bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutrophil Volume, Conductivity and Scatter Parameters to Diagnose Neonatal Sepsis
Authors: Celik Istemi Han , Demirel Gamze , Sukhachev Dmitry , Erdeve Omer , Dilmen Ugur 
Published: October 2019

Abstract
Abstract 4726

INTRODUCTION:
Neonatal sepsis remains an important clinical syndrome despite advances in neonatology. Early diagnosis and adequate antibiotic treatment are required because of high rates of mortality and morbidity. Current hematology analysers can determine cell volume (V), conductivity for internal composition of cell (C) and light scatter for cytoplasmic granularity and nuclear structure (S) and standart deviations. This VCS measurements were used to diagnose sepsis particularly in adults and higher levels of V and lower levels of S were seen in septic patients. We investigated these parameters in diagnosis of neonatal sepsis.

METHOD:
We investigated these parameters in secreening of neonatal sepsis, defined as a systemic infection, validated by a positive blood culture, diagnosed beyond the first day of life. We used LH780 hematological analyzer (Beckman Coulter, Fullerton, CA). We combined these parameters with interleukin-6 (IL-6) and C-reactive protein (CRP), and developed models to diagnose sepsis by Effective Modelling of Moleculer Activity (EMMA). It uses combinatorial algorithm of the selection parameters for regression equation based on modified stepwise procedure. It allows compute a number of “best” regression equations with different combinations of parameters.

RESULTS:
A total of 237 newborn, 61 proven sepsis, 108 clinical sepsis and 68 control, were enrolled the study. Gram positive and negative bacteria including 12 types of microorganism were isolated from 34 and 27 patients, respectively. Mean neutrophil volume (MNV) and volume distribution width (VDW) were found to be statistically increased both in proven and clinical sepsis groups. We developed models using MNV, VDV, IL-6 and CRP (Table 1). We used these models both for proven sepsis and sepsis (proven and clinical sepsis) groups. Table 2 and 3 summarize the cut-off levels of MNV, VDV, IL-6, CRP and models' performance of proven sepsis and sepsis groups. These models gave more sensitivity and specificity than usage of MNV, VDW, IL-6 and CRP alone.

CONCLUSION:
We suggest to use combination of MNV and VDW with markers such as CRP and IL-6, and use diagnostic models created by using EMMA including these markers.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of STAT5 Phosphorylation by Anti-GM-CSF Monoclonal Antibodies: Validation of a New Whole Blood Assay
Authors: Grivel Karine , Magnenat Laurent , Moulard Maxime 
Published: October 2019

Abstract
Abstract 4728

Introduction.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (CD116) is expressed on primary monocytes and mediates cell signaling through STAT5 phosphorylation. Therefore STAT5 is central to GM-CSF action. BioCytex has developed a new semi-quantitative flow cytometry (FC) assay to measure the phosphorylation status of STAT5 in human primary monocytes and lymphocytes in whole blood samples. The assay was used to investigate the potency of recombinant monoclonal antibodies (MAbs) for the neutralization of human GM-CSF.

Methods.
Engineered anti-GM-CSF MAbs were generated and characterized by Merck Serono. Several clones and preparation were tested, including MAbs 343 (low and high LPS), 467; 340; 339; 366, and MAb 285 as isotype control. Their different in vitro potencies for blocking recombinant GM-CSF activity in stable cell lines was determined using the TF-1 proliferation and IL-8 secretion in U937 cell assays. Whole blood FC assay was designed and validated at BioCytex using either a rabbit MAb or a rabbit polyclonal antibody specific for phospho-STAT5 (Tyr694). Monocytes were gated using a PE-labeled anti-CD14 probe. Inhibition of STAT5 phosphorylation by anti-GM-CSF MAbs was performed using recombinant GM-CSF at EC90.

Results.
EC90 of GM-CSF was determined at 2.3 pM from n=5 healthy volunteers. All GM-CSF specific MAbs inhibited STAT5 phosphorylation in monocytes, whereas the control (MAb 285) did not. More interestingly, there was a marked effect of LPS content since inhibition of STAT5 phosphorylation was not completed in presence of high LPS load (MAb 343). IC50 and IC90 for MAbs 339 and 366 were 10-fold increased as compared to MAbs 467 and 340. IC50 were ranging from 0.12 to 0.19 nM for clones 343 (low LPS), 467 and 340. IC50 and IC90 for clone 339 were 0.016 and 0.034 nM, respectively.

