigates the hypothesis that interaction of MSCs with macrophages could play a significant role in their anti-inflammatory/immune modulatory effects. All studies were approved by IRB of University of Wisconsin School of Medicine and Public Health. MSCs were culture expanded from discarded bone marrow filters after bone marrow harvest from normal healthy sibling HLA-matched donors. We used passages -35 for our experiments. Ex vivo culture expanded MSCs were characterized by their cell surface phenotype (positive for MSC markers: CD29, CD44, CD73, CD90, and CD105; and negative for hematopoietic markers: CD31, CD34, and CD45), and their differentiation potential into bone, fat and cartilage. Monocytes were isolated from peripheral blood mononuclear cell fraction of healthy donors using CD14+ Miltenyi magnetic bead cell separation method. We cultured human CD14+ monocytes for seven days without any added cytokines to generate macrophages, and then co-cultured them for three more days with culture-expanded MSCs. We used cell surface antigen expression and intracellular cytokine expression patterns to study the immunophenotype of macrophages at the end of this co-culture period, and phagocytic assays to investigate their functional activity in vitro. Macrophages co-cultured with MSCs consistently showed high level expression of CD206, a marker of alternatively activated macrophages, in addition to being positive fro CD14 marker. Using CD1a and CD209 staining we did not detect presence of any dendritic cells either at the end of seven days culture of monocyte-derived macrophages or at the end of co-culture period. Furthermore, macrophages that were co-cultured with MSCs expressed high levels of IL-10 and low levels of IL-12, as determined by intracellular staining, typical of alternatively activated macrophages. However, macrophages co-cultured with MSCs also expressed high levels of IL-6 and low levels of TNF-α, compared to controls. Functionally, macrophages co-cultured with MSCs showed a higher level of phagocytic activity using Alexa 488-conjugated E. coli phagocytic assay. In summary we describe a novel type of human macrophage generated in vitro after co-culture with MSCs that assume an immunophenotype defined as IL-10 high, IL-12 low, IL-6 high and TNF-α low secreting cells. These MSC-educated macrophages may be a unique and novel type of alternatively activated macrophages with potentially significant role in tissue repair.

Disclosures:
No relevant conflicts of interest to declare.
A Role for the Transcriptional Coactivator PRC1 in Mitochondrial Biogenesis During Erythroid Development.
Authors: Xu Xiuling , Friedman Jeff S. 
Published: October 2019

Abstract
Abstract 3642
Poster Board III-578
Red cell development requires unparalleled mitochondrial metabolic and biosynthetic capacities to support hemoglobin formation. In addition to meeting the energetic requirements of the erythroblast and reticulocyte, mitochondria are the site of multiple steps in heme biosynthesis and are essential for iron utilization. As such, perturbation of mitochondrial function in hematopoietic cells appears to differentially affect red cell development. For instance, we show that the drug chloramphenicol (an inhibitor of mitochondrial protein synthesis) preferentially inhibits erythroid colony formation with relatively little effect on other types of myeloid progenitors. Similarly, we show that knockout of the mitochondrial superoxide dismutase (Sod2) preferentially inhibits BFU-E, CFU-E and GEMM colonies. Clinically, several mutations in mitochondrial DNA or in nuclear encoded mitochondria-localized proteins preferentially disrupt erythroid development resulting in sideroblastic anemia. In order to better understand regulation of mitochondrial activity during red cell development/differentiation, we have investigated expression of transcription factors and coactivators implicated as regulators of mitochondrial biogenesis in other tissues including PGC-1 α and β, ERR proteins, NRF 1 and NRF 2 in normal and Sod2 deficient murine erythroblasts. Microarray and qPCR data suggest that neither of the classic transcriptional coactivators PGC-1 α or β are expressed at appreciable levels in erythroblasts. However, a PGC-1 related coactivator, PRC1 is detectable at both the transcript and protein level in developing erythroid cells. In the Sod2 deficiency model, one of the most prominent transcriptional changes we have observed in erythroblasts is a broad down regulation of expression of nuclear encoded mitochondria localized protein transcripts including multiple components of the electron transport chain, TCA cycle enzymes and mitochondrial ribosomal proteins. We show that PRC1 expression is down regulated in Sod2 deficient cells—suggesting that this coactivator plays a key role in mitochondrial biogenesis during erythroid development. Further, using the MEL cell line, we show that PRC1 expression is turned on following induction of differentiation with DMSO. These results identify PRC1 as a novel, and perhaps exclusive regulator of mitochondrial biogenesis during erythroid development. A better understanding of regulation of PRC1 expression and identification of transcriptional targets of PRC1 during erythroid development will improve our understanding of how perturbations in mitochondrial physiology affect the red cell compartment.

