bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.
Authors: Fierro Fernando , Illmer Thomas , Jing Duhoui , Le Coutre Philip , Ehninger Gerhard , Boxberger Sabine , Bornhaeuser Martin 
Published: October 2019

Abstract
Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC.
Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay.
In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.
Genomics Analysis of Freshly-Purified Human Marrow Stromal Cells (MSCs): Comparison with Hematopoietic Lineage Cells and Culture-Expanded MSCs.
Authors: Jones Elena A. , Kinsey Sally E. , English Anne , Field Sarah , Rawstron Andy , Jones Richard A. , Strazsynski Liz , Rundlett Stephen , Jain Selina , Deans Robert , Emery Paul , Ponchel Frederique , McGonagle Dennis 
Published: October 2019

Abstract
Gene and antigen expression profile of bone marrow MSCs in culture has been investigated, but these characteristics of MSCs in vivo are poorly understood. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (f-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (e-MSCs). Following enrichment with Anti-Fibroblast MicroBeads, f-MSCs were purified by cell sorting as CD45lowCD235a-LNGFR+ [1] and HLCs as CD45+CD235a+LNGFR-. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct comparison with qPCR. Duplicate hybridizations were used to define 3 relative zones of sensitivity and select appropriate thresholds for significant changes in expression. The microarray data were validated by qPCR using several mesenchymal lineage genes (including Sox9 and Runx2) and by flow cytometry for several known MSC surface markers (including CD73, CD90, CD146, CD10, CD63 and CD130). Expression data were further searched for gene ontology using a custom-made, Perl-based program. Compared to HLCs, f-MSCs over-expressed genes encoding extracellular matrix proteins (including versican, fibronectin and collagen I), stromal cytokines/chemokines (including SDF-1, IL-7 and CXCL10), and molecules associated with cell junctions and communication with endothelium (including connexin 43, VCAM-1 and angiopoietin-1). Osteogenesis-related genes (BMPs and their receptors, alkaline phosphatase, osteonectin, osteocalcin, osteomodulin and others) were more abundant in f-MSCs than in HLCs. Some mature fat tissue products were also quite abundant in f-MSCs (particularly lipoprotein lipase and adipsin), suggesting the presence of cells committed to adipogenesis. Of note, the expression of stroma- and fat-related transcripts was significantly reduced in e-MSCs compared to f-MSCs. Novel surface antigens of f-MSCs identified by microarray, Junctional Adhesion Molecule 2 (normally expressed on vascular endothelial cells), Leptin receptor (implicated in fat and bone metabolism), and CD81 (tetraspanin family adhesion molecule) were confirmed at the protein level by 6-colour flow cytometry. Altogether, this work confirmed the CD45lowCD235a-LNGFR+ phenotype f-MSCs in human bone marrow, revealed a unique expression profile of f-MSCs in vivo and described new surface markers of f-MSCs. These data have implications for our understanding of the in vivo biology of non-hematopoietic bone marrow stromal cells and their role in hematopoiesis and skeletal remodelling in health and disease.
A Role for DNA Double Strand Breaks in the Sensitivity of AML Cells to Clofarabine: Enhanced In Vitro Sensitivity in Primary AML Cells with FLT3 Mutations.
Authors: Pallis Monica , Grundy Martin , Seedhouse Claire , Pimblett Heather , Russell Nigel 
Published: October 2019

