bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Transcription Factor Pbx1 Is Required for the Development of Double Positive Thymic T Cells.
Authors: Sanyal Mrinmoy , Tung James W. , Dejbakhsh-Jones Sussan , Strober Samuel , Herzenberg Leonore A. , Cleary Michael L. 
Published: April 2020

Abstract
It has become increasingly apparent that transcription factors originally discovered at the sites of chromosomal translocations in acute leukemias serve critical roles for hematopoietic cell development and function, which require fine orchestration of gene expression. Pbx1 is one such transcription factor, which forms a chimeric oncoprotein, E2a-Pbx1, resulting from t(1;19) chromosomal translocations between the E2a and Pbx1 loci in a subset of the pediatric pre-B cell acute lymphoblastic leukemias. Previously, our laboratory has shown that mice nullizygous for Pbx1 die in utero with substantial myelo-erythroid defects associated with profound fetal anemia. To determine whether Pbx1 is also required for normal lymphoid development, we performed Rag1-deficient blastocyst complementation assays using Pbx1 homozygous null embryonic stem (ES) cells. Interestingly, analysis of resulting chimeric mice displayed a partial rescue of the Rag1 phenotype as evidenced by T cell-restricted lymphocyte development. Pbx1 null ES cells produced circulating CD4 and CD8 single positive T cells, however in contrast to the typical naive T cell phenotype (CD62LhighCD44loCD45RBhigh) in control mice they exclusively displayed a characteristic memory T cell phenotype (CD62LlowCD44highCD45RBlow). Analysis of the thymi of the chimeric mice for T cell developmental precursors showed the complete absence of CD4/CD8 double-positive intermediates. Rather, T cells in the thymus, and all secondary lymphoid organs, were single positive CD4 or CD8 cells that exclusively displayed a similar memory T cell phenotype typical of T cells derived from bone marrow resident committed T cell progenitors (CTP). The striking absence of classical CD4/CD8 double-positive cells in the thymus indicates that Pbx1 deficiency interrupts the intrathymic T cell developmental pathway. Furthermore, the phenotypic resemblance of Pbx1 null T cells with T cells derived from bone marrow resident CTP strongly suggests that T cell development in the absence of Pbx1 may occur via an extrathymic pathway.
Differential Sensitivity of Normal and CML-Derived CD34+ Cells to Inhibition of SHP-2 Gene Expression by RNA Interference (RNAi).
Authors: Scherr Michaela , Battmer Karin , Chaturvedi Anuhar , Schultheis Beate , Ganser Arnold , Eder Matthias 
Published: April 2020

Abstract
RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of &gt; 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.
Temporal Gene Expression after BCR-Crosslinking Based upon Zap70 Related Genomic Features.
Authors: Vallat Laurent D. , Park Yuhyun , Rassenti Laura , Gribben John G. 
Published: April 2020