Disclosures:
No relevant conflicts of interest to declare.
Epigenetic Control of C/EBPa by Noncoding RNAs.
Authors: Di Ruscio Annalisa , Ebralidze Alexander K , D'Alò Francesco , Voso Maria Teresa , Leone Giuseppe , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 3644
Poster Board III-580
Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels.

Disclosures:
No relevant conflicts of interest to declare.
Molecular Mechanisms of the Pre-TCR Alpha Chain (pTa) Enhancer Transcriptional Regulation by Notch1.
Authors: Tremblay Cedric Stevens , Tremblay Mathieu , Herblot Sabine , Hoang Trang 
Published: October 2019

Abstract
Abstract 3649
Poster Board III-585
The Notch1 pathway and pre-TCR signalling are critical regulators of thymocyte development that have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Although the pre-TCR is required for Notch-dependent T-ALL, the role of pre-TCR alpha (pTa) as a Notch1 target remains controversial. Previous work in our laboratory has shown that the pTa gene is a target gene of the E2A-HEB transcription factors and that the SCL and LMO1 or LMO2 oncogenic transcription factors inhibits E2A activity and pTa expression, thereby causing thymocyte differentiation arrest during the pre-leukemic stage. SCL and LMO1/2 are implicated in 25% of T-ALL cases and induce aggressive T-ALL in transgenic (tg) mice when inappropriately expressed in the thymus. To define the mechanism regulating pTa expression by Notch1 and SCL, we used pTa regulatory sequences as a tool. To this end, we have performed transcriptional assays by co-delivering a luciferase reporter gene construct driven by the 200 bp pTa enhancer sequence with or without SCL or Notch1 intracellular domain (N1ICD), the functional fragment of the Notch1 receptor. Sequence analysis of the pTa enhancer revealed the presence of four E boxes that are potential binding sites for E2A-HEB and a CSL consensus sequence that can recruit the CSL/RBP-Jk transcription factor. Transcriptional assays in double negative thymomas indicated that the integrity of E box 2 and 3 (that recruit E2A-HEB) are essential for pTa enhancer activity in DN thymocytes, confirming the importance of E2A-HEB for pTa expression in these cells. In contrast, the CSL consensus sequence is important but not essential for pTa enhancer activity in these cells, consistent with previous results based on a conditional Notch1 knock out. In the presence of N1ICD, pTa enhancer sequences are strongly activated, either in fibroblasts or in double negative T cell lines in a dose dependent manner. This activation requires the integrity of the CSL consensus sequence but not of the E boxes. Moreover, we found that N1ICD strongly overrides the inhibitory activity of SCL on the pTa enhancer. Inhibition by SCL requires the integrity of E2 and E3 but not the CSL, confirming that SCL inhibits E2A-HEB activity, without affecting N1ICD. Together, our observations indicate that the regulation of pTa enhancer activity by Notch1 and E2A/HEB occur though independent mechanisms.