Abstract
Clofarabine is a purine nucleoside analogue which has been incorporated into several therapeutic trial protocols for the treatment of leukaemias including acute myeloid leukaemia (AML). The aims of the study are to ascertain mechanisms of clofarabine action in AML using cell lines and presentation samples from patients. We measured apoptosis by TdT assay, cytochrome C release and flow cytometric assays of the mitochondrial membrane potential probe DiOC6. To study the effects of clofarabine on DNA synthesis and DNA double strand breaks, we used bromodeoxyuridine (BrdU) and H2AX assays respectively. Equitoxic doses were established that caused approximately 20% cell death in the AML cell lines HL-60 (0.3 μM), KG1 (1 μM) and MV4.11 (1 μM) after 6 hours of continuous exposure. At these doses, clofarabine induced apoptotic DNA nicks, as measured by the TdT assay, and Cytochrome C release in all three cell lines. However, clofarabine-induced mitochondrial hyperpolarisation and depolarisation were found to be cell-type specific, occurring in HL-60 but not MV4.11 or KG1 cells, i.e. mitochondrial membrane depolarisation is not an essential part of the mechanism of clofarabine-induced apoptosis. Following clofarabine treatment, DNA synthesis was reduced by 91% within 1 hour in KG1 cells and by 80% in HL-60, but by only 9% in MV4.11. Out of the three cell lines, MV4.11 cells, which have a FLT3 internal tandem duplication (ITD), were the only ones to completely repair DNA double strand breaks following clofarabine removal. In 12 samples from patients with AML, the proportion of cells which incorporated BrdU in a 45 minute assay - a measure of the rate of cell cycling - differed considerably between samples, from 0.8% to 23%, median 13%. Cell death induced by clofarabine was correlated with the cycling rate of untreated cells (P=0.016). All 12 samples showed inhibition of DNA synthesis within 60 minutes of clofarabine treatment (range 20%–92% inhibition, median 54%). Mitochondrial membrane hyperpolarisation and depolarisation were not observed in patient cells. However, DNA double strand breaks were induced by clofarabine in patient cells. Paradoxically, i.e. in contrast to the results seen with the MV4.11 cell line, toxicity was greatest in samples with a FLT3 mutation (P=0.007). In conclusion, the FLT3 mutant MV4.11 cell line effectively repairs clofarabine-induced double strand breaks. Cell death in patient cells cultured with clofarabine is correlated with the presence of a FLT3 mutation. As we have previously established that AML samples with FLT3 mutations have upregulated DNA repair activity, this paradox might be explained by cycles of attempted DNA repair frustrated by renewed clofarabine incoporation into DNA, thus increasing the toxicity of the drug.
Genetic and Biochemical Biomarkers of IMPDH Inhibition in Phase I Dose Escalation of AVN-944 for Hematologic Malignancies.
Authors: Strovel Jeffrey W. , Chakiath Marion , Natarajan Pachai , Lawrence Tammy , Zuck Karina M. , Urbani Lenore , Mitchell Beverly S. , Bol David K. 
Published: October 2019

Abstract
AVN-944 is an orally bioavailable inhibitor of inosine monophosphate dehydrogenase 1 and 2 (IMPDH). IMPDH2 is highly upregulated and required for the de novo production of guanine nucleotides in many malignancies, including hematologic cancers. AVN-944 is currently in a repeat-dose, dose escalation trial in patients with advanced hematologic cancers including AML, CLL, Multiple Myeloma, and lymphomas. In addition to the objective of defining the maximum tolerated dose and repeat dose pharmacology of AVN-944 (given bid for 21 days on a 28 day cycle), we have examined a comprehensive set of pharmacodynamic biomarkers. We have isolated peripheral blood samples from all patients in this trial both before and after receiving AVN-944. These samples are being used to determine the effects of the drug on GTP pools and IMPDH enzyme activity, and to correlate these changes to the genetic response of the circulating cells using gene expression detection technologies. We previously identified a set of 32 genes that respond to AVN-944 exposure in a multitude of laboratory settings including different cell types, different concentrations and timepoints. These genes relate directly to cellular pathways and functions dependent upon, or required for, guanine nucleotide biosynthesis, including purine and pyrimidine biosynthesis, mitochondrial functions, metabolic pathways and energy production, cell proliferation and the apoptotic cascade. Examination of the behavior of these genes in patient samples from this phase I trial show a dynamic response of subsets of these genes depending on
the type of malignancy, the predominant cell type in the peripheral blood, and the dose of AVN-944 received.
One set of 9 disease-related genes show changes in expression following drug administration in leukemia patients, while another set of 6 genes designed to report pharmacodynamic effects in normal cells consistently shows changes in expression in the peripheral blood lymphocytes of multiple myeloma patients. In total, 27 of 34 selected genes examined by qPCR showed changes following dosing of AVN-944 at the starting dose of 25 mg p.o. Increasing doses resulted in increases in the number of genes changing per individual, and/or an increase in the magnitude of change in a gene-dependent manner. These genetic markers, correlated with biochemical effects of the drug on protein function and guanine nucleotide levels, can be used as important biomarkers in selecting the optimal dose with which to initiate phase II studies with this drug.
Urinary Hepcidin Excretion in Patients with Myelodysplastic Syndrome (MDS) and Myelofibrosis (MF).
Authors: Winder Asher , Lefkowitz Rafi , Ghoti Hussam , Leiba Merav , Ganz Tomas , Nemeth Elizabeta , Rachmilewitz E.A. 
Published: October 2019