Conclusion.
Inhibition of GM-CSF mediated monocyte activation by neutralizing MAbs can be monitored in a whole blood assay. STAT5 phophorylation could be viewed as a potential biomarker for GM-CSF receptor activation. The cell-signaling inhibition potency in primary cells correlated with in vitro potency using stable cell lines. In summary, the new validated FC could be used for research and clinical development purposes.

Disclosures:
Grivel: LFB Biotechnologies: Consultancy. Magnenat:Merck Serono S.A.: Employment. Moulard:BioCytex: Consultancy, Employment.
mTOR Signaling Is Required for the Survival and Proliferation of Hematopoietic Stem/Progenitor Cells
Authors: Xiao Xia , Luo Hongmei , LaRue Amanda C. , Schulte Bradley A. , Wang Yong 
Published: October 2019

Abstract
Abstract 4787
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates protein synthesis, gene transcription, cell growth and cell proliferation. Previous studies have demonstrated that abnormalities in the Pten-mTOR pathway may contribute to the development of leukemia and lead to premature exhaustion of hematopoietic stem cells (HSCs). These findings suggest a role for mTOR in the regulation of HSC self-renewal and cell transformation. The aim of this study was to investigate the involvement of mTOR signaling in the survival and proliferation of HSCs and hematopoietic progenitor cells (HPCs). Immunofluorescent staining with antibodies against phosphorylated mTOR and S6 kinase revealed that Thrombopoietin (Tpo) activates mTOR in cultured mouse bone marrow (BM) lineage negative cells, suggesting that Tpo may promote the survival and proliferation of HSCs/HPCs via activation of the mTOR signaling pathway. Further studies revealed that treatment with an mTOR specific small molecule inhibitor (Ku-69734) significantly suppressed the colony-forming ability of HPCs as evidenced by a dose-dependent decrease in the production of CFU-GM, BFU-E and CFU-GEMM. We also examined the clonogenic function of HSCs using cobblestone-area forming cell (CAFC) assays and found that Ku-69734 treatment markedly reduced the number of CAFCs in long-term BM culture. Moreover, immunophenotyping and flow cytometric analyses showed that inhibition of mTOR induced apoptosis primarily in HSCs and to a lesser degree in HPCs, indicating that mTOR inhibition may suppress the clonogenic function of HSCs and HPCs via the induction of apoptosis. Together, these data demonstrate that mTOR signaling is required for the survival and proliferation of HSCs and HPCs. Given that many mTOR inhibitors are currently in clinical trials for the treatment of cancers, our findings provide the rationale to further evaluate the potential toxicity of mTOR inhibitors in HSCs/HPCs in vivo.

Disclosures:
No relevant conflicts of interest to declare.
A Wharton's Jelly Mesenchymal Stromal Cell Derived 3D Osteogenic Niche Allows for Cord Blood Stem Cell Attachment
Authors: Aljitawi Omar S. , Keefe Peggy , Ott Lindsey , Li Dandan , Zhang Da , Abhyankar Sunil , Garimella Rama , McGuirk Joseph , Detamore Michael 
Published: October 2019

Abstract
Abstract 4813

Background:
Most of umbilical cord blood (UCB) stem cell ex vivo expansion methods have led to UCB stem cell differentiation instead of their self-renewal. Evidence suggests that bone osteoblastic cells are responsible, through physical contact with hematopoietic stem cells (HSCs), for the maintenance of long-term HSCs.

Aims:
To develop a 3 dimensional (3D) osteogenic structure that provides a niche for UCB stem cells and secondarily, assess the ability of this structure, in addition, to a cocktail of cytokines to expand UCB stem cells.>

Methods:
Mesenchymal stromal cells (MSCs) isolated form Wharton's Jelly (WJ) were seeded into biodegradable scaffolds followed by osteogenic differentiation induction using osteogenic differentiation media for up to 4 and 6 weeks to develop a 4-week and a 6-week osteogenic scaffolds, respectively. CD34+ selected UCB stem cells were expanded on a monolayer of WJMSCs using a cocktail of cytokines for one week, following which the monolayer and expanded CD34+ UCB stem cells were trypsinized and added to the 4-week and 6-week 3D osteogenic scaffolds (3D conditions), or plated again in culture flask (2D conditions). Pre- and post-expansion total nucleated cell counts were determined and flow cytometry was used to assess the phenotype of the expanded population.

Results:
Osteogenic differentiation was successfully induced in 3D scaffolds as evident by Alizarin-red staining, scanning electron microscopy (SEM) and molecular testing. UCB stem cell attachment to the osteogenic scaffold was verified by SEM. TNCs were expanded 10X in 2D and 200X in 3D conditions. However, the percentage of CD34+ cells was 5.31% and 4.91% in 2D, and 2.98% and 1.14% in 4-week and 6-week 3D conditions, respectively.

