bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphatidylserine-Positive Erythrocytes Modulate Tissue Factor Activity In Vivo and In Vitro in Sickle Cell Disease.
Authors: Setty Yamaja B.N , Key Nigel S. , Stuart Marie J. 
Published: April 2020

Abstract
Evaluation of fluid phase markers reveals activated blood coagulation in sickle cell disease (SCD), with enhanced in vivo thrombin generation. Studies have shown that whole blood tissue factor (WTBF) activity is elevated, and that both monocytes and endothelial cells (ECs) contribute to WBTF. In a recent preliminary study, we have found an unexpected and interesting positive correlation between WBTF and the levels of both reticulocytes (R=0.6) and phosphatidylserine (PS)-positive red cells (R=0.4) in SCD and an inverse correlation with hemoglobin (R=-0.4). We have therefore evaluated potential mechanisms for erythroid modulation of WBTF. Reticulocytes being PS-positive, we initially evaluated the effects of PS-positive red cells on endothelial TF using human lung microvascular ECs and ionophore-treated PS-exposing control erythrocytes, since sickle erythrocytes also express other adhesion markers. Following incubation with appropriate treatments for 24 hours, ECs were analyzed for TF expression (with E-selectin as a positive control for EC activation). Co-incubation of PS-positive red cells (15% ±) with ECs increased endothelial expression of TF in a hematocrit-dependent manner. PS-positive cells increased TF expression by 140, 200, 260 and 320% at hematocrits of 2, 5, 10 and 20%, respectively, compared to media controls. Results similar to those observed with TF were also noted with E-selectin. PS-positive sickle erythrocytes have been classified as either type I or type II cells. Type I express low levels of PS (reticulocytes), while type II are highly PS-positive and include sickle dense cells. We next evaluated whether both type I and type II PS-positive red cells triggered endothelial TF expression. Type II cells were prepared by incubating control erythrocytes with calcium ionophore, and type I cells were generated by incubating type II cells with predetermined amounts of annexin V (100 μg annexin V per 25X106 PS-positive cells). FACS analyses demonstrated appropriate PS levels in each PS positive category. We show that type I cells had no effect. TF expression occurred following incubation of ECs with type II erythrocytes (0.21±0.06 units TF with type II vs 0.10±0.06 with PS-negative cells at a 2% hematocrit, n=5, P&lt;0.01). In final patient related studies we have observed a marked correlation between WBTF and type II PS-positive red cells (R=0.5, P&lt;0.00001, n=81). No such correlation was noted with type I. We conclude that while the reticulocyte component of the hemolytic anemia does not appear to affect WBTF, type II PS cells (including potentially dense cells) modulate WBTF activity in SCD with implications for both the thrombophilic and adhesive pathophysiologic complications of this disease entity. These studies in addition have potential implications for other disease states where erythrocyte PS exposure occurs.
Mechanisms of Activation of the FIP1L1-PDGFRα Fusion Kinase.
Authors: Stover Elizabeth H. , Cools Jan , Gilliland D. Gary 
Published: April 2020

Abstract
We recently identified the FIP1L1-PDGFRα fusion protein as a frequent cause of hypereosinophilic syndrome (HES), a disorder characterized by persistent eosinophilia and organ dysfunction. ~60% of HES patients who are sensitive to imatinib, a selective tyrosine kinase inhibitor, harbor the FIP1L1-PDGFRα fusion. The fusion is expressed as a consequence of an interstitial chromosomal deletion of human chromosome 4 that fuses a novel protein FIP1L1 to the cytoplasmic domain of the tyrosine kinase PDGFRα, a type III receptor tyrosine kinase (RTK). The fusion protein is a constitutively active kinase and has transforming properties in vitro and in vivo that are inhibited by imatinib. In order to understand the mechanism of activation of the FIP1L1-PDGFRα fusion kinase, a series of deletions of FIP1L1 were fused to PDGFRα and the fusions were tested for transforming activity. The data suggested that the FIP1L1 moiety was in fact dispensable for PDGFRα activation. For example, substitution of FIP1L1 with the Myc-epitope fused to PDGFRA still resulted in PDGFRα autophosphorylation; transformation of Ba/F3 cells to IL-3 independent growth; and induction of a myeloproliferative disease in a murine bone marrow transplant assay. Structural analyses of other type III RTKs, such as FLT3, have identified an autoinhibitory function of the juxtamembrane (JM) domain. Thus, the lack of requirement for FIP1L1 suggests that activation of the FIP1L1-PDGFRa fusion kinase might be caused by disruption of an autoinhibitory JM domain in PDGFRα. Indeed, in patients with the FIP1L1-PDGFRα fusion, the breakpoints in PDGFRA are tightly clustered within exon 12, which encompasses the JM domain. Whereas activation of most known fusion kinases relies on enforced dimerization by a fusion partner, disruption of an autoinhibitory JM domain may be an alternative mode of fusion kinase activation.
Clonal Origin of Circulating Endothelial Progenitor Cells in Patients with Myelofibrosis with Myeloid Metaplasia.
Authors: Bergamaschi Gaetano , Pramparo Tiziano , Rosti Vittorio , Ramajoli Isabella , Gregato Giuliana , Massa Margherita , Marchetti Monia , Zuffardi Orsetta , Cazzola Mario , Barosi Giovanni 
Published: April 2020