Abstract
One of the main features of patients with Myelodysplastic Syndrome (MDS) is severe anemia which requires multiple blood transfusions with consequent iron overload. Moreover, iron metabolism is impaired particularly in refractory anemia with ring sideroblasts (RARS). Hepcidin is a small, cysteine-rich cationic peptide produced by hepatocytes, secreted into plasma and excreted in the urine. Hepcidin control iron absorption in the gut, release of iron from macrophages and is a key regulator of iron metabolism. Patients with iron overload have elevated urinary hepcidin levels but patients with anemia or increased erythropoietic precursors have suppressed hepcidin. The purpose of this study was to measure hepcidin excretion in the urine of patients with MDS and Myelofibrosis (MF). The assay of urinary Hepcidin was performed by the method of Nemeth et al (J. Clin.Invest113:1271–1276, 2004) and expressed per mg urinary creatinine. The samples were obtained from 20 patients, 16 with MDS and 4 with MF. Their subtypes and age, ranging from 54 to 87 years, are shown in the table. Nine MDS patients received less than 10 units of blood and the rest, between 13 and 147 units. Seven MDS patients had ferritin levels above 1000 ng/ml and their transferrin saturation in most of them was more than 50%. The urinary excretion of hepcidin was low (less then 1 ng/mg creatinine) in 3 MDS and 3 MF patients, while in the rest, with the exception of 2 patients, the levels were less than 50 ng/mg creatinine. Although the degree of iron overload was not related directly to the subtype and to the number of blood units, in some patients mainly with RARS it was quite severe. More importantly, the urinary hepcidin excretion had a more consistent pattern of low levels in all the patients even in those with high ferritin or high transferrin saturation levels. Hepcidin concentrations correlated well with serum ferritin (r2 = 0.62 on a log-log plot) but the slope of the correlation was only 0.05 (normal ~ 1) indicating marked suppression of hepcidin relative to ferritin. Suppression of hepcidin in MDS and MF suggests that hepcidin deficiency could worsen the maldistribution of iron from the nontoxic form in macrophages to toxic forms in parenchymal cells, and that intestinal iron absorption as well as transfusional iron could contribute to their iron overload.
Age Gender Diagnosis/ MDS WHO subtype No. of transfused PC Transferrin saturation % Ferritin ng/ml Hepcidin ng/mg creat 76 F RA 7 65 422 32 76 M RA 2 28 87 10 76 M RA 0 44 1350 20 65 F RA 0 43 420 24 69 M RARS 147 87 5600 137 69 F RARA 76 45 2100 49 84 F RARS 87 57 4450 16 77 F RCMD 4 16 90 <1 75 M RCMD 4 88 307 21 77 F RCMD 65 48 2870 138 72 M RAEB I 5 44 102 <1 87 M RAEB I 62 87 3190 48 77 M RAEB II 13 20 152 20 79 M RAEB II 0 26 202 <1 75 F RAEB II 4 9 104 23 71 M RAEB II 80 53 2100 35 68 F MF 17 17 111 13 73 M MF 26 27 618 27 71 F MF 7 5 19 <1 54 F MF 0 12 42 <1
Expression of DNMT Isoforms Is Differentially Associated with Aberrant Promotor Methylation in MDS Hematopoietic Progenitor Cells during Lineage Specific Differentiation.
Authors: Hopfer Olaf J. , Komor Martina , Koehler Ina S. , Schulze Matthias , Freitag Claudia , Hoelzer Dieter , Thiel Eckhard , Hofmann Wolf-Karsten 
Published: October 2019

Abstract
Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.
Low Doses of Thalidomide (Thal) in Low Risk MDS with Transfusion-Dependant Anemia: The GFM THAL-SMD-200 Trial.
Authors: Tamburini J. , Elie C. , Vassilief D. , Quarre M.C. , Beyne-Rauzy O. , Gardembas M. , Aouba A. , Berthou C. , Sanhes L. , Guerci A. , Stamatoullas A. , Bauduer F. , Sotto J.J. , Legros L. , Vaultier S. , Fenaux P. , Dreyfus F. , Bouscary D. 
Published: October 2019

