bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Mechanism Based Pharmacokinetic/Pharmacodynamic Model of the Effect of a Prolyl Hydroxylase Inhibitor on Erythropoietic Response In Mice
Authors: Singh Indrajeet , Krzyzanski Wojciech , Singh Pratap 
Published: October 2019

Abstract
Abstract 4769

Background:
The oxygen sensing pathway is one of the major controlling mechanisms of erythropoiesis in humans and other mammals. The transcription factor, Hypoxia-Inducible-Factor (HIF) regulates the local gene expression of the hormone erythropoietin (EPO). Degradation of HIF1α is regulated via oxygen dependent pathway by Prolyl Hydroxylases (PHDs) enzymes and the inhibition of PHD enzyme activity stimulates the erythropoietic response. The purpose of this study was to develop a mechanistic Pharmacokinetic/Pharmacodynamic (PK/PD) model to describe the effect of a Prolyl Hydroxylase Inhibitor (PHI) on the erythropoietic process in mice.

Methods:
PHI was given orally at three single doses of 10, 30 and 100mg/kg to male BALB/C mice (N=3-6, per time point). The blood plasma and kidney tissue samples were collected upto 96hrs. The PD markers were: kidney HIF1α concentration, kidney EPO mRNA expression, EPO plasma concentration and reticulocyte count. A two compartment model with first order absorption was fitted to describe the PK in plasma. Two different dose dependent apparent volume of distribution was assumed for the central (plasma) compartment at lower and higher doses. For the PD model, under quassi steady state assumption the enzymatic hydroxylation leading to degradation of HIF1α by PHDs is assumed to be an irreversible reaction. The drug present in plasma stimulates the removal of PHDs which results in accumulation of HIF1α and further EPO stimulation. This stimulation of kidney EPO mRNA, EPO protein level and reticulocytes were described by a cascade of indirect response models. ADAPT5 was used for all model fittings.

Results:
At higher PHI dose levels, ~30-40 fold increase in HIF1α was observed which caused ~1000 fold increase in EPO mRNA and EPO plasma concentrations within 6–8 hrs whereas the reticulocytes level increased by 3–4 fold within 3–5 days of single dose administration. Model parameters such as PHI elimination rate (kel, 0.43hr-1), the dose dependent apparent volume of distribution (V/F, 2–8L), the drug potency (IC50, 0.68μ M) for the degradation of PHDs; rate of loss (kout, 0.26hr-1) of HIF1α; the depletion rate of EPO mRNA (kd1, 0.48hr-1), plasma EPO (kd2, 0.25hr-1); and half maximal effective concentration (EC50, 7.01 ng/mL) of EPO for reticulocyte stimulation were estimated with high precision levels. Overall, the PK/PD model adaquetly captured the experimental data for all three doses.

Conclusion:
The erythropoietic process follows a cascade of biological events which is well described by the presented PK/PD model. Our model uses the systems biology approach to vertically integrate the intracellular HIF1α and EPO mRNA responses with in vivo markers of erythropoiesis such as EPO plasma levels and reticulocyte counts.

Disclosures:
No relevant conflicts of interest to declare.
Whole Genome Transcriptional Analysis of Cd4+ and Cd8+ T-Lymphocyte Immunomodulated by Mesenchymal Stromal Cells
Authors: Saldanha-araujo Felipe , Panepucci Rodrigo , Araujo Amélia , Covas Dimas , Zago Marco A. 
Published: October 2019

Abstract
Abstract 4777

Introduction.
Recently, several researchers have showed that mesenchymal stromal cells (MSCs) are useful for preventing and treating the graft versus host disease, however the molecular mechanisms involved in immunomodulation of T lymphocytes by MSCs remains to be explored.

Aims.
With that in mind, we performed a whole genome microarray study to explore transcriptional differences in activated TCD4 and TCD8 lymphocytes immunomodulated or not by MSCs.