Abstract
Chronic myeloproliferative diseases are clonal disorders of the hematopoietic stem cell. Among these conditions myelofibrosis with myeloid metaplasia (MMM) is characterized by increased numbers of circulating CD34+ cells compared to other Ph-negative myeloproliferative disorders and normal controls. Increased numbers of circulating CD34+ cells are likely due to mobilization of primitive hematopoietic stem cells/progenitor cells (HSC/HPC) from the bone marrow of patients with MMM following activation of proteases and disruption of adhesion interactions, which normally result in the retention of HSC/HPC within the marrow. A more detailed characterization of circulating CD34+ cells in MMM suggested that a cell fraction which included VEGFR-2+, CD34+VEGFR-2+, CD133+, and CD34+CD133+ cells contained endothelial progenitor cells.
In the present work we cultured peripheral blood mononuclear cells (PBMNC) from three patients with MMM (one male and two females), from one female patient with essential thrombocythemia (ET) and from three normal females in the presence of fibronectin and vascular endothelial growth factor (VEGF) for 7 days, in order to obtain endothelial cell colonies. The endothelial origin of colonies was assessed by VE-cadherin and CD31 staining. Clonality of endothelial cell colonies was evaluated in female subjects by X-chromosome inactivation studies based on determination of differential DNA methylation at the two alleles of X-linked polymorphic loci (Humara or PGK). Such evaluation was performed on at least 8 single colonies and on a pool of 7–8 colonies from each subject. The male patient with MMM had a 7q34 deletion in 50 % of metaphases. The deletion resulted in loss of heterozygosity at the polymorphic marker D7S684 which was therefore evaluated on both endothelial and hematopoietic colonies grown from PBMNC. Normal subjects showed a polyclonal pattern of X-chromosome inactivation in endothelial colonies, whereas MMM and the ET female patients showed selective inactivation of the same chromosome X which was inactivated in clonal peripheral blood polymorphonuclear cells. Loss of heterozygosity at the polymorphic marker D7S684 was observed in 6/10 and 4/9 endothelial and hematopoietic colonies, respectively, from the male patient with the 7q34 deletion. Our results suggest that in MMM, and possibly in other chronic myeloproliferative diseases, circulating endothelial progenitor cells are clonal and the disease is due to transformation of a hematopoietic stem cell which also retains endothelial differentiation potential.
Aberrant DNA Methylation Patterns in Chronic Idiopathic Myelofibrosis.
Authors: Jost Edgar , Gehbauer Gerald , Maintz Christoph E. , Jousten Peter , Habets Leo , Herman James G. , Osieka Rainhardt , Galm Oliver 
Published: April 2020