Disclosures:
No relevant conflicts of interest to declare.
PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.
Authors: Bartholdy Boris , Yamamoto Yukiya , Evans Erica , Crispino John , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 3651
Poster Board III-587
The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line.
Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Diagnostic Value and Limitations of FDG-PET for the Staging of Childhood Lymphomas.
Authors: Kabickova Edita , Votrubova Jana , Sumerauer David , Mejstrikova Ester , Hrusak Ondrej , Kyncl Martin , Kodet Roman , Stary Jan , Belohlavek Otakar 
Published: October 2019

Abstract
Abstract 3658
Poster Board III-594

Introduction
Positron emission tomography with fluorine-18-fluorodeoxyglucose (FDG-PET) has the potential to detect malignant cells by their increased glycolysis. PET can detect early changes, before they are apparent on anatomic imaging. Whole-body PET can also detect lesions at unexpected sites. However, physiologic FDG uptake in non-malignant conditions limits the specificity of PET. PET is predominantly used in adult lymphoma patients, limited number of studies were published dealing with PET and PET/CT in childhood lymphomas. The aim of our study was to assess the usefulness of PET for initial staging of pediatric lymphomas and to evaluate benign pathologic causes of FDG uptake.

Patients and methods
Over a period of 5 years 86 children and adolescents with lymphoma (58 with Hodgkin's lymphoma, 28 with non-Hodgkin's lymphoma) had complete staging work-up including FDG-PET or PET/CT and were included into this prospective study. Patients were aged 4-19 years, 57 (66%) were boys. No patient had CNS lymphoma infiltration. PET findings were correlated with conventional staging methods (CSM) including CT, ultrasound, and bone marrow examination. Discordant findings were verified by MRI and follow-up radiographic studies including PET.

Results
PET revealed 40 additional lymphoma manifestations in 34% (26/86) of studies and correctly upstaged 15% (13/86) of patients. Only 2% (2/86) of children were not accurately staged by PET, when PET failed to visualize diffuse bone marrow infiltration (extent of 15% cells) in 1 patient, and missed small pulmonary metastases (≤6 mm) in 1 child. Compared with CSM, PET had significantly higher sensitivity (97% vs. 83%), specificity (100% vs. 89%), and significantly higher accuracy (98% vs. 84%). Physiologic thymic FDG uptake was observed in 15% (13) of patients; diffuse increased FDG bone marrow activity had 22% (19) of children, and intense FDG activity in fatty tissue occurred only in 8% (7) of patients.

Conclusions
PET imaging was highly sensitive in detecting all subtypes of pediatric lymphomas and has potential to accurately define the disease stage except the lungs, where CT has excellent sensitivity. Understanding of the physiologic FDG biodistribution and benign pathologic causes of FDG uptake is essential for accurate scan interpretation.
Supported by grants: IGA NS/9997-4, NS/10480-3, MSM 0021620813 and MZO FNM 2005

Disclosures:
No relevant conflicts of interest to declare.
B Cell Depletion Reduces the Number and Function of T- Regulatory Cells and Enhances the Anti Tumor response
Authors: Tadmor Tamar , Zhang Yu , Dunn Robert , Shin Seung-uon , Cho Hyung-Mee , Rosenblatt Joseph 
Published: October 2019