Materials and Methods.
Peripheral blood CD3+ T lymphocytes from 3 individuals were activated by anti-CD3/CD28 beads and cultured either in the absence or in the presence of MSC (5 to 1 ratio). Following a 5 day period, CD4+ and CD8+ lymphocytes cultivated or not with MSCs were immunomagnetically purified to over 95% purity (flow cytometry) and profiled by whole genome microarrays. Differentially expressed genes were analyzed in search of regulatory networks and levels of selected transcripts were evaluated by RT-PCR.

Results.
T lymphocytes proliferation was inhibited significantly by MSC (BrdU incorporation assay) and induced the expression of classical regulatory T cell genes (IL10, FOXP3, CTLA-4 and GITR). Our microarray analyses revealed that lymphocytes cultured with MSCs displayed several transcriptional modifications related to distinct signaling pathways. Among them, TCR signaling, cell cycle (G1/S progression), NF-kB pathway, mTOR signaling and in enzymes and factors related with chromatin modification and remodeling. An insignificant number of transcript behaved distinctly (eg. with changes in opposite direction) when TCD4+ and TCD8+ lymphocytes where compared.

Conclusion.
Our results clearly show that both CD4+ and CD8+ T lymphocytes are immunomodulated by MSCs by similar mechanisms and this effect occurs by means of distinct molecular mechanisms. Supported by FAPESP, CNPq and FINEP.

Disclosures:
No relevant conflicts of interest to declare.
Human Umbilical Cord Blood Derived IPS Cells as a Source of Hematopoietic Progenitors Cells
Authors: Moraghebi Roksana , Rönn Roger Emanuel , Parker Aaron , Lutz Margaret , Berggren Travis , Woods Niels-Bjarne 
Published: October 2019

Abstract
Abstract 4790
The ability to generate hematopoietic stem cells (HSCs) from an unlimited source of cells, such as from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable HSCs for therapeutic purposes. Umbilical cord blood is an ideal source of fetal/neonatal cellular material for iPS reprogramming due to the proliferative capacity of the cells as well as the reduced exposure of these cells to environmental factors compare to commonly used skin fibroblasts. In addition, it has been proposed that the cellular starting material imparts an epigenetic memory to the iPS cell line that influences lineage predisposition upon its differentiation. This project seeks to evaluate human umbilical cord blood as a starting cell source for generating iPS cells, with the ultimate goal to generate transplantable HSCs. We have isolated, cultured, and characterized an adherent cell fraction from the hemato-endothelial lineage. These cells were found to have an endothelial progenitor phenotype with CD45neg, CD133neg, VE-Cadherinhi, VEGFR2med, CD31hi, CD34hi cell surface markers as determined by FACS and formed tubules in the matrigel tubular assay. The iPS cell lines were generated using a 5-factor cocktail of inducible lentiviral vectors with an efficiency of 0.02%. The iPS cell lines were then evaluated for blood cell lineage differentiation capacity using our state-of-the-art ES/iPS-to-blood differentiation protocol. From the 5 iPS lines we tested, we saw 30 +/− 20% hematopoietic (CD45+) cells in our differentiation cultures. Moreover, the percentage of hematopoietic progenitors (CD45+ CD34+) of the hematopoietic cell fraction was 19.8+/− 0.6%, and also showed the presence of the more primitive CD45+ CD34+ CD38- progenitor fraction. Clonogenic progenitor cell counts determined by methylcellulose colony assay showed 44 +/− 3 colony forming units per 10,000 plated CD45+ cells. This is in the range of colonies obtained from umbilical cord blood mononuclear cell isolates (mean= 35+/− 2). The efficiency of hematopoietic generation for the lines ranged from 8 to 60% CD45+ suggesting that there are significant differences between the lines in terms of the completeness of reprogramming towards the pluripotent state. Further investigation into the epigenetic status of these lines is being performed. These data demonstrate the utility of human umbilical cord blood derived iPS cells for generating and expanding hematopoietic progenitors. This project advances iPS technologies towards treating life threatening hematological malignancies and diseases.