Abstract
Chronic idiopathic myelofibrosis (CIMF) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, angiogenesis and extramedullary hematopoiesis. No specific genetic defect underlying the disease has been identified so far. The spectrum of cytogenetic abnormalities in CIMF includes del (13q), del (20q) and partial trisomy 1q as well as abnormalities of chromosomes 1, 7 and 9. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function in cancerogenesis. In order to investigate the role of DNA methylation changes in the pathogenesis of CIMF, we have analyzed the methylation status of the promoter-associated CpG islands of 13 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in peripheral blood cells from 20 adult patients with CIMF. The frequency of aberrant methylation among the patient samples was 25.0 % (5/20) for SOCS-1 and 5.0 % (1/20) for E-cadherin, MGMT, p73 as well as TIMP-2. There was no hypermethylation of p15, p16, RARbeta, DAP kinase 1, SOCS-3, hMLH1, TIMP-3 and RASSF1A. We detected at least one hypermethylated gene promoter region in 35.0 % (7/20) of the primary patient samples. Our data indicate that hypermethylation of tumor suppressor genes is a common event in CIMF. Epigenetic modification of genes regulating growth factor signaling, cell adhesion and DNA repair may, in addition to genetic aberrations, contribute to the malignant phenotype in CIMF. The exploration of our growing knowledge about epigenetic aberrations in tumorigenesis may help develop novel strategies in diagnosis and treatment of CIMF for the future.
PKC412 Effectively Inhibits Constitutively Activated FGFR3 Mutants in Murine Leukemia Models and t(4;14) Myeloma Cell Lines.
Authors: Chen Jing , Lee Benjamin H. , Williams Ifor R. , Kutok Jeffery L. , Duclos Nicole , Cohen Sarah , Adelsperger Jennifer , Okabe Rachel , Coburn Allison , Moore Sandra , Huntly Brian J.P. , Mitsiades Constantine S. , Anderson Kenneth C. , Griffin James D. , Fabbro Doriano , Gilliland D. Gary 
Published: April 2020

Abstract
Reccurent translocation t(4;14) associated ectopic expression of FGFR3, sometimes containing the activation mutation K650E (TDII), has been identified in 25% of human multiple myeloma (MM) patients and cell lines. However, current empirically-derived cytotoxic chemotherapy does not effectively treat this disease. One potential therapeutic strategy of treating MM is to inhibit the tyrosine kinase activity of FGFR3. In this report, we evaluated the efficacy of PKC412 (N-benzoyl-staurosporine), a small molecule tyrosine kinase inhibitor, for the treatment of FGFR3 mutants induced diseases. PKC412 effectively inhibits the tyrosine kinase activity and activation of downstream effector pathways of FGFR3 TDII or the constitutively activated TEL-FGFR3 fusion that was reported in a subtype of human peripheral T-cell lymphoma (PTCL), as well as proliferation of hematopoietic Ba/F3 cells transformed by the FGFR3 mutants. Furthermore, PKC412 drastically inhibits proliferation of four different multiple myeloma-derived primary cell lines that are associated with t(4;14) and expression of dysregulated FGFR3. Moreover, oral-gavage treatment with PKC412 resulted in statistically significant prolongation of survival in the murine bone marrow transplant (BMT) models of FGFR3 TDII-induced pre-B cell lymphoma or TEL-FGFR3 fusion-induced myeloproliferative disease, which suggests suitable pharmacokinetic and toxicity profiles of PKC412 for clinical use. Together, our data establish the small molecule inhibitor PKC412 as a molecularly targeted therapy for multiple myeloma and other human malignancies expressing activated FGFR3.
Effects of the Indazolylpyrimidine GW786034 on Angiogenesis and MM Cell Growth: Therapeutic Implications.
Authors: Podar Klaus , Simoncini Melissa , Le Gouill Steven , Tai Yu-Tzu , Kumar Shaji , Tassone Pierfrancesco , Catley Laurence P. , Pandite Lini N. , Kumar Rakesh , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C. 
Published: April 2020

Abstract
Vascular endothelial growth factor (VEGF) and its receptors play an important role in the pathogenesis of multiple myeloma (MM). Specifically, VEGF, which is present in the MM bone marrow (BM) microenvironment, induces neovascularization; triggers tumor cell growth, survival, and migration; inhibits dendritic cell maturation; and induces osteoclastogenesis. Therefore, the VEGF receptor provides a potential novel therapeutic target in MM. Previous studies showed that GW654652, a pan VEGFR inhibitor, inhibits growth and migration of MM tumor cells (Podar et al. Blood. 2004; 103:3473–3479.). Here we describe the effects of GW786034, a derivative of GW654652, on MM cells to support its potential clinical evaluation in MM patients. GW786034 inhibits VEGF- triggered Flt-1 phosphorylation and activation of downstream signaling molecules, induces concentration-dependent MM cell and HUVEC apoptosis, and inhibits VEGF- triggered MM cell migration. Specifically, GW786034 induces cleavage of caspase-8 and PARP, but not caspase-9, and induces downregulation of the pro-apoptotic molecules survivin, cIAP1, 2, and Mcl-1. Furthermore, GW786034 decreases proliferation of MM cells induced by their adhesion to BMSCs, and inhibits VEGF-induced upregulation of ICAM-1 and VCAM-1, thereby abrogating their adhesion to HUVECs. Consistent with these results, GW786034 inhibited both tubuli formation and accumulation of tumor cells at vascular branching points in a Matrigel tube forming assay. Finally, GW786034 sensitizes both MM cells alone and tumor cells bound to BMSCs or HUVECs to melphalan and bortezomib. Taken together, these studies support clinical evaluations of GW786034 either alone or in combination with other agents in MM patients.
Effect of Thalidomide and Revlimid on the Gene Expression That Reveals Molecular Circuitries Involved in T Cell Co-Activation.
Authors: Prabhala Rao , Cheema Puneet , Shammas Masood A. , Song Weihua , Blanchard Elizabeth , Chauhan Dharminder , Neri Paola , Tassone Pierfrancesco , Stirling David , Anderson Kenneth , Munshi Nikhil C. 
Published: April 2020