Abstract
Abstract 3668
Poster Board III-604
Increasing evidence suggests that B lymphocytes play a central role in inhibiting the immune response against certain tumors, but the underlying mechanisms by which B cells facilitate tumor growth are still poorly understood.
In this study, we investigated how the presence or absence of B cells affects expansion and function of T- regulatory cells (‘Tregs’) in a murine tumor model (EMT-6). We compared tumor growth, and the number and function of T- regs cells in wild type immune competent mice ( ICM), B cell deficient mice ( BCDM) and /or in BALB-C mice following B- cell depletion induced by injection of anti murine CD20 antibodies (mCD20 Ab, 18B12, mouse IgG1,k, Biogen-IDEC) Mice were either tumor-naïve or implanted with EMT6 mammary adenocarcinoma cells.
Absence of B cells as in BCDM completely inhibited tumor growth in the majority of mice, while B cell depletion in normal mice substantially slowed the growth of EMT-6 tumors compared to wild type mice (ICM). Substantial T regs expansion, as defined by CD4+/CD25+/FOXP3+ cells, was evident on day 26 post tumor inoculation in EMT-6 tumor bearing ICM in comparison to the non- tumor bearing mice ( 15.2 +/− 1.2. % and 11.9 +/− 1.1% respectively), isolated from spleen as compared to naïve or tumor bearing BCDM (10.1+/− 0.2% and 10.8+/− 1.2%) The percentage and absolute number of T-regs in the spleen, tumor draining lymph nodes and tumor bed were significantly reduced in the BCDM and/or B cell depleted ICM compared to tumor bearing ICM (10%+/−0.8, 13.9+/− 1.23% and 17+/− 1.3% respectively p<0.01. data from single cell suspensions isolated from spleens on day 20 post tumor inoculation). Similar effects of B cell depletion on the numbers of T-regs were observed in the setting of pre-established EMT6 mammary tumors. In contrast to tumor bearing mice, differences in T-reg number and function were minimal in tumor free B cell deficient or in B cell depleted naïve mice compared to ICM.
T-reg function, measured by suppression assay and proliferation assays, was also markedly reduced in tumor bearing BCDM compared to ICM. Combining B cell and T-reg depletion using i.p. injection of anti CD 25 antibody (PC61 or PBS) resulted in similar rates of tumor regression in B cell depleted mice as were seen in BCDM suggesting that the combination of B cells depletion and further depletion of Tregs augmented anti-tumor response.
In conclusion, our studies indicate that B cell depletion may play a useful role in augmenting the T cell anti-tumor response, in part due to their effects on T-regulatory cell biology.

Disclosures:
Dunn: Biogen IDEC: Employment.
Involvement of Fatty Acid Synthase in Azacytidine-Induced Cytotoxicity in Waldenstrom's Macroglobulinemia.
Authors: Xu Lian , Patterson Christopher J , Sun Jenny , Yang Guang , Zhou Yangsheng , Hunter Zachary , Hatjiharissi Evdoxia , Ciccarelli Bryan , Gong Ping , Liu Xia , Tseng Hsiuyi , Ioakimidis Thea , Manning Robert , Hanzis Christina , Cao Yang , Lewicki Megan , Treon Steven 
Published: October 2019

Abstract
Abstract 3731
Poster Board III-667
Azacytidine is a potent demethylation agent and has been used for the treatment of myelodysplastic syndrome. Despite a role in reactivating epigenetically silenced tumor-suppressor genes, the mechanisms of action for azacytidine are not well understood. The relationships between the reactivation of tumor-suppressor genes and the significance for clinical responses also remain to be established. Azacytidine can incorporate into both RNA and DNA strands and may therefore act through multiple pathways. We therefore investigated the therapeutic potential of azacytidine in Waldenstrom's Macroglobulinemia (WM), and characterized certain molecular changes associated with azacytidine. We found that azacytidine exhibited significant dose-dependent cytotoxicity against WM cell lines, and primary WM cells. Treatment of BCWM.1 WM cells with 2 uM of azacytidine rapidly induced cell cycle arrest at G1, and also induced apoptosis at 48 hours. Cleavage of caspase 3, 7, 8, 9 and PARP-1 suggested an involvement of mitochondrial and death receptor pathways in azacytidine-induced apoptosis. The BH3 proteins Puma and Bim, and the pro-apoptotic protein Bax were up-regulated while the anti-apoptotic proteins Bcl-2 and Bcl-xl remained unchanged after treatment with azacytidine. In addition, azacytidine did not induce Akt and NFκB pathways. To further elucidate molecular changes associated with azacytidine treatment, we performed genome-wide microarray expression in BCWM.1 WM cells treated with azacytidine. We observed that the gene encoding fatty acid synthase (FASN) was among the most down-regulated of targets. FASN has been linked to tumor cell proliferation, and its expression was observed by us to be upregulated by microarray studies in WM patients, and further validated as a highly over-expressed target by quantitative RT-PCR analysis in WM patients versus healthy donors. Moreover, by these studies we show that knockdown of FASN by small interfering RNA resulted in growth arrest, and also induced apoptosis in BCWM.1 WM cells. Cerulenin, a specific inhibitor of FASN derived from a natural product, induced strong cytotoxicity against BCWM.1 and primary WM cells at 20 uM in 24 hours. In addition, a synergistic induction of apoptosis in BCWM.1 WM cells was observed in the presence of both azacytidine and cerulenin. The results of this study support an important role for FASN in azacytidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as a novel target for drug therapy in WM. A clinical study investigating the activity of azacytidine in WM is planned.

