bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.
Authors: Chen Jichun , Young Neal S. 
Published: April 2020

Abstract
Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals.
Figure Figure
Marked Improvement in Short- and Long-Term Survival in Severe Aplastic Anemia Patients Treated with Immunosuppression in the Past 18 Years.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal S. 
Published: April 2020

Abstract
Forty years ago severe aplastic anemia (SAA) was almost universally fatal disease, but now most patients can be successfully treated with hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). Since the majority of patients are not suitable candidates for allogeneic HSCT from a histocompatible sibling, IST is usually the treatment of choice. From the introduction of the standard anti-thymocyte globulin (ATG) + cyclosporine (CsA) regimen in the 1980s, the hematologic response rate has been 60–70% despite efforts to improve on the ATG+CsA platform. Across many protocols, there is a strong link between response to IST and survival. We examined short and long term survival of patients who were treated with initial IST at the Clinical Center of the National Institutes of Health from 1989 to 2006; a total of 372 patients were analyzed in 3 separate cohorts: those who received IST from 1989–1995; 1996–2001 and 2002–2006. Thirty-two deaths occurred within the 6-months period of IST with 15 being attributed to a fungal infection. The distribution of early deaths (&lt; 6 months) in relation to the different time periods were 15% (1989–1995); 9% (1996–2001); and 5% (2002–2006) (p = 0.003). Survival beyond 6 months also improved (1989–1995 and 2002–2006, 65% vs. 85%, respectively; p &lt; 0.01). Surprisingly, the survival benefit was most striking among patients who did not respond to IST at 6 months. Among the total 116 non-responders, the number of patients who received a second course of IST was 0% (1989–1995), 37% (1996–2001) and 51% (2002–2006); those who underwent HSCT were 19% (1989–1995), 23% (1996–2001) and 18% (2002–2006); and those who did not receive a second course of IST or HSCT were 83% (1989–1995), 29% (1996–2001) and 11% (2002–2006). We draw two major conclusions from our data analysis. First, improvement in short term survival over the last two decades is most likely due to advances in supportive care, as the response rate to horse ATG + CsA has remained largely unchanged during the same time period. The marked decline in deaths due to invasive Aspergillosis infection suggests that the introduction of effective and relatively nontoxic antifungal drugs is of prime importance (e.g., voriconazole in 2002 and caspofungin in 2004). Second, the utilization of secondary therapies--a repeat course of IST and allogeneic HSCT for older adults and matched unrelated transplants for younger individuals--have salvaged patients who are primary ATG-failures. The advances in supportive care also impact long term survival in those who receive salvage therapies and in those who fail repeated courses of IST and are not suitable candidates for HSCT, who now can be adequately maintained on growth factor and transfusion support, often for years. Current survival rates in SAA should be weighed when in considering treatment algorithms for patients who fail initial IST.
Identification of Alternative Splicing Changes in Erythroid Differentiation Using Human Exon Arrays.
Authors: Yamamoto Miki L. , Kang Jeong-Ah , Arribere Josh A. , Wickrema Amittha , Conboy John G. 
Published: April 2020