Disclosures:
No relevant conflicts of interest to declare.
Differential Expression of Transforming Growth Factor Beta Receptor 2 (TGFβR2) In Diabetic CD34+ Cells: Implications for Vascular Repair
Authors: Bhatwadekar Ashay , Stepps Valerie , Grant Maria B , Bartelmez Stephen H. 
Published: October 2019

Abstract
Abstract 4795
We recently reported that the vascular reparative ability of diabetic CD34+ cells can be restored by transient inhibition of transforming growth factor β1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (TGF-β1-PMO) (Bhatwadekar 2010). TGF-β1 mediates its action by binding to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-βR1 subunits. We investigated whether healthy or diabetic CD34+ cells differentially express TGFβR2 receptor and whether inhibition or knock-out of endogenous TGF-β1 expression would affect the surface expression of TGF-βR2.
TGF-βR2 expression of murine bone marrow (BM) lin- Sca-1+/− cells from TGF-β1 knockout mice or wild type mice was studied using flow cytometry. Unexpectedly, TGF-β-R2 expression was essentially absent in BM lin- Sca-1 +and - cells from TGF-β1 knockout mice compared to wild type cells. Because the absence of endogenous and exogenous TGF-β1 resulted in complete down regulation of TGF-βR2 in primitive murine hematopoietic cells, we explored the relationship of endogenous TGF-β1in human mature and immature CD34+ cells in blood, bone marrow and cord blood.
Human CD34+CD45+ CD38+ or - cells from healthy and diabetic individuals were FACS sorted based on surface expression the analysed for TGF-βR2 expression. At the same time, TGF-βR2 and TGF-β1 mRNA expression was determined for sorted populations using quantitative real time RT-PCR.
A comparison of TGF-βR2 surface expression between mature progenitors (lin- CD34+ CD45+ CD38+) from diabetic and healthy cord blood revealed that TGF-βR2 expression did not differ between these groups. In contrast, TGF-βR2 surface expression in immature cells (CD34+ CD45+ CD38-) was very low in diabetic patients compared to healthy controls (p = 0.001).
Next, we inhibited endogenous TGF-β1 in healthy CD34+ cells which resulted in a significant (p<0.01) reduction in TGF-β-R2 mRNA expression. However, in diabetic CD34 cells, TGF β1-PMO treatment abolished TGFβR2 expression.
Our study suggests that healthy and diabetic CD34+ cells differentially regulate TGF-βR2 expression which may be the result of the elevated intracellular and extracellular levels of TGFb1 in diabetic individuals. A reduction in TGF-βR2 expression in diabetic CD34+ CD38- cells may physiologically enhance proliferation of the primitive CD34+ CD38- cell compartment, an outcome that may be necessary to keep up with increased demand for CD34+ cells in the diabetic individual.

Disclosures:
Stepps: BetaStem Therapuetics: Employment. Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Hematopoietic Stem Cell Expansion Leads to Monocytosis In Animal Models of Atherosclerosis
Authors: Akhtari Mani , Murphy Andrew J , Pagler Tamara A , Tall Alan R 
Published: October 2019