Conclusion:
Attachment of CD34+ UCB stem cells to 3D osteogenic scaffold allowed for their expansion, though the majority of the expanded cells lost their CD34 expression possibly secondary to their differentiation.

Disclosures:
No relevant conflicts of interest to declare.
Prognostic Factors for Survival in 60 Patients with Classical Hodgkin Lymphoma From Peru
Authors: Beltran Brady E , Alva Jose C , Morales Domingo , Quinones Pilar , Miranda Roberto N. , Castillo Jorge J 
Published: October 2019

Abstract
Abstract 4863

Introduction:
Hodgkin lymphoma (HL) is an uncommon lymphoma with a good prognosis. However, the characteristics of HL in Peruvian patients have not been previously reported. HL is thought to be related to chronic Epstein Barr virus (EBV) infection, and new biological markers such as EBV, CD68 and FOXP3 expression could be of potential biological interest in this entity. Our study aims to identify prognostic factors for survival in Peruvian patients with classical HL.

Methods:
This study was approved by the IRB at our institution. Between January 2001 and December 2009, patients diagnosed with classical HL were selected for this study. HIV-negative patients were excluded. Clinical data were reviewed retrospectively and patient's biopsies were analyzed for the immunohistochemical expression of CD20, PAX5, CD30, CD15, FOXP3 and CD68. Samples were also analyzed for the presence of EBV-encoded RNA (EBER) using an in situ hybridization (ISH) technique. Clinicopathological characteristics will be presented using descriptive statistics. Overall survival (OS) estimates were calculated using the Kaplan-Meier method and compared using the log-rank test. For the multivariate survival analysis, Cox proportional-hazard regression test was used. P-values <0.05 were considered statistically significant.

RESULTS:
A total of 60 patients with a pathological diagnosis of classical HL were included in our study. The median age was 55 years (range: 18–98 years) with a 7:3 male predominance. Advanced stages (stage 3 and 4) were seen in 52%, ECOG performance status >1 in 15% and B symptoms in 67%. White blood cell (WBC) counts >15,000 cells/mm3 was seen in 7%, lymphocyte count <600 cells/mm3 in 12%, albumin levels <4 g/dl in 70% and hemoglobin <10.5 g/dl in 40% of the patients. FOXP3 expression >25% was seen in 45%, CD68 >25% in 48% and EBER 2–3+ in 53%. ABVD was the preferred therapy, accounting for 90% of the cases and a complete response to ABVD was obtained in 78%. With a median follow-up 34 months, the median overall survival (OS) has not been reached and the 5-yr OS was 80%. In the univariate analysis, ECOG >1 (p=0.0001), elevated LDH levels (p=0.04) and lymphocyte count <600 cells/mm3 (p=0.05) were adverse prognostic factors. Age, sex, B symptoms, WBC, albumin and hemoglobin levels were not associated with survival. In the multivariate analysis, only ECOG >1 (p=0.01) was an independent adverse prognostic factor in Peruvian patients with classical HL.

CONCLUSION:
ECOG is a best prognostic factor in Peruvian patients with a diagnosis of classical Hodgkin lymphoma. CD68 expression, EBV status and FOXP3 expression by the malignant cells were not prognostic factors for OS in our cohort of patients.

Disclosures:
Castillo: GlaxoSmithKline: Research Funding; Millennium Pharmaceuticals: Research Funding.
Cytogenetic Analysis of 1863 Chineses Patients with Chronic Myeloid Leukemia
Authors: Mu Qitian , Ma Qiuling , Wang Yungui , Tong Xiangmin , Chen Zhimei , Lou Jiyu , Cheng Yizhi , Lu Ying , Ni Wangmao , Chen Feifei , Jin Jie 
Published: October 2019

Abstract
Abstract 4888

Background:
Cytogenetic analyses of chronic myeloid leukemia (CML) have been performed previously in a large number of reports, but systematical research based on large sample sizes is seldom available. In order to further elucidate the cytogenetic nature of CML, we analyzed retrospectively the cytogenetic profiles of 1863 Ph/BCR-ABL-positive CML patients from a research center in China.