Abstract
Terminal erythroid differentiation is accompanied by extensive structural remodeling as the cell enucleates and eventually assumes the biconcave disk morphology of the mature cell. Previous studies have documented many changes at the transcriptional level essential for erythroid differentiation. Changes in erythroid gene expression also occur at the level of pre-mRNA alternative splicing: the activation of 4.1R (EPB41) exon 16 splicing in late erythroblasts increases 4.1R affinity for spectrin-actin and mechanically strengthens the plasma membrane. We hypothesize that analogous changes in alternative splicing affect the structure and function of other erythroid proteins. To identify additional alternative splicing switches in erythroid genes, a genome-wide exon expression analysis was carried out using the new Affymetrix Human Exon 1.0 ST Array. Unlike traditional gene expression microarrays, this array has single exon resolution and can detect changes in expression due to alternative splicing. Samples for array analyses were prepared from RNA of human erythroid progenitor cells grown in culture for 7, 10, and 14 days, corresponding to basophilic, polychromatic, and orthochromatic stages. Analysis of this exon array data confirmed that 4.1R exon 16 splicing was activated in day 14 cells, and that a known inhibitor of exon 16 splicing, hnRNP A1, was down-regulated in coordination with the 4.1R splicing switch. As another positive control, we confirmed in array data the expression of a known erythroid-specific 3′ end in beta-spectrin mRNA in all three time points of erythroblasts, while array data from muscle tissue showed expression of only the non-erythroid 3′ end of beta-spectrin. Array data is now being analyzed to identify new cases of alternative splicing during erythropoiesis, and confirmation of several candidate splicing switches by RT-PCR and quantitative PCR is under way. A number of genes, including PIK3R1, SLC12A6, and TNPO2, show changes in alternative 5′ first exon usage during late erythropoiesis. A splicing change involving an internal cassette exon in MBNL2, which encodes a splicing regulator, was identified by array data and confirmed by RT-PCR. In addition, overall gene expression analyses confirm up-regulation of known genes expressed during erythroid differentiation, including Band 3, GLUT1, ALAS2, and BCL2L1. This preliminary analysis demonstrates the application of exon arrays toward the identification of splicing switches that occur during differentiation of human erythroblasts. Further validation of putative alternative splicing events is in progress, and investigation of the regulation of the validated events and the physiological implications of the predicted changes in the proteins will be pursued in the future.
High Resolution HLA Matched Unrelated Donor Versus HLA Identical Sibling Transplantation for Chronic Phase CML.
Authors: Arora Mukta , Spellman Stephen , Haagenson Michael , Klein John P. , Hurley Carolyn , Selby George , Antin Joseph , Kernan Nancy , Kollman Craig , Nademanee Auayporn , Weisdorf Daniel J. , Horowitz Mary M. , McGlave Philip , Petersdorf Effie 
Published: April 2020

Abstract
Current criteria for the selection of adult volunteer unrelated donors (URD) include matching for HLA alleles determined by high resolution typing methods. However, the impact of URD matching for 8/8 HLA alleles (HLA-A, B, C, DRB1) on survival compared to HLA identical sibling donor bone marrow transplant (BMT) remains undefined. We tested this hypothesis in a homogenous population of patients with good-risk chronic myeloid leukemia (CML) in first chronic phase (CP1) where a strong allogeneic effect and hence a lower risk of relapse is anticipated. In total, 939 URD bone marrow transplant recipients with CML in CP1 were retrospectively typed for HLA -A, B, C, DRB1, DQ and DP alleles. Outcomes were compared to 3514 HLA identical sibling donor bone marrow transplant recipients (CML, CP1). Five-year survival after 8/8 matched URD HCT was slightly, but significantly worse than that after sibling BMT (54% versus 63%). [RR 1.36 (95% CI 1.17–1.59), p=0.0001]. In multivariate analysis (model adjusted for time from diagnosis to transplant, CMV serostatus of donor and recipient, year of transplant, and T-depletion, and stratified on donor and recipient gender mismatch and radiation use), a single allele mismatch at any class I locus or at HLA-DRB1 led to significantly poorer survival. Risks for single class I versus single DRB1 mismatches were similar. Two locus mismatching was associated with higher risk compared to matched or single locus mismatching. In this homogenous cohort of good risk patients with CML in CP1, 5 year survival after transplant from 8/8 allele-matched donors begins to approach the results of sibling donor transplantation. Additive effects of multi-locus mismatching are not well tolerated and should be avoided if possible.
N Relative risk (95% CI) P-value 5 year survival * Antigen or allele mismatch, **allele mismatch HLA identical siblings 3514 1.00 63% (61–64%) 8/8 matched 531 1.36 (1.17–1.59) 0.0001 54% (50–58%) 7/8 A mismatch* 68 1.90 (1.36–2.65) 0.0002 43% (32–55%) 7/8 B mismatch* 34 2.77 (1.81–4.23) &lt;0.0001 30% (15–48%) 7/8 C mismatch* 113 1.88 (1.45–2.43) &lt;0.0001 42% (33–51%) 7/8 DRB1 mismatch* 37 2.07 (1.37–3.12) 0.0005 37% (23–54%) 6/8 Class I mismatches* 128 2.66 (2.11–3.35) &lt;0.0001 33% (25–42%) 6/8 Class I mismatch* and DRB1 mismatch** 28 3.40 (2.23–5.19) &lt;0.0001 21% (8–37%) 7/8 DRB1 mismatch** vs. 7/8 Class I mismatch* 37/215 1.03 (0.67–1.61) 0.88N/A
Functional Polymorphisms of Notch 4 and the Risk of Aplastic Anemia.
Authors: Kim Hee Nam , Kim Yeo-Kyeoung , Lee II-Kwon , Yang Deok-Hwan , Shin Myung Geun , Kook Hoon , Hwang Tai Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim HyeoungJoon 
Published: April 2020