Abstract
Abstract 4797
Atherosclerotic cardiovascular disease is associated with leukocytosis. Hypercholesterolemic animal models like apolipoprotein E deficient (Apoe-/-) mice develop leukocytosis and monocytosis. However, the mechanisms linking hypercholesterolemia to leukocytosis are poorly understood. Recent studies in our laboratory have shown increased proliferation of hematopoietic stem and progenitor cells (HSPCs) and leukocytosis in mice deficient in efflux of cholesterol to HDL. Apoe also appears to be highly expressed in HSPCs, suggesting a role in cholesterol efflux. We hypothesized that leukocytosis in Apoe-/- mice might be associated with increased proliferation and expansion of HSPCs and that lowering their cholesterol levels with atorvastatin would suppress this expansion. Wild type (WT), LDL receptor-deficient (Ldlr-/-), and Apoe-/- mice were fed a Western-type diet (WTD) for 0, 10 and 20 weeks (8 mice per group per timepoint). After 10 weeks of WTD, 8 Apoe-/- received WTD with atorvastatin (0.01% wt/wt) for the remainder of the study. On chow diet Apoe-/- mice had monocytosis (+66% vs WT, P<.01), neutrophilia (+90%, P<.0001), and expanded HSPCs (+46% compared to the other groups, P<.05). After 10 weeks of WTD, leukocytosis developed further in Apoe-/- mice, along with increased bone marrow proliferation (+86%, P<.01) and more granulocyte-macrophage (GM)-colony formation (110% more, P<.01). After 20 weeks, all mice had developed monocytosis, neutrophilia, and expansion of the HSPCs compared to their chow starting point. Atorvastain reduced plasma cholesterol by 60% (P<.01) and attenuated monocytosis (30% reduction, P<.05) and neutrophilia (35% reduction, P<.05), but did not reduce the HSPCs levels in Apoe-/- mice. In conclusion, leukocytosis and expansion of HSPCs in Apoe-/- mice is observed on a chow diet and develops earlier than in the other models on the WTD, consistent with a role of APOE in modulating HSPCs cholesterol content. However, dietary hypercholesterolemia eventually led to HSPCs expansion paralleling leukocytosis in all groups, suggesting this is a general response to dietary hypercholesterolemia. Leukocytosis was attenuated with atorvastatin treatment through a mechanism unrelated to the reversal of HSPCs expansion.

Disclosures:
Tall: CSL: Consultancy; Merck: Consultancy; Roche: Consultancy; Arisaph: Consultancy.
Evaluation of Possible Risk Factors for Avascular Necrosis of the Femoral head in patients with Sickle Cell Disease
Authors: Al-Momen Abdul Kareem , Owaidah Tarek , Naqshabandi Nizar , Hasanato Rana , Cana Sri-Halima , Qayum Abdul , Zamzami Marwan , Aleem Aamer 
Published: October 2019

Abstract
Abstract 4819
Sickle cell disease is one of the most common causes of avascular necrosis (AVN) of the femoral head. many previous studies have investigated the risk factors for AVN with conflicting conclusions. We hereby report the results of a total of 44 adult patients (26 males and 18 females, age 18–56 years) with severe sickle cell disease seen in our hematology clinic who were investigated for the presence of AVN. Out of these patents 24 (55%) had AVN of the femoral head (6 bilateral and 18 unilateral). All of these patients had further investigations including methelene tetrahydrofolate reductase (MTHFR) gene mutation by molecular technique, along with Hb F level, Hb A2 level and MCV. Six of the 24 AVN patients (24%) were positive for either heterozygous or homozygous for C677T variant compared with four out of the 20 who did not have AVN (the difference is not statistically significant). HbF level was statistically higher in patients with AVN (P- value 0.03). In conclusion, we confirm here that patients with sickle cell disease and AVN have high prevalence of MTHFR mutation and higher level of Hb F, both of which which could be contributing factors for AVN in patients with Sickle Cell Disease.

Disclosures:
No relevant conflicts of interest to declare.
Prevalence of Sickle Cell Anemia In Eastern India
Authors: Chakraborty Abhijit , Basak Jayasri , Majumdar Deboshree , Mukhopadhyay Soma , Chakraborty Sagnik , Mukhopadhyay Ashis 
Published: October 2019

