bluuebunny/crossref_metadata_2025_split huggingface dataset:
NOD-Rag1nullPrf1null Mice Support Long-Term Engraftment of Human Hematopoietic Stem Cells.
Authors: Minamiguchi Hitoshi , Livingston Anne G. , Wingard John R. , Shultz Leonard D. , Ogawa Makio 
Published: April 2020

Abstract
NOD-scid mice have been widely used as recipients in the xenograft assay for human hematopoietic stem cells (HSCs). One major problem with the strain is the low level of engraftment except when large numbers of cells are injected. This is probably caused by the presence of residual natural killer (NK) cell cytotoxic activity. NOD-scid/beta 2 microglobulin (B2m)null mice have been reported to have reduced NK cell cytotoxic activity and support higher levels of human cell engraftment. However, use of this strain of mice is limited by their difficulty in breeding and short life span caused by early development of lymphomas, which is accelerated by irradiation. Another immune-incompetent mouse model, NOD-recombination activating gene (Rag1)null mice allow longer observation of human cell engraftment than NOD-scid mice and are easier to breed. Genetic crossing of perforin (Prf) structure gene-targeted mutation onto NOD-Rag1null strain results in absence of NK cell cytotoxic function. In these mice, NK cells are not capable of killing target cells because of the absence of Prf, the major mediator of cytotoxic activity. We have tested the use of NOD-Rag1nullPrf1null mice as recipients of long-term xenograft assay for human HSCs by adopting Yoder’s method of conditioning newborn mice with minor modifications. Pregnant NOD-Rag1nullPrf1null dams were treated with 22.5mg/kg busulfan in 20% dimethylsulfoxide in Hank’s BSS on day 17.5 and 18.5 pc via subcutaneous injection. On the day of delivery, mononuclear cells (MNCs) were isolated from human cord blood (n=3) by density gradient centrifugation and T cell-depleted MNCs were separated by using mouse anti-human CD3, CD4, and CD8 antibodies and sheep anti-mouse IgG immunomagnetic beads to prevent preferential T cell engraftment. The busulfan-exposed pups were transplanted with 4–5 million T cell-depleted MNCs via the facial vein. At 6 months post-transplantation, human cells were detected in the bone marrow of 4 out of 10 transplanted mice. The levels of human CD45+ cells in the bone marrow of engrafted mice were 79.9, 69.8, 60.5, and 7.4%, and those in the peripheral blood were 6.3, 5.8, 4.1, and 1.3%. Multilineage engraftment was confirmed by phenotypic analysis. Next, we tested the hypothesis that human cord blood HSCs have dye efflux activity by injecting T cell-depleted Rhodamine 123 (Rho)− or + cells into conditioned newborn NOD-Rag1nullPrf1null mice. Six-month engraftment was found only with the Rho− cells. Thus, conditioned newborn NOD-Rag1nullPrf1null mice provide an excellent model for assaying long-term engrafting human HSCs.
Recombinant Tpo Stimulates Maturation of Megakaryocytes Derived from Adult Peripheral Blood- but Not Cord Blood-CD34+ Cells.
Authors: Pastos Karen M. , Slayton William B. , Rimsza Lisa M. , Sola Martha C. 
Published: April 2020

Abstract
Umbilical cord blood (CB) is a valuable source of stem cells for transplantation. However, platelet engraftment is slow, taking approximately 70 days for CB transplants versus 20 days for mobilized adult peripheral blood (PB) transplants. This time is not significantly shortened by the administration of recombinant thrombopoietin (rTpo). The cause for the delay in platelet engraftment following CB transplant is unknown. We hypothesized that developmental differences in size and ploidy of neonatal versus adult megakaryocytes (MKs) contribute to this delay. To mimic these two types of transplant in vitro, we compared CB to PB CD34+ cells cultured in adult bone marrow stromal-conditioned media (CM) or unconditioned media (UCM) for 14 days. Increasing doses of rTpo were added to the CM, and the resulting MK maturation was compared with that of UCM with maximal rTpo concentration. MK number and ploidy were determined by flow cytometry using CD41-FITC and propidium iodide, respectively. Increased ploidy levels were expressed as percentage of MKs with a ploidy ≥ 8N. Results represent an average of three independent experiments.
Figure Figure
As seen in the figure, PB-derived MKs reached highest ploidy levels in the presence of UCM + 100 ng/ml rTpo. When cultured in CM, they exhibited lower ploidy levels, regardless of Tpo concentration. In contrast, CB-derived MKs exhibited higher ploidy levels in response to CM with either 0 or 0.1 ng/ml (physiologic concentration) of rTpo, as compared to higher rTpo concentrations or UCM + 100 ng/ml rTpo. MK numbers increased in response to rTpo in a dose-response manner, regardless of the source of the MKs (data not shown). These results indicate that intrinsic differences between CB- and PB-derived megakaryocytes exist, and that maturation is regulated differently in neonatal versus adult MKs. While Tpo is a potent stimulator of MK maturation in PB-derived MKs, it appears to inhibit this process in CB-derived MKs. These differences may be relevant to understanding the delayed platelet engraftment following CB transplants.
Homing Efficiency in Two Models of Submyeloablative Conditioning and Correlation with Long-Term Engraftment.
Authors: Meyers Stephen E. , Meyers Justin L. , Goebel W. Scott 
Published: April 2020