Abstract
Aplastic anemia (AA) is a heterogeneous disease characterized by bone marrow aplasia and peripheral blood pancytopenia, a profound deficit of hematopoietic stem and progenitor cells. Notch4 is structurally distinct from Notch1 through Notch3. Notch4 signaling plays a role in lineage commitment in hematopoiesis. Therefore, we hypothesized that Notch4 polymorphisms could potentially influence the expression level of Notch4 protein and associate with the risk of AA. To investigate the role of the two Notch4 rs2071282 (+2763 C&gt;T, Pro204Leu) and 422951(+3323 A&gt;G, Thr320Ala) polymorphisms in susceptibility to AA, we genotyped in 151 AA patients and 552 healthy control subjects using PCR-RFLP and quantified expression of Notch4. We previously reported that Notch4 +2763 CT genotype is significantly associated with increased risk for AA (odds ratios (OR) = 1.77; 95% confidence intervals (CI) = 1.05–2.98, p=0.032) and the haplotype CG is significantly associated with the risk of AA (OR= 0.70 95% CI 0.50–0.98, p= 0.037) (The American Society of Hematology 2006. 12. poster). With quantified expression level of Notch4 using quantitative SYBR green methods, we identified a significant positive correlation between +2763 C&gt;T and +3323 A&gt;G each genotype and transcription level (p&lt;0.0001) in AA. With quantified expression level of Jagged 1, we also identified a significant positive correlation between +2763 C&gt;T and +3323 A&gt;G each genotype and transcription level (p&lt;0.0001) in AA. To the effect of the SNP on transcription, we also carried out allelic imbalance using allelic quantification mode pyrosequencing method. The mean ratio was 1.7, higher than that of controls cDNA and DNA amplicons of AA (ratio=1.3, P &lt; 0.001) in the 16 heterozygotes AA and 25 contrrols with genotype +3323 A/G, but there was no significance correlation in heterozygotes with genotype +2763 C&gt;T. In conclusion, our results suggest that regulation of expression Notch4 by two polymorphisms may play an important role in the susceptibility to AA.
Inhibition of Factor VIIa-Tissue Factor by Protein C Inhibitor.
Authors: Fortenberry Yolanda , Cook Anne B. , Church Frank C. 
Published: April 2020

Abstract
Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that inhibits several proteases in blood coagulation including thrombin, thrombin-thromobomodulin and activated protein C. Although PCI has been shown to inhibit these key blood coagulation proteases, a clearly defined hemostatic role for PCI remains to be elucidated. Preliminary results from our laboratory show that exogenously added PCI prolongs venous thrombus formation upon blood vessel injury in a murine model of vascular injury. These results suggest that PCI may be responsible for inhibiting other proteases in blood coagulation, possibly including factor VIIa. Blood coagulation is initiated when factor VIIa binds to TF on cell surfaces. We investigated the ability of PCI to inhibit factor VIIa, both in the presence and absence of TF in vitro, and in a cellular environment. It has been shown previously and we have confirmed that the serpin antithrombin (AT) inhibits both factor VIIa and factor VIIa/TF; however, the ability of PCI to inhibit this protease has not been fully investigated. PCI and AT inhibit factor VIIa in the presence of TF and heparin (AT: k2=1.1 × 104 M−1 s−1; PCI: k2=7.2 × 103 M−1s−1). In the absence of heparin the inhibition of factor VIIa/TF by PCI is reduced 10-fold. Interestingly, PCI is unable to inhibit VIIa in the absence of TF with or without heparin. Furthermore, both PCI and AT form SDS-stable complexes with factor VIIa (in the presence of TF). We also investigated whether PCI is able to inhibit VIIa-mediated cell migration of the breast cancer cell line MDA-MB-231 that constitutively expresses TF. AT and PCI are able to inhibit cell surface derived factor VIIa/TF. Interestingly, the rate of factor VIIa inhibition on MDA-MB-231 cells is 1.2 and 0.4 × 103 M−1s−1 for PCI and AT, respectively. The factor VIIa-mediated cell migration of MD-MB-231 cells is attenuated in the presence of PCI, but not in the presence of the PCI inactive mutant (T341R). Given that both AT and PCI are able to inhibit factor VIIa/TF both in vitro and in a cellular environment, suggests that more potent derivatives (prepared by site-directed mutagenesis) of these serpins may be useful alternatives as novel therapeutics anticoagulants that target the initial step in the coagulation pathway.
The Effect of Aquaporin 2 Gene Variations on Plasma Levels of von Willebrand Factor and Factor VIII and on the Risk of Venous Thrombosis.
Authors: Nossent Anne Yael , Ellenbroek Johanne H. , Frolich Marijke , Rosendaal Frits R. , Bertina Rogier M. , Knoers Nine V.A.M. , Eikenboom Jeroen C.J. 
Published: April 2020

