bluuebunny/crossref_metadata_2025_split huggingface dataset:
Live Attenuated Salmonella Carrying Platelet Factor 4 cDNAs as Radioprotection and Anti-Tumor Angiogenesis Agents.
Authors: Han Zhong Chao , Liu Bin , Zhao Lihua 
Published: October 2019

Abstract
In cancer therapy, specific radioprotection of normal tissue and antiangiogenesis are the ways to increase the therapeutic gain. Here we describe a novel gene therapy, which uses attenuated salmonella SL3261 as oral vectors carrying with cDNA of platelet factor 4 (PF4) or that of a truncated PF4. After oral administrations of attenuated salmonella carrying with cDNA of PF4 or truncated PF4, the survival rate of mice which received sublethal total body irradiation was improved by 50%, In comparison with the control mice, the bone marrow cells obtained from the mice of experimental group increased (13.2±8.3, 15.7±1.5 vs 4.1 ± 2.0 P<0.05) at day 7 after TBI, and the number of HPP-CFC of bone marrow cells also increased significantly (15.7±9, 11.7±5 vs 4.3±4.1 P<0.05) at day 7, suggesting a stimulating effect of PF4 on hematopoietic recovery. This gene therapy also caused significant tumor regression. The microvessel density (MVD) of tumors was significantly decreased in the group of treated mice compared to controls (4.25±0.96, 4.08±0.56 vs 11±0.83 P<0.05). Analysis TUNEL kit revealed an increase in the number of apoptosis cells in tumors of mice treated by SL3261 carrying with cDNA of PF4 or a truncated PF4. GFP expression and gene integration were detected in the liver, kidney, spleens, intestine, peripheral blood, bone marrow and tumors samples of the SL3261 treated mice, and the expression of GFP was higher in tumors than that in other tissues. These data demonstrate for the first time a dual biological function of PF4 against tumor growth and radiation injury. These results also demonstrate that attenuated salmonella can be used in vivo as a DNA delivery vector
E2F4 Plays a Critical Role in Early B-Cell Development.
Authors: Smith Clayton , Glozak Michelle , Gasparetto Maura , Rempel Rachel , Domens Jos , Cress Doug , Nevins Joseph 
Published: October 2019

Abstract
The E2Fs are important mediators of cell cycle control, DNA synthesis and apoptosis in many cell types. Recently E2F4 has been shown to play a role in hematopoietic cell growth and development (Rempel et al. Mol Cell, 6 p293, 2000). Here we report the effects of loss of E2F4 specifically on B-cell development. E2F4−/− mice have a partial block in early B-cell development prior to immunoglobulin gene rearrangement. The block is intrinsic to B-cell progenitors rather than secondary to micro-environmental effects since it occurs following transplant of E2F4−/− marrow into wild type recipients. Increases in apoptosis and abnormalities in cell cycle progression were found in B220+CD43+ B-cells of E2F4−/− mice indicating that E2F4 plays an important role in these processes in early B-cells. Expression of a variety of genes important in B-cell development including E2A, RAG, IL-7, EBF and Pax-5 were decreased in early E2F4−/− B-cells. In contrast, Id1 and Id2, regulators of a variety of genes critical to B-cell development, were relatively over-expressed in early E2F4−/− B-cells while Id3 was relatively under-expressed in these cells. E2F binding sites were identified in the Id2 and Id3 promoters and E2F4 was found to directly bind to these promoters in splenic B-cells. These findings suggest that E2F4 may also regulate early B-cell development by directly and indirectly modulating expression of the genes critical to B-cell differentiation. Together, these observations indicate that E2F4 is a critical mediator of early B-cell development via its effects on multiple pathways including those involved with apoptosis, cell cycle progression and differentiation. These findings also suggest that the E2Fs may serve to link cell survival and proliferation pathways to differentiation pathways in early B-cells and perhaps other cells aswell.
Correction of a Murine Model of Von Willebrand Disease by Gene Transfer.
Authors: Pergolizzi Robert G. , Dragos Rachel , Chan Diane , Wagner Denisa D. , Lenting Peter J. , Crystal Ronald G. 
Published: October 2019