Abstract
Thalidomide (Thal) and its immunomodulatory analog, Revlimid (Rev) have demonstrated anti-myeloma activity in phase II and III studies and part of their activity is immune-mediated. We have previously demonstrated co-stimulatory effects of Thal and Rev on T cells mediated via B7-CD28 activation pathway. To understand the molecular events involved in T cell co-stimulation, we investigated the effects of Thal and Rev on the genome-wide gene expression profile of T cells with or without anti-CD3 activation. Normal PBMC were incubated with Thal or Rev in the presence or absence of anti-CD3 antibodies (200ng/ml) for 3 or 24 hours at 37oC and total RNA was analyzed using U133 GeneChip (Affymetrix). Although Thal and Rev do not show significant effect on T cell proliferation without CD3 stimulation, we observed up-regulation (&gt;2-folds) of 56 genes and down-regulation of 42 genes at 3 hours, and up-regulation of 41genes and down-regulation of 35 genes at 24 hours with Rev. Significant up-regulation of IFN-α (10-folds) was observed at 3 hours. Up-regulation of chemokine CXC5 (9 fold); IFN-β (5 fold), IL-7 (5 fold) and IL-2 (3 fold); and signaling genes like JK 3 (10 folds) and MAPKK5 (5 folds) was observed at 24 hours with Rev. Following anti-CD3 activation, Rev induced significant upregulation of 59 and 48 genes and down-regulation of 126 and 61 genes at 3 and 24 hours respectively. These include down-regulation of Th2 cytokine genes IL-5(5 folds) and TGF-β (2 folds), and apoptotsis related genes, TNFSF12 (4 folds) and TNFRSF 6b (4 folds). Genes involved in B7-CD28 activation pathway and up-regulated include CXC 5 (9 folds), IL-8 (5 folds) and IL-3 (4 folds). Interestingly, p52, which is a key molecule in CD28 signaling, was elevated 18 folds. Additionally less than 2 fold increase was observed in other CD28 signaling genes like IKK, Vov, and RelA (p65). Rev also down-regulated, at 24 hours, the expression of IL-10 (6 folds), IL-17 (3 folds) and TGF-β (3 folds) which allows sustained co-stimulation. Similar changes with lesser magnitude were observed with Thal. These results provide insight into early molecular changes induced by Thal and Rev that is important for co-stimulation and suggest new molecular targets to generate effective immune responses. Moreover, these observations facilitate the pre-clinical rationale for the use of these immunomodulatory compounds to improve clinical outcome in myeloma.
CD133-Expressing Cells with High Aldehyde Dehydrogenase Activity Represent a Primitive Human Hematopoietic Stem Cell Population with Enhanced Repopulating Potential.
Authors: Hess David A. , Wirthlin Louisa , Craft Timothy P. , Meyerrose Todd E. , Nolta Jan A. 
Published: April 2020