Abstract
High levels of von Willebrand factor (VWF) and factor VIII (FVIII) are a risk factor for thrombosis. Determinants of high VWF and FVIII levels are poorly understood. Secretion of VWF from endothelial storage pools is regulated by vasopressin type-2 receptor (V2R). Previously, we have shown that a V2R variant, which has increased binding affinity for its ligand vasopressin (AVP), is associated with increased levels of VWF and FVIII1. Nephrogenic Diabetes Insipidus (NDI) is a disorder characterized by renal insensitivity to AVP, caused by mutations in the genes encoding V2R or the aquaporin 2 water channel (AQP2). AQP2 expression is enhanced by stimulation of V2R. Patients with NDI are unable to concentrate their pre-urine. We hypothesized that carriers of AQP2 mutations compensate excess fluid loss by up-regulating AVP release and V2R expression, resulting in increased VWF and FVIII secretion. To test this hypothesis, we set up the Factor Eight in Nephrogenic Diabetus Insipidus study (FENDI), which includes 13 NDI families: 14 NDI patients (12 V2R- and 2 AQP2-linked), 14 carriers (9 V2R- and 5 AQP2-linked) and 25 unaffected family members, as well as 48 unrelated healthy individuals. In addition, we looked at effects of common AQP2 gene variations in a case-control study on venous thrombosis, the Leiden Thrombophilia Study (LETS), which consists of 474 patients with a first deep vein thrombosis and 474 healthy controls, sex and age matched to the patients. In the FENDI, no differences were observed between NDI patients, carriers and unaffected individuals in markers for fluid homeostasis such as hematocrite, serum osmolality and blood pressure. AVP reached detectable levels in all carriers of AQP2 mutations, compared to 27% and 56% in unrelated and related unaffected individuals, respectively. AVP levels, were, when detectable, elevated in all patients and carriers. VWF propeptide, a measure of the VWF secretion rate, VWF antigen and FVIII activity were also highest in carriers of AQP2 mutations. In the LETS, we sequenced a 6.6 kb long genomic region around the AQP2 gene in 25 selected individuals. We identified 18 single nucleotide polymorphisms (SNPs), of which 16 were genotyped in the entire LETS. Although reliable haplotypes could not be formed, due to recombination, the SNPs were linked within 5 clusters. In three of these clusters, up to 2.5-fold increases in thrombosis risk were observed. In these same clusters we observed associations of the AQP2 SNPs with arterial blood pressure. However, none of the AQP2 SNPs were associated with VWF or FVIII levels in healthy controls of the LETS. In conclusion, increased AVP levels in carriers of NDI-causing AQP2 mutations appear associated with increased VWF secretion. Furthermore, in the LETS, common AQP2 gene variations are associated with the risk of venous thrombosis.
LDL Receptor Mediates Effective Endocytosis of Activated Factor V.
Authors: Meijer Alexander B. , Roosendaal Sigrid D. , Limburg Vincent , van der Zwaan Carmen , Rodenburg Kees W. , Mertens Koen 
Published: April 2020

Abstract
The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.