Abstract
Von Willebrand disease (VWD) is the most common inherited bleeding disorder affecting 1 to 3% of the U.S. population. It is caused by defects in expression and processing of Von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis, that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. Since it is established that 1–5% of normal levels of VWF is sufficient to ameliorate the bleeding phenotype, the possibility that corrective levels of circulating VWF could be produced using a gene transfer strategy was investigated. However, since the normal sites of VWF production, endothelial cells, megakaryocytes and platelets, are difficult to target with traditional gene transfer methodologies, we explored the strategy of using gene transfer to the liver, an easily transduced ectopic site for VWF production. The study employed a murine “knockout” model of VWD created by disrupting the naturally occurring VWF gene (VWF-KO mice) which exhibit defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events closely mimicking severe human VWD. Transected-tail wounds in untreated VWF-KO mice will bleed to death unless cauterized, whereas similar wounds in WT mice stop bleeding in <3 min. To investigate the impact of VWF gene transfer on this phenotype, a plasmid containing the complete murine VWF cDNA under the control of a cytomegalovirus immediate/early promoter/enhancer, was delivered to the VWF-KO mice intravenously. An identical plasmid containing the cDNA for green fluorescent protein (GFP) and saline were used as controls. Bleeding time after wounding was determined 24, 48 or 72 hr after transfer of 10 to 250 μg of plasmid. Bleeding time was normalized by 48 hr after delivery of 50 μg or more of VWF cDNA, but not by GFP cDNA. Western analysis of plasma from animals receiving VWF cDNA, but not GFP cDNA, revealed high-molecular weight VWF multimers similar to the pattern observed in wild type mice. These data suggest that it may be possible to correct VWD using gene therapy delivered to tissues other than the naturally occurring sites of VWF manufacture, and that the liver is capable of expressing, processing and secreting VWF in a physiologically active form.
Eicosapentaenoic Acid (EPA) Attenuates the Anemia Due to Ribavirin/Interferon a Treatment in Patients with Chronic Hepatitis C.
Authors: Seki Ritsuko , Okamura Takashi , Ide Tatsuya , Maruyama Toru , Fujino Takehiko , Uyesaka Nobuhiro , Sata Michio 
Published: October 2019

Abstract
Ribavirin(RBV)is a water soluble synthetic guanosine analog that exerts antiviral activity against DNA and RNA viruses after intracellular phosphorylation. Current studies indicate that combination therapy with RBV and Interferon a (IFNa) is associated with higher rates of sustained virological and biochemical response compared to IFN a monotherapy for chronic hepatitis type C (CHC). Hemolytic anemia is the major side effect of the therapy, with 67% of treated patients developing anemia. The exact mechanism for RBV-related anemia is unknown, but it is thought to be decreasing deformability of eythrocytes resulting from accumulation of phosphorylated RBV in erythrocytes. Highly purified eicosapentaenoic acid (EPA) is widely used for the treatment of hyperlipidemia and atherosclerosis in Japan. We have investigated that EPA has a beneficial effect in patients with RBV-related anemia (Int J Mol Med11:729–732, 2003).
Our aim of this study is to establish the safer treatment of RBV/IFNa using EPA for CHC patients and elucidate the mechanism of EPA action for improving anemia.
[Patients and methods] Thirty three patients with CHC were randomized to RBV/IFNa therapy (800mg/6MU/day) with or without EPA (1800mg/day) for 12 weeks (14:+EPA; 19:-EPA). Erythrocyte filterability (Nickel mesh filtration method; Jpn J Physiol53: 481-486, 2003) was measured before and after 12 weeks of treatment in 8+EPA and 9 -EPA patients. Statistical analysis was performed by Wilcoxon and ANOVA test.
[Result] As shown in Fig. 1, hemoglobin (Hb) levels decreased in both groups by the treatment. However, decrease of Hb levels significantly attenuated by the addition of EPA at 4 and 8 weeks after initiation of the treatment. This effect continued for 12 weeks. Initial mean erythrocyte filterability (%) at study entry was 65.3% and 68.1% in the + and -EPA group, respectively. After 12 weeks treatment, improvement of erythrocyte filterability occurred in 6/8 patients in the +EPA group. In contrast, 7/9 patients resulted in the decrease of erythrocyte filterability in the -EPA group. As shown in Fig 2, the mean erythrocyte filterability (%) for the +EPA group was 71.0%, against 51.4% in the -EPA group (P=0.01) after 12 weeks.
[Discussion and Conclusion] EPA significantly attenuated the anemic effect of RBV/IFNa therapy for CHC patients. Erythrocyte filterability decreased in the -EPA group. On the contrary, decrease of erythrocyte filterability was not found in the +EPA group. Therefore, EPA improved the anemic side effect of RBV/IFNa via the stabilizing action of the membrane by amelioration of erythrocyte filterability. This study would be useful for clinical application.
Stimulation of Transferrin Receptor Expression by Enhanced Heme Biosynthesis in Murine Erythroleukemia Cells.
Authors: Lok Chun-Nam , Ponka Prem 
Published: October 2019