Abstract
Submyeloblative conditioning regimens for bone marrow transplantation (BMT) are desirable to reduce host toxicity in many settings. We previously demonstrated that hosts conditioned with 160 cGy or with two sequential doses of the antimetabolite 5-fluorouracil (5-FU) displayed ~50% and ~35% donor chimerism 6 months after transplantation of 20 x 106 fresh congenic bone marrow (BM) donor cells, respectively. Donor chimerism in 160 cGy- or 5-FU-conditioned mice was proportional to the reduction in host long-term repopulating ability (LTRA) caused by the conditioning regimen (~80–90% vs. ~55%, respectively). In this study, we sought to determine if donor marrow homing efficiency also correlated with long-term engraftment in these models of submyeloablative conditioning. We hypothesized that sequential doses of the cell cycle-specific agent 5-FU (5-FU x 2) may damage the marrow microenvironment less than low-dose radiation, resulting in more efficient homing in 5-FU x 2-conditioned mice. C57Bl6/J (Bl/6) hosts were conditioned with either 5-FU x 2 administered on days -5 and -1 (150 mg/kg IP) or with 160 cGy given the day of transplantation (day 0). Bl/6 (CD 45.2+) hosts were transplanted with 20 x 106 PKH-26 stained Bl/6 or with Boy J (CD 45.1+) whole BM cells via tail vein injection. The percentage of homed cells 20 hours after BMT in 5-FU x 2-conditioned mice was 3.53 % ± 3.1% (n = 13 from 3 independent experiments), while the percentage of homed cells in the 160 cGy group was 8.67 % ± 2.1% (n = 12; p = 0.0375). The fraction of donor cells which homed to the spleen or remained in the peripheral blood after 20 hours was similar between conditioning regimens. This difference in homing efficiency led us to consider other possible explanations as to why BM cells home less efficiently in 5-FU x 2-conditioned hosts than in low-dose irradiated mice. We hypothesized that 5-FU x 2 may have a more injurious effect on the marrow microenvironment than previously thought. Examination of marrow stromal cell potential using a colony-forming unit-fibroblast (CFU-F) assay revealed that marrow from 5-FU x 2-treated mice (n = 3) contained 1.1 ± 0.4 colonies/marrow femur equivalent (FE), whereas marrow from 160 cGy-treated mice (n = 3) contained 33.8 ± 13.4 colonies/FE. In comparison, marrow from untreated mice (n = 3) averaged 95.6 ± 27.5 colonies/FE. These data suggest that homing efficiency correlates with donor chimerism in these two models of submyeloablative conditioning, and establish at least two potential mechanisms (a lesser reduction of host LTRA, and decreased donor cell homing efficiency) for the lower level of long-term donor chimerism observed in 5-FU x 2- compared to 160 cGy-conditioned hosts. Furthermore, 5-FU x 2 conditioning appears to disrupt the marrow microenvironment to a greater degree than low-dose radiation. Current efforts are aimed at exploring the molecular interactions of homed donor cells with conditioned marrow.
Donor CD8+ T Cells Facilitate Induction of Chimerism and Tolerance without GVHD in Autoimmune NOD Mice Conditioned with Anti-CD3 mAb.
Authors: Liang Yaming , Huang Tammy , Zhang Chunyan , Todorov Ivan , Atkinson Mark , Kandeel Fouad , Forman Stephen , Zeng Defu 
Published: April 2020