Abstract
Human hematopoietic stem cells (HSC) are commonly purified by the phenotypic expression of cell surface markers such as CD34. We have recently characterized a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Lin− cells with high ALDH activity (ALDHhiLin−) represented approximately 0.1% of total UCB mononuclear cells and demonstrated enriched expression of the primitive HSC markers CD34 (91.0±2.9%) and CD133 (70.9±4.0%). Most notably, clonogenic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin− population. Here, we have further purified the ALDHhiLin− population based on the expression of CD133, or prominen, a non-restricted surface molecule expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133− and ALDHhiCD133+ cells, sorted to &gt;95% purity, represented 14.7±2.1% and 23.2±4.3% of the total human UCB Lin− population respectively (n=6). Both ALDHhiCD133−Lin− and ALDHhiCD133+Lin− cells demonstrated clonogenic progenitor function in vitro. However, total colony production was significantly enhanced (p&lt;0.05) in ALDHhiCD133−Lin− cells (1 CFU in 3.5 cells, n=5) when directly compared to ALDHhiCD133+Lin− cells (1 CFU in 10 cells, n=6). Human hematopoietic repopulation was consistently observed in the bone marrow, spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 103 ALDHhiCD133+Lin− cells, whereas transplantation of up to 2x105 ALDHhiCD133−Lin− cells produced no detectable human engraftment. BM repopulation at limiting dilution demonstrated increased NOD/SCID repopulating ability elicited by ALDHhiCD133+Lin− cells when directly compared to CD133+Lin− cells not selected for ALDH activity. Repopulating ALDHhiCD133+Lin− cells differentiated into cells expressing markers for mature myeloid (CD33, CD14) and B-lymphoid (CD19, CD20) cells. ALDHhiCD133+Lin− cells also supported the maintenance of primitive cell phenotypes up to 8 weeks post-transplantation (2.4±0.7% CD34+CD38−, 5.5±0.6% CD34+CD133+, n=6) and the repopulating function of these cells are currently being confirmed by secondary transplantation. We are also investigating the ability of ALDHhiCD133+Lin− cells to mediate tissue repair in non-hematopoietic organs. Fractionation of human HSC based on combined expression of CD133 and high ALDH activity provides a rigorous selection of purified hematopoietic stem and progenitor cells that maintain primitive characteristics after transplantation, and may be considered a potential alternative to CD34+ cell isolation.
In Vitro Activity of a Novel Fully Human Anti-CD40 Antibody CHIR-12.12 in Chronic Lymphocytic Leukemia: Blockade of CD40 Activation and Induction of ADCC.
Authors: Tong Xia , Georgakis Georgios V. , Li Long , Susan O’Brien , Anas Younes , Mohammad Luqman 
Published: April 2020

Abstract
B-cell chronic lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5+ B cells. However when cultured in vitro CLL cells die quickly by apoptosis. Protection from apoptosis in vivo is believed to result from supply of survival signals provided by cells in the microenvironment. We and others have previously reported that CLL cells express CD40 receptor, and that CD40 stimulation of CLL cells may rescue CLL cells from spontaneous and drug-induced apoptosis in vitro. These observations suggested that blocking CD40-CD40L pathway might deprive CLL cells from survival signals and induce apoptosis. To test this hypothesis, we have generated a fully human anti-CD40 blocking monoclonal antibody in XenoMousemice (Abgenix, Inc.). The antibody CHIR-12.12 was first evaluated for its effect on normal human lymphocytes. Lymphocytes from all 10 healthy blood donors did not proliferate in response to CHIR-12.12 at any concentration tested (0.0001 mg/ml to 10 mg/ml range). In contrast, activating CD40 on normal B-lymphocytes by CD40L induced their proliferation in vitro. Importantly, CHIR-12.12 inhibited CD40L- induced proliferation in a dose dependent manner with an average IC50 of 51 ± 26 pM (n=10 blood donors). The antagonistic activity of CHIR-12.12 was then tested in primary CLL samples from 9 patients. CHIR-12.12 alone did not induce CLL cell proliferation. In contrast, primary CLL cells incubated with CD40L, either resisted spontaneous cell death or proliferated. This effect was reversed by co-incubation with CHIR-12.12 antibody, restoring CLL cell death (n=9). CHIR-12.12 was then examined for its ability to lyse CLL cell line EHEB by antibody dependent cell mediated cytotoxicity (ADCC). Freshly isolated human NK cells from normal volunteer blood donors were used as effector cells. CHIR-12.12 showed lysis activity in a dose dependent manner and produced maximum lysis levels at 0.1 mg/ml. When compared with rituximab, CHIR-12.12 mediated greater maximum specific lysis (27.2 % Vs 16.2 %, p= 0.007). The greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on CLL cell line compared to CD20 molecules. The CLL target cells expressed 509053 ±13560 CD20 molecules compared to 48416 ± 584 CD40 molecules. Collectively, these preclinical data suggest that CHIR-12.12 monoclonal antibody may have a therapeutic role in patients with CLL.