Abstract
In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.
Novel Iron Chelators Alleviate Symptoms of Thalassemia.
Authors: Szuber Natasha , Buss Joan L. , Trudel Marie , Felfly Hady , Ponka Prem 
Published: October 2019

Abstract
Thalassemia is a blood disorder requiring lifelong transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction. We hypothesized that removing this proximate iron compartment as a primary treatment using novel iron chelators, could prevent cell death and alleviate the need for transfusion. Novel, highly cell permeable iron chelators, pyridoxal isonicotinoyl hydrazone and pyridoxal ortho-chlorobenzoyl hydrazone (o-108) were compared to the present mainstay, desferrioxamine and deferiprone, in vitro and in vivo. Treatment of human model b-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron (P < 0.002) and reduced oxidative stress, as indicated by methemoglobin levels (P < 0.01). When administered to β-thalassemic mice, iron chelators mobilized erythrocyte membrane iron (P < 0.05), reduced cellular oxidation (P < 0.03), and prolonged erythrocyte survival. Consistently, these mice showed substantial correction of hematological abnormalities (P < 0.05). A beneficial effect as early as the erythroid precursor stage, was also determined by normalized proportions of mature versus immature reticulocytes (P < 0.05). Remarkably, all four compounds reduced iron accumulation in target organs (P < 0.05). Most importantly, o-108 revealed superior activity compared to desferrioxamine, further decreasing iron in spleen and liver by ~2-fold and ~5-fold, respectively (P < 0.05). Our study demonstrates that iron chelators ameliorate thalassemia in a human and murine model, and validates their primary use as an alternative to transfusion therapy.
ABCG2 Expression as Prospective Marker for Hematopoeitic Stem Cell Purification.
Authors: Tadjali Mehrdad , Zhou Sheng , Rehg Jerold , Sorrentino Brian P. 
Published: October 2019

Abstract
ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.
CD43 Defines Early Hematopoietic Progenitors Derived from Human Embryonic Stem Cells (hESC).
Authors: Vodyanik Maxim A. , Thomson James A. , Slukvin Igor I. 
Published: October 2019