Abstract
Prevention of autoimmune diabetes and induction of islet transplantation tolerance in NOD mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI)-conditioning of the recipients. The toxicity of radiation and potential for graft versus host disease (GVHD) prevents its clinical application. Donor CD8+ T cells play a critical role in facilitation of engraftment, but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8+ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 mAb. The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (i.e. TNF-α), high-level production of anti-inflammatory cytokines (i.e. IL-4 and IL-10), and confining of the donor CD8+ T cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor specific tolerance and reversal of insulitis. These results demonstrate that donor CD8+ T cell-mediated facilitation of engraftment can be separated from GVHD in non-irradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.
GATA-1, NF-E2 and EKLF/SP1 Binding Elements Cooperate To Specify Patterns of Histone Hyper-Acetylation at β-Globin LCR HS Core Elements.
Authors: Ackley Catherine J. , Lowrey Christopher H. 
Published: April 2020

Abstract
The active elements of the β-globin LCR are necessary for high-level expression of the linked globin genes. These active elements are contained within regions of unique, erythroid-specific chromatin structure characterized by the formation of positioned nucleosomal arrays, areas of approximately 150–200 bp which are highly accessible for protein-DNA interactions (DNase I HSs) and hyperacetylation of histones H3 and H4. At a DNA level, each of the LCR HS cores contain NF-E2, EKLF/Sp1 and tandem, inverted GATA binding elements. Our lab has shown that all of these transcription factor-binding sites are necessary for HS formation. We have previously proposed a model of HS formation in which Sp1 and/or EKLF first bind to the core regions and alter local chromatin structure allowing GATA-1 and NF-E2 to bind and stabilize the HS core structure. Additional factors could then bind to the core and flanking regions. An important aspect of HS core formation that our experiments have not previously addressed is the mechanism of histone acetylation. Based on our previous findings and the fact that GATA-1 and NF-E2 can both interact with histone acetyltransferases (HATs), we hypothesized that histone acetylation at the HS requires the binding of EKLF/Sp1 followed by GATA-1 and/or NF-E2 and therefor should be a late event in HS formation. To test this hypothesis, we have generated a series of 11 artificial HS core elements based on the structure of human HS4. These plasmid-based constructs contain different combinations of the six binding sites in their normal arrangement and spacing: (EKLF/Sp1)-(NF-E2)-(EKLF/SP1)-(GATA-1 x 2)-(EKLF/Sp1). These constructs were stably transfected into MEL cells and assayed for their ability to mediate histone H3 and H4 acetylation as determined by the ChIP assay. The endogenous murine β-globin LCR HS3 served as a positive internal control for histone acetylation. Previous results from our lab showed that all six factor-binding sites within the HS core are required for HS formation. However, when assayed for histone acetylation, the NF-E2 and the tandem inverted GATA sites are able to independently direct H3 and H4 acetylation to between 10 and 50% of that seen at the native HS site. Combination of the NF-E2 and GATA-1 sites results in 89% of normal H4 acetylation. Full HH4 acetylation (98% of the wild-type HS) is only seen with all six factor binding sites. These results indicate that NF-E2 and GATA-1 direct most of the acetylation of LCR HS core histones but that EKLF binding is also likely to contribute. They also indicate that extensive histone acetylation can occur without HS formation. Finally, in contrast to our model of HS formation, GATA-1 and NF-E2 appear able to direct the acetylation of HS core histones without the presence of binding sites for Sp1 or EKLF.
Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.
Authors: Sterner Lauren , Miyazaki Toru , Swift Larry , Dean Ann , Little Jane 
Published: April 2020

Abstract
We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels &lt;1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n&gt;=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p&lt;.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress.
Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs
% Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p &lt; .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p &lt; .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p &lt; .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns
Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of GATA-1, NF-E2, and RNA Polymerase II.
Authors: Wang Jin , Liu Hui , Epner Elliot 
Published: April 2020

Abstract
The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. A chicken chromosome marked by insertion of a selectable marker gene into the upstream folate receptor gene locus or 5′ HS1 followed by Cre recombinase mediated removal of the selectable marker gene served as control “wild type” chromosomes. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Transcriptional activation of the ρ and βH genes correlated with hyperacetylation of histones H3 and H4, loss of methylated K9 histone H3, and binding of RNA polymerase II to the gene promoters. The status of methylated CpG dinucleotides at the promoters did not correlate with the transcriptional status of the genes. The phenotype of the 5′ HS1, and HS2 deletions were surprisingly similar to that of the intergenic enhancer, which suggested these elements may function either in the same pathway or complex. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18.
Long-Range Enhancement of β-Globin Gene Expression Is Dependent on Gt Motifs Residing in the HS3 Core Element of the Locus Control Region.
Authors: Navas Patrick A. , Stergachis Andrew B. , Sheffer Hadar H. , Ye Xin , Stafford Mary , Stamatoyannopoulos George 
Published: April 2020