Abstract
Abstract 4822

Background:
Sickle cell disease is an inherited disorder of hemoglobin synthesis. This is due to replacement of Valine for Glutamic Acid in position six of the Beta globin chain of hemoglobin. This genetic alteration yields unstable RBC which lasts for 10–20 days. In stressful conditions the cells become sickle shaped and get lysed. There are about 20 million people with sickle cell disease in India. During January 2009- May2010 camps were held in various parts of West Bengal, Jharkhand, Chattisgarh. Along with various mutations of thalassemia, we also observed sickle cell anemia among them. This triggered our interest to study the spectrum of the sickle mutation co-inheritant with different mutations such as Homozygous Sickle Cell, Sickle Cell-Beta0 Thalassemia, Sickle Cell-Beta+ Thalassemia, Severe β+ thalassemia genes, Moderate β+ thalassemia genes, Mild β+ thalassemia genes Sickle cell-HbE Thalassaemia, Sickle cell-HPFH Thalassaemia, in said part of India. Since Indian patients with SS disease had higher hemoglobin, red cell counts and higher HbF levels and lower HbA2, MCHC, MCV, and reticulocyte counts, hence a high hemoglobin is a risk factor for painful crises and may also be a risk factor for avascular necrosis of the femoral head, proliferative sickle retinopathy, and acute chest syndrome.

Methods:
We have screened 332 individuals in eastern part of India during the period January 2009- May 2010. 3ml of peripheral blood was collected in EDTA vial from each individual. NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) was performed on spot. Then Complete Blood Count was done within 24 hours of collection. HPLC (High Performance Liquid Chromatography) was performed to identify the samples for confirmation. In our observation in case of sickle cell anaemia HbF (Fetal haemoglobin), Hb (haemoglobin), MCV (mean corpuscular volume) values ranges between 0–10 %, ≤7-10g/dl, 65–90fl respectively. ARMS (Amplification Refractory Mutation System) PCR (polymerase chain reaction) was done to confirm the mutation.

Result:

Conclusion:
Of the total samples collected in the camps held at various places of Jharkand, Chattisgarh & West Bengal 87 of them was carriers of sickle cell anemia. There was 7 homozygous (SS), 14 sickle beta, 12 double heterozygous for HPFH (High Persistance of Fetal Hemoglobin) & sickle cell anemia. In conclusion, the manifestations of sickle cell disease are influenced by a variety of other genetic and environmental factors. The occurrence of the disease against different genetic and environmental backgrounds provides experimental models that contribute to understanding the variability in clinical and hematological expression of the disease.

Disclosures:
No relevant conflicts of interest to declare.
Gain of the Centromeric Region of Chromosome 8, a New Germline Alteration In Juvenile Myelomonocytic Leukemia
Authors: Steinemann Doris , Ripperger Tim , Tauscher Marcel , Praulich Inka , Göhring Gudrun , Pabst Brigitte , Teigler-Schlegel Andrea , Schlegelberger Brigitte , Niemeyer Charlotte M , Flotho Christian 
Published: October 2019