Abstract
We have demonstrated that co-culture of hESCs with OP9 bone-marrow stromal cells induced efficient hESC differentiation into hematopoietic colony-forming cells (CFCs). During hESC/OP9 co-culture, the first CFCs were detectable 1–2 days after emergence of CD34+ cells (day 4–5 of co-culture) and 3–4 days before generation of CD45+ cells, indicating that early CFCs arose in the CD45- population. To define phenotype of early hematopoietic progenitors, we examined the expression of CD41a, CD43, CD61 and glycophorin A (CD235a) on the CD34+ cells in the course of hESC/OP9 co-culture. The first detectable CD34+ cells expressed a high level of VEGF-R2 (KDR) and were CD41a, CD43, CD61 and CD235a negative. Appearance of CFCs was accompanied with down-regulation of KDR and almost simultaneous expression of CD41a, CD43 and CD235a on the CD34+ cells. CD43+ cells gradually increased thereafter, whereas the expression of CD41a was retained on the subset of CD43+ cells that co-express CD235a and CD61. CD45+ cells emerged within CD43+ population and the most of CD45+ cells did not express CD235a, CD41a or CD61. The expression of CD34 was decreased progressively on CD43+CD41a+CD235a+CD61+ cells, but retained by CD43+CD45+ cells. To assay hematopoietic potential of defined subsets, CD34+ cells were isolated by magnetic sorting and further separated into CD34+ CD43+ and CD34+CD43− cell fractions. In addition, CD34−CD43+ cells were isolated from CD34− fraction. CD34+ selection markedly enriched all types CFCs except of E-CFCs. All GEMM-CFCs were found within CD34+ population and none within CD34−. However, CD34− cell fraction contained E-CFCs and minimal numbers of M-CFCs and GM-CFCs. CD34+ cells were also heterogenous in morphologic appearance and contained endothelial cells that were capable to endocytose Ac-LDL. Positive selection of CD43+ cells resulted in total recovery of all types of CFCs. E-CFCs, which consistently detected in the CD34- fraction, were also recovered by CD43 selection. Morphologically, CD43+ cells comprised a homogenous blast-like population and were lack of endothelial-like cells. Thus, expression of CD43 molecule (leukosialin/sialophorin) is detected on the earliest clonogenic hematopoietic progenitors before expression of CD45, persists on developing hematopoietic cells after their lineage specification and most likely defines the divergence of hematopoiesis from endothelial cells during early development in humans.
Mitochondrial DNA (mtDNA) Sequence Heterogeneity among and within Single Human CD34 Cells, T Cells, B Cells and Granulocytes.
Authors: Ogasawara Yoji , Nakayama Kazutaka , Tarnowka Magdalena , McCoy J. Philip , Molldrem Jeffrey J. , Levin Barbara C. , Kajigaya Sachiko , Young Neal S. 
Published: October 2019

Abstract
Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood101:3118 [2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells’ mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.
Tmtsp, a Novel Gene Encoding a Cell Surface Protein Highly Specific to Hematopoietic Stem and Endothelial Cells.
Authors: Takayanagi Shin-ichiro , Hiroyama Takashi , Yamazaki Satoshi , Iwama Atsushi , Nakauchi Hiromitsu 
Published: October 2019

Abstract
In the adult mouse bone marrow, hematopoietic stem cells (HSC) are highly enriched in the CD34 (−/low), c-Kit (+), Sca-1 (+), and lineage marker (−) (CD34 (−) KSL cell population. Using in silico data base search against NCBI EST database, we identified a novel cell surface molecules specific to CD34 (−) KSL HSCs. We cloned its full-length cDNAs of both mouse and human counterparts from total embryo (E15) and human spleen cDNAs, respectively. They were 2815-bp and 2953-bp long and the deduced amino acid sequences consisted of 851 and 852 amino acids, respectively. They contain a signal sequence, three Ig-like domains, a Thrombospondin type 1 (TSP1) domain, and a transmembrane region. Its domain composition shows high similarity to that of Unc5h3, a receptor for netrin, which plays a key role in neuronal axon guidance. Therefore, we designated this gene as Transmembrane Molecule with Thrombospondin Module (Tmtsp). BLAST search against the mouse genome database displayed that the mouse Tmtsp is composed of four exons and spanning over 26kbp on the chromosome 8qA2. Tmtsp was translated into a 170 kDa transmembrane protein. An alternative splicing which splices out the 3rd exon was detected in all major organs tested, leading to in-frame translation of a variant protein without TSP1 domain. RT-PCR analysis of hematopoietic cells demonstrated that Tmtsp is confined to KSL cells in expression and shows even higher expression in CD34 (−) KSL HSCs. On the other hand, Northern blot analysis detected its expression in all major organs tested and the highest expression in the lung. To specify the cells that express Tmtsp, we generated monoclonal antibodies against the extracellular domain of Tmtsp. Immunohistochemical analysis using anti-Tmtsp monoclonal antibodies revealed that its expression is tightly restricted to endothelial cells in tissues. These findings indicate that Tmtsp is a novel cell surface transmembrane molecule specific to both hematopoietic stem/progenitor cells and endothelial cells. Among a list of molecules shared by HSC and endothelial cells, Tmtsp is the first one that possesses the TSP1 domain implicated in cell adhesion. In this context, structure and expression of Tmtsp strongly suggest important roles in the development and maintenance of HSCs and endothelial cells including HSC homing and formation of vascular network.