Abstract
Normal expression of the human β-globin genes is dependent on a powerful regulatory element residing upstream of the globin gene cluster referred as the locus control region (LCR), physically characterized by five DNAseI-hypersensitive sites (HS1 to HS5). Of particular interest is HS3, which is characterized by a 225 bp core element region containing seven GT motifs (GT1 to GT7) that alternate with four GATA-1 binding sites. The GT motifs are bound by a family of Kruppel-like factors and are important for proper expression of many housekeeping and tissue-specific genes. We previously demonstrated in transgenic mice carrying a 248 kb human β-globin yeast artificial chromosome in which the GT6 motif (GT6m β-YAC) was mutated resulted in a decrease in ε- and γ-globin gene expression during embryonic erythropoiesis and a decrease in γ-globin expression during definitive erythropoiesis, thus, providing evidence that a single transcriptional motif distantly located can have profound effects on gene expression. We have used the same β-YAC transgenic mouse model system to analyze the function of the remaining six GT motifs of the HS3 core element. Currently we have produced transgenic mice carrying either a mutation of GT1/2 (GT1 and GT2 motifs overlap) or GT3. Total RNA was isolated from yolk sac, liver and blood samples of transgenic F2 embryos at 12- and 14-day postconception and adult blood, and subjected to RNAse protection analysis. The GT1/2m β-YAC transgenic mice exhibited normal ε- and γ-globin gene expression during embryonic erythropoiesis, but during definitive erythropoiesis γ-globin gene expression was reduced 4 to 5 fold in day 12- and day 14- fetal livers. In the two GT1/2m β-YAC transgenic lines produced, β-globin gene expression was reduced and levels varied from 12.3% ± 2.9% to 63.2% ± 5.8% of endogenouse murine α-globin indicating that β-globin expression is strongly influenced by the position of integration of the transgene. In contrast, the GT3m β-YAC transgenic mice the mutation to GT3 had no effect on globin gene expression during development. Our results suggest that multiple GT motifs within the same highly complex regulatory element contribute differently to the enhancement of the downstream genes.
Keratinocyte Growth Factor (KGF) Is Required for Post-Natal Thymic Regeneration.
Authors: Alpdogan Onder , Hubbard Vanessa M. , Muriglan Stephanie J. , Kochman Adam A. , Eng Jeffrey M. , Zakrzewski Johannes L. , Goldberg Gabrielle L. , Smith Odette M. , Waldman Elisha , van den Brink Marcel R.M. 
Published: April 2020

Abstract
KGF is a fibroblast growth factor family member (FGF-7) that mediates epithelial cell proliferation and differentiation in a variety of tissues, including thymic epithelial cells. Recent studies have demonstrated that KGF administration (before conditioning) to the recipients of allogeneic bone marrow transplantation (BMT) can enhance T cell and thymic reconstitution. Therefore, we studied the role of KGF on T cell development under normal and stress conditions (such as irradiation) by using KGF (−/−) mice in experimental murine models. Phenotypic analysis of KGF (−/−) mice at varying ages demonstrated that the bone marrow, thymus and lymph node cellularity and the cell distribution among KGF (−/−), wild type (WT) and KGF (+/−) mice (6–11 weeks of age) were similar. However, splenic cellularity and splenic T cell numbers were slightly lower than WT and littermate controls. KGF (−/−) mice are more vulnerable to sublethal irradiation (450–600 cGy) and a more than six fold decrease was found in thymic cellularity when analyzed on day 28 after irradiation, whereas there was no delay in the erythroid, myeloid and platelet recovery after irradiation.
We used bone marrow transplantation models to assess the relative contribution of KGF produced by thymocytes or thymic epithelial cells during thymic regeneration after irradiation. T cell reconstitution was impaired in syngeneic or allogeneic KGF (−/−) BMT recipients in comparison to littermate controls but there was no difference in the distribution of thymocyte subsets or splenic T cell content. The recipients of KGF (−/−) bone marrow had decreased thymic and splenic cellularity after allogeneic BMT. These data demonstrate that both donor and host derived KGF play a role in thymic regeneration. Finally, KGF administration to young and old mice (10 month old) enhanced thymopoiesis when analyzed 28 days after KGF administration. Pre-BMT KGF administration to the recipients of syngeneic and allogeneic BMT also resulted in a 2–6 fold increase in the thymic cellularity compared to the control group.
We conclude that KGF produced by both thymocytes and epithelial cells is not required for normal and post-natal thymic development, but plays a role in post-natal thymic regeneration after irradiation. These data support the potential use of KGF to protect or restore thymic damage after chemo/radiotherapy.