Results:
Of 1266 newly diagnosed CML patients, the median age was 41 years, which is younger than the median age of diagnosis in western populations. The incidence of additional chromosome abnormalities(ACAs) was 3.1% in newly-diagnosed chronic phase(CP), 9.1% in CP after therapy, 35.4% in accelerated phase(AP) and 52.9% in blast phase(BP), reflecting cytogenetic evolution with CML progression. 5.3% patients harbored a variant Ph translocation. A higher prevalence of ACAs was observed in variant Ph translocations than in classical t(9;22) in the disease progression, especially in BP(88.2% vs. 50%, p=0.002). Moreover, a hyperdiploid karyotype and trisomy 8 were closely correlated with myeloid blast crisis(BC) while a hypodiploid karyotype and monosomy 7 were associated with lymphoid-BC. Among subsets of myeloid-BC, compared with myeloid-BC with granulocytic differentiation or monocytic differentiation, myeloid-BC with minimal differentiation had higher ACAs rate (80% vs.46.8%, p=0.009 and 80% vs. 42.9%, p=0.006).

Conclusion:
CML tends to afflict younger population in China. In the disease progression, the incident of ACAs was higher in variant Ph translocations than in classical t(9;22). Among subsets of myeloid-BC, myeloid with minimal differentiation had distinct cytogenetic features.

Disclosures:
No relevant conflicts of interest to declare.
Biomarker for Benzopyrene Exposure Is Mitochondrial Mass and Mitochondrial DNA Copy in Blood Cells and Hematopoietic Tissues
Authors: Shin Myung-Geun , Kim Hye-Ran , Choi Hyun-Jung , Kim Hwan-Young , Han Dong-Kyun , Baek Hee-Jo , Moon Jai Dong , Kook Hoon , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook 
Published: October 2019

Abstract
Abstract 4889
Benzopyrenes are well known pollutants and carcinogens. They can intercalate into DNA and interfere transcriptions, resulting in causing various human diseases. However, biomarkers of benzopyrene toxicity have not been comprehensively studied in blood and leukemia cells. The current study was investigated to discover biomarkers for benzopyrene exposure in blood cells and leukemia cell lines. Peripheral blood, peripheral blood hematopoietic stem cell and leukemia cells (THP-1, K562, Molt-4 and HL-60) were cultured in RPMI 1640 media with adding 0, 50, 100 and 200μM of benzopyrene. Viability and apoptosis were assessed by tryptophan blue dye exclusion test and flowcytometry using annexin V. Hydrogen peroxide was measured using enzyme immunoassay. Mitochondrial mass, membrane potential and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, Red probes and real time PCR, respectively. The number of cell remained constant for three weeks culture. Viability of four cell lines disclosed significant decrease after two weeks of benzopyrene treatment. Apoptosis was increased in time- and dose-dependent manner after two weeks of benzopyrene treatment. Mitochondrial contents and membrane potentials were dramatically increased in three-week culture at dose dependent manner. Hydrogen peroxide level was significantly elevated after two weeks treatment of benzopyrene compared to non-benzopyrene treatment group. The number of mtDNA copy increased gradually after exposure to benzopyrene. These results indicated that increased mitochondrial mass and mtDNA copy number were biomarkers for direct exposure of benzopyrene in blood cells and hematopoietic tissues.

Disclosures:
No relevant conflicts of interest to declare.
CCL19 Induced Responses Are Differentially Regulated by Atypical Chemokine Receptor CRAM and Its Classical Chemokine Receptor CCR7 in B-CLL Cells
Authors: Burger Nathalie , Haerzschel Andrea , Leick Marion , Hartmann Tanja Nicole , Catusse Julie , Burger Meike 
Published: October 2019

Abstract
Abstract 4896

Introduction:
Chemokines are known to play an important role in the migration and survival of B-CLL cells. The non-signalling chemokine receptors, including DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner, and to variable degrees on B-CLL cells. We have recently shown that it competitively binds CCL19 and that this binding is not followed by classical chemokine responses. CCL19 and its signalling receptor CCR7 are centrally involved in B cell localisation and maturation within the secondary lymphoid tissues. CCR7 is also highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21 which have been shown to be present at higher concentrations in serum of patients with lymphadenopathia compared to patients without. In this study we investigate the influence of CRAM on the CCL19 dependent responses of B-CLL cells and potential correlations to clinical data with a specific focus on lymphadenopathia.