Abstract
Abstract 4830
Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myeloproliferative disorder characterized by the clonal hyperproliferation of myelomonocytic cells. The pathogenesis of JMML involves disruption of signal transduction through the RAS pathway, which may be an early event during leukemogenesis. Additional genetic lesions may be necessary for full malignant transformation. We applied microarray BAC/PAC- and high resolution 244k oligo-arrayCGH to bone marrow samples from 21 JMML patients in order to identify subtle genomic alterations. In 8 of13 JMML patients with normal karyotype and 2 of 8 patients with monosomy 7, additional copy number alterations were identified. A recurrent deletion of around 1.4 Mb at 17q11.2 targeting the NF1 gene was identified in 2 patients with clinical diagnosis of neurofibromatosis due to germline mutations and LOH in the tumor as described previously. In addition to the monosomy 7, one patient (D600) with a somatic mutation in PTPN11 showed an additional marker chromosome. The origin of this marker chromosome was resolved by arrayCGH. The BAC/PAC array indicated a gain of the centromeric region: arr8p12-q11.21(37,204,000-49,686,000)×3. Breakpoints were refined to 8p12-q11.21(36,673,794- 50,142,678) by high resolution oligo-aCGH. Interestingly, in patient D703 with a heterozygous CBL germline mutation and homozygosity in hematopoietic cells, we detected a nearly identical gain arr8p12-q11.21(40,076,917-49,622,319)×3 not accompanied by -7 and with slightly different breakpoints. The gain was confirmed by means of FISH using the centromere-specific probe for chromosome 8 CEP8 showing three signals. This patient was also found to have a small additional marker chromosome. In order to rule out that the marker chromosome was inherited, we performed chromosome analyses in the parents of patient D703. Both had a normal karyotype and did not carry an extra copy of the region 8p12-q11.21. However, FISH on buccal epithelial mucosa cells of the patient D703 revealed a mosaic constellation with 34% cells positive for trisomy 8. Likewise, in the second patient, the dup 8p12-q11.21 (D600) was constitutional as 38% of fibroblasts showed 3 CEP8 FISH signals. There has been a longstanding discussion as to whether mosaic trisomy 8 may be constitutional. Our findings provide evidence that at least partial trisomy 8 may indeed be present as a germline mutation. Future work is needed to determine how constitutional partial trisomy 8 might contribute to leukemogenesis in JMML with different mutational subtypes.

Disclosures:
No relevant conflicts of interest to declare.
Concordance of CNS Leukemia Detection Between Morphology and Flow Cytometry Prior to Stem Cell Transplantation In Patients with MDS, AML, ALL and CMML
Authors: Smith Kelly M. , Cherian Sindhu , Wood Brent , Sandmaier Brenda M. , Pagel John M. , Estey Elihu H. 
Published: October 2019

Abstract
Abstract 4842
Accurate diagnosis of CNS involvement is essential for optimizing treatment and maximizing outcomes in patients with leukemia prior to hematopoietic stem cell transplantation (HCT). The most common methods of detecting CNS involvement include cytomorphic analysis and multiparametric flow cytometry (MFC). While cytomorphic analysis still has diagnostic and prognostic importance, MFC may be more than twice as sensitive as standard cytology analyses, raising the question of whether MFC might be able to detect CNS disease in cases where cytology did not.1 We retrospectively evaluated cerebral spinal fluid (CSF) results among 109 continuous patients with MDS, AML, ALL and CMML who underwent a reduced-intensity HCT at the Fred Hutchinson Cancer Research Center between May 2006 and May 2010. Ninety-seven of these patients had a pre-HCT lumbar puncture on arrival to evaluate for CNS disease. Thirty-five patients had CSF sent for morphology alone, 2 patients had CSF sent for MFC alone and 60 patients had CSF sent for both morphology and MFC. Of the 60 patients with both MFC and morphology results, CSF notable for blasts by concordant morphology and MFC was detected in 6 patients. These patients received treatment with intrathecal chemotherapy with or without craniospinal radiation. One patient had a single atypical cell with blast-like morphology seen by cytology with negative MFC. Fifty-three patients had both negative CSF by morphology and MFC. This data suggests that there is high concordance between morphology and MFC, however, it is difficult to determine the concordance with much confidence due to the small number of patients with CNS disease. Given the infrequency of CNS disease in this patient population, more patients will need to be studied in order to fully assess the value of MFC in addition to cytomorphic analysis. Because it is also difficult to be sure that our patient population was representative, a prospective large study, for example, in consecutive patients presenting for treatment of AML, might be worthwhile.
1J.E.C. Bromberg, MD, D. A. Breems, MD, PhD, J. Kraan, BSc, G. Bikker, B. van der Holt, MSc, P. Sillevis Smitt, MD, PhD, M. J. van den Bent, MD, M. van't Veer, MD, PhD and J. W. Gratama, MD, PhD. CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies. Neurology 2007;68:1674-1679.

Disclosures:
No relevant conflicts of interest to declare.