Disclosures:
No relevant conflicts of interest to declare.
Phase II Trial of 90y-Ibritumomab Tiuxetan Treatment as Consolidation After 6th R-CHOP Chemotherapy in Patients with Limited-Stage, Bulky Diffuse Large B Cell Lymphoma.
Authors: Yang Deok-Hwan , Kim Won Seog , Suh Cheolwon , Kwak Jae-Yong , Jung Joo-Sep , Kim Sung Hyun , Kim Jin Seok , Lee Je-Jung , Kim Hyeoung-Joon 
Published: October 2019

Abstract
Abstract 3751
Poster Board III-687
Limited-stage non-Hodgkin's lymphomas with bulky mass are known to have a poor prognosis as like to those with advanced disease. There are very limited data to compare the results of chemotherapy alone with those of combined modality treatment. However, defining local failure at bulky sites as one component of relapse, radioimmuotherapy (RIT) appears to reduce failure rates at sites of bulky disease. We assessed the clinical efficacy and toxicity of 6th R-CHOP chemotherapy and followed by ibritumomab tuixetan (Zevalin¢ç) consolidation in patients with bulky diffuse large B cell lymphoma (DLBCL).

Patients and Methods
This prospective, multi-center phase II clinical trial included the patients following as: i) patients with newly diagnosed CD20+ DLBCL ii) patients with stage I/II and bulky disease defined by maximum diameter greater than 10 cm or any mediastinal mass exceeding 1/3 the maximum trans-thoracic diameter (more than 8 cm) iii) patients that achieved a complete (CR) or partial response (PR) after 6th R-CHOP chemotherapy iv) aged over 18 years and ECOG performance status 0-2. Conventional, single dose of ibritumomab tuixetan (0.4 mCi/kg) was administered after 6th R-CHOP. Clinical response was evaluated after completion of 3rd and 6th R-CHOP and at 8 and 20 weeks after RIT, and assessed according to revised International Workshop Criteria.

Results
Twenty patients were treated with ibritumomab tuixetan as consolidation. Median age was 45.5 years and median size of bulky mass was 10.0 × 6.6 at diagnosis. 5 patients (35%) presented with high-intermediate or high risk by IPI and 4 patients underwent debulking operation. After a median of 17.5 months follow-up, the overall response rate was 95%, with a 80% CR and 15% PR. Two patients with PR after R-CHOP chemotherapy improved to CR after consolidation. However, four patients (20%) experienced a relapse with one of them progressed within 1 month after RIT. 2-year probability of progression-free survival (PFS) was 78.9 ±9.1%. The adverse events were mainly hematologic toxicities (more than grade3); neutropenia (60%) and thrombocytopenia (35%). Only one patient experienced a febrile neutropenia.

Conclusions
Consolidation treatment with Ibritumomab tuixetan resulted in a favorable response with tolerable toxicity in patients with bulky DLBCL.

Disclosures:
No relevant conflicts of interest to declare.