Results:
We demonstrate that B cells from patients with B-CLL present high, but variable degrees of CCR7 and CRAM expression. Patients with compared to patients without lymphadenopathia show a higher CRAM expression level whereas the CCR7 expression is not significantly different. In single samples showing extremly high CRAM expression the migration towards CCL19 is reduced compared to patients with lower CRAM expression. These observations confirm results in the B-CLL cell line MEC-1 showing increased migration toward CCL19 when CRAM expression is reduced using CRAM-siRNA. On the other hand, CRAM seems to be a chemokine presenter as we can show that it does not degrade its chemokine ligand but presents it on the surface of polarised cell layers. Thus, we assume that CRAM plays a role for cell migration, possibly transmigration and cell localisation within lymph nodes of B-CLL cells.

Conclusions:
We show that CRAM can act as an integrator of different recruitment and activation factors. It is associated to CCR7 driven recruitment of B cells by regulating CCL19 availability. Expression of CRAM differs in B cell malignancies for which CCL19 and CCL21 have already been shown to be implicated in lymphadenopathia. We therefore suggest that CRAM is an additional player in the localisation and differentiation/maturation processes of malignant B cells of B-CLL patients.

Disclosures:
No relevant conflicts of interest to declare.
Erythrokinetics in Cooley’s Anemia
Authors: STURGEON PHILLIP , FINCH CLEMENT A. 
Published: October 2019

Abstract
Blood production and destructions have been measured in four patients with Cooley’s anemia. Methods employed included the determination of erythroid/ myeloid ratio of the marrow, reticulocyte count, plasma iron turnover and red cell utilization, Cr51 survival and fecal urobilinogen. Rates of production obtained by these measurements have been compared to normal.
Patients with Cooley’s anemia have been shown to have an increased turnover of hemoglobin constituents comparable to the maximal response seen in other hemolytic anemias. There is, howvever, a marked decrease in maximal delivery of erythrocytes to the peripheral blood amounting to about 50 per cent in the mildly anemic patients and 85 per cent in severely anemic patients. The rate of destruction of circulating erythrocytes was similar in the three patients studied. The severity of anemia was therefore largely related to the production defect.
It was concluded that the defect in Cooley’s anemia is not in total hemoglobin synthesis, but in the fabrications of circulating erythrocytes, which in turn have the associated manifestations of hypochromia, increased percentage of fetal hemoglobin and shortened survival time.
Mixed Agglutination of Leukocytes and Erythrocytes in Relation to Studies of Leukocyte Antigens
Authors: BAKEMEIER RICHARD F. , SWISHER SCOTT N. 
Published: October 2019

Abstract
Serologic experiments are described in which was studied the agglutination of leukocytes from donors representing various ABO, Rh, and Lewis erythrocyte groups an certain canine blood groups, in the presence of corresponding antisera.
Appropriate mixing of antisera with leukocytes and erythrocytes from donors of different groups was seen to produce clumping of leukocytes which did not conform to the reactions of the erythrocytes from the leukocyte donor. When viewed under phase microscopy, certain of these leukocyte clumps, which appeared homogeneous with ordinary light microscopy were found to be clumps of leukocytes mixed with ghosts of erythrocytes reacting with the antibody present.
Factors contributing to this apparently immunologically non-specific clumping were the presence of complement-fixing, potentially hemolytic antibody and thermolabile components of serum. A possible relationship with erythrophagocytosis is suggested.
These observations indicate that certain results of this and other "leukoagglutination" technics, which have been interpreted as demonstrating the presence of A and B antigens on human leukocytes, deserve re-evaluation and emphasize the importance of developing methods of preparing homogeneous leukocyte suspensions.
The Postnatal Synthesis of Fetal Hemoglobin. I. Some Studies in Newborn Goats Using Radioactive Fe59 and Glycine-2-C14
Authors: VISSER H. K. A. , HUISMAN T. H. J. , WOLDRING M. G. 
Published: October 2019

Abstract
Postnatal synthesis of fetal and adult hemoglobin was studied with radioactive iron (Fe59) and glycine-2-C14 in newborn goats.
At birth about 60 per cent of Hb-F is present in newborn goats; after 60 days only about 5 per cent is found. Two goats were treated with "exchange transfusions" with blood of the mother. Using an alkali denaturation method the fetal hemoglobin was not demonstrable during the first few days after this treatment, but 20 days later about 20 per cent was present in circulating blood.
Maxima of radioiron-specific activity in adult hemoglobin occurred in untreated newborn goats at the 12th day, of C14 at the sixth day; in "exchange goats" for radioiron at the 8th day. The fetal hemoglobin maxima of specific activity occurred in the untreated goats resp. after 32 and 24 days, in "exchange goats" after 8 days.
The reappearance of Hb-F in "exchange goats" and the specific activity of Hb-F are direct indications for a synthesis of this component after birth in this species.