Abstract
Background: Thal has shown some efficacy in low risk MDS, but with dose-limiting toxicity requiring discontinuation in > 50% pts (Bouscary D, BJH, 2005, 131, 609 and other groups). Thus we assessed Thal at lower dose in such MDS.
Methods: From Jan 2003 to Oct 2005, we performed a prospective study of low dose Thal in 87 MDS pts with transfusion-dependant anemia (THAL-SMD-200 trial). The first 59 pts received Thal at 200 mg/d, whereas the last 28 pts received 50 mg/d. Responses (IWG) were evaluated at week 12.
Results: 48 M and 39 F pts were included. Median age 73.6 years. IPSS Low 34 pts, INT-1 44 pts, INT-2 9 pts. Mean number of RBC units transfused within 2 months of inclusion 6 (2–16). Median ANC, plts, Hb were 1960/μl (0–24160), 164 000/μL (1 000–789 000) and 8,2 (5,1–14,7) resp. 14 (16%) patients had del 5q. Thirty two (37%) pts stopped Thal before week 12, 19 (32%) and 13 (46%) patients in the 200mg/d and the 50mg/d groups resp. Reasons for interruption were: important toxicity in 23 (72%) (mainly dizziness, fatigue, sleepiness), patient’s decision in 6 (19%), progression to AML in 3 (9%) and death in 1 (3%). 3 pts developed deep venous thrombosis in the 200mg/d group (none in the 50mg/d group). 55 patients completed 12 weeks of treatment, 22 of them (40%, ie 25% of pts included) had an erythroid response, including 12 major responses (HI-EM) and 10 minor responses (HI-Em). We observed 17 (43.5% of pts who completed 12 weeks, 28% of all pts) and 5 (29.4% of pts who completed 12 weeks, 18% of all pts) erythroid responses in the 200mg/d and the 50mg/d group respectively, not statistically different (p = 0,27). Erythroid response was seen in 15 (44%) patients with IPSS low, versus 6 (14%) in INT-1 and 1 (11%) in INT-2 resp (p = 0.005), whereas no other prognostic factor of response was found. No neutrophil or platelet response was seen.
Conclusion: Thal at 200mg/d improved erythropoiesis in 40% of the pts who reached week 12 (28% of included pts). Side effects leading to treatment interruption < week 12 occurred in 32% of pts, ie a similar efficacy and somewhat better tolerance than in our experience with higher doses. Most erythroid responses occurred in IPSS Low pts. On the other hand, Thal at 50mg/d gave a similar incidence of side effects as 200mg/d, with slightly lower but not statistically different erythroid response rate, and thus brought no obvious benefit over 200mg/d. Longer follow up of pts who continued Thal will be presented.
Targeting Hepcidin with Antisense Oligonucletides Improves Anemia Endpoints in Mice.
Authors: Crosby Jeffrey R. , Gaarde William A. , Egerston Jarrett , McKay Robert , Sun Yingqing , Freier Susan , Watt Andy , Hung Gene P. , Monia Bret P. 
Published: October 2019

Abstract
Anemia is one of the more common blood disorders and is associated with a number of diseases, including chronic kidney disease, chronic inflammation, and certain types of cancer. Under these conditions iron is essential as it is required for erythroid progenitor cell proliferation and red cell function. Hepcidin is a liver-derived growth factor that regulates iron absorption in the GI tract and iron absorption and release in tissues. Furthermore, hepcidin overexpression has been strongly linked mechanistically as a mediator of decreased iron availability and anemia. We have utilized an antisense approach to investigate the role of hepcidin in animal models of anemia and as a potential therapeutic approach for the treatment of this disorder in humans. Second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotides (ASOs) were screened in isolated primary mouse hepatocytes, followed by in vivo screening in mice, for the ability to reduce hepcidin mRNA levels. ASO treatment resulted in a reduction of hepcidin mRNA in liver which was associated with a significant increase in serum iron levels. The best hepcidin ASO was then tested in a mouse model of turpentine induced hypoferremia and anemia to determine the role of hepcidin in regulating serum iron and anemia endpoints. Mice were treated (I.P. twice/weekly) with hepcidin or control ASO for two weeks at varying doses prior to a single subcutaneous injection of turpentine. Turpentine treatment 16 hours post-injection produced a significant reduction in serum iron levels and at two weeks resulted in reduced RBC numbers, hematocrit and hemoglobin levels. Treatment with the hepcidin ASO resulted in a dose dependent improvement in all of these endpoints while the control oligonucleotide had no effect. Studies are in progress to further characterize the pharmacological activity of hepcidin ASO in additional models of anemia and results from these on-going studies will also be presented.