Abstract
Ig mutation status discriminates aggressive from more indolent chronic lymphocytic leukemia (CLL). Several gene expression studies distinguished CLL from other leukemia but failed to discriminate these two types of patients. Nevertheless, differentially expressed genes including zap70 were identified and further validated as an important prognostic factor. Zap70 is a normal component from the T cell receptor pathway, and may have a critical role in the B cell receptor (BCR) signaling pathway in CLL. We questioned whether zap70 might alter response to BCR signaling in CLL cells and explain the difference in biologic characteristics of this disease. To examine this we crosslinked the BCR in CLL cells from 12 patients and B cells from 6 healthy donors. We examined the response to BCR stimulation on cell proliferation, apoptosis and gene expression profile. Here we analyzed the temporal gene expression after BCR crosslinking at different time points, in addition to the basal level of gene expression. Based on the results of a pilot study examining multiple different time points, four time points were analyzed in all cases ranging from 60 min to 390 min and gene expression of stimulated (S) and unstimulated (US) CLL and control cells analyzed for a total of 170 HU133 plus2.0 DNA-chips. We defined the expression data from the logarithmic ratio log(S/US) for each time point. In keeping with previous findings, the basal gene expression distinguished CLL samples from healthy B-cells, but did not distinguish mutated from unmutated patients. We defined the linear combination first for the initial time point and then for all four time points to score for expression over time. This temporal gene profile of BCR engagement was based on filtering of 2850 genes that now distinguish healthy B-cells from CLL-cells, but is now capable also of distinguishing a specific BCR signature of a group of patients. Of note, this distinguished patients based less upon Ig gene mutation status but on zap70 protein level, with a mean protein expression difference of 9 fold between these two groups. This more aggressive pattern reveals changes in gene expression in important genetic pathways associated with BCR crosslinking, including JUN and NFkB. Stimulating a crucial pathway for CLL biology identified temporal gene expression pathways linked with the aggressiveness of this leukemia. This suggests that a fuller understanding of the biologic differences in leukemia cells may be identified by a more dynamic approach to gene profiling by examination of response to specific stimuli, rather than examination at the basal level. Based upon these results, ongoing studies examine the biologic impact of gene silencing of zap70 using RNAi on BCR stimulation in CLL cells.
Decrease in Sensitivity of PIG-A-Mutant Cells to Natural Killer Cells Conferred by Missing of Stress-Inducible Membrane Proteins ULBPs.
Authors: Hanaoka Nobuyoshi , Kawaguchi Tatsuya , Horikawa Kentaro , Nagakura Shoichi , Ishihara Sonoko , Mitsuya Hiroaki , Nakakuma Hideki 
Published: April 2020

Abstract
The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand to manifest symptoms is still unknown. PNH cells with PIG-A mutations do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of a series of GPI-linked membrane proteins. Blood cells with PNH phenotype (GPI− cells) expand in patients with bone marrow (BM) failure syndromes including PNH, aplastic anemia, and myelodysplastic syndromes. The diseases share an immune-mediated BM injury possibly by cytotoxic lymphocytes such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL). It is suggested that the BM injury allows PNH clones survive selectively. Indeed, we have shown that human leukemic cells (K562) decrease their sensitivity to NK cells in vitro when K562 cells acquire PIG-A mutations (Nagakura et al, Blood 2002). In the present study, we show that the decrease in NK sensitivity of PIG-A mutant cells is ascribable to the deficiency of GPI-linked membrane proteins, ULBPs. ULBPs bind cytomegalovirus protein UL-16, consist of ULBP1-3, and emerge in cell membrane when cells are infected or transformed. They trigger off a NK activation signal, which overrides an inhibitory signal from MHC class I (Cosman et al, Immunity 2001). ULBPs also activate CTL besides NK cells. As target cells, a pair of GPI+ control and GPI− mutant cell lines was prepared from a GPI− K562 cell line bearing a PIG-A mutation by transfection with a PIG-A cDNA and a vector alone, respectively. Flow cytometry detected ULBP1-3 on the surface of GPI+ but none of GPI− K562 cells. As effector cells, we used cultured human NK cells (KHYG-1) deficient in Fc γR type III (CD16). GPI− cells were more resistant than GPI+ cells to the killing by NK cells in the 51Cr-release assay. GPI+ cells decreased their sensitivity to NK cells to the level of GPI− cells in the presence of antibodies to both ULBP1 and ULBP2, while antibodies to ULBP3 exerted no effects. None of the antibodies to ULBPs showed any effects on the killing of GPI− cells, while antibodies to NKG2D, which is a NK receptor for both ULBPs and MICA/B, inhibited the killing of both GPI+ and GPI− cells. Thus, GPI− cells that lack membrane ULBPs show a survival advantage in the setting of immune attack in vitro. Of clinical interests, ULBPs were detected on the cell surface of GPI+ but none of GPI− granulocytes of patients with PNH. GPI+ granulocytes of healthy individuals were negative for ULBPs. There appears pressure to induce membrane ULBPs in patients with PNH, leading to BM injury. These findings suggest that the failure of membrane expression of ULBPs permits selective expansion of PNH clones in patients with PNH and other BM failure syndromes.
Nuclear Localization of Codanin-1, a Product of a Newly Identified Gene Mutated in Congenital Dyserythropoeitic Anemie Type I.
Authors: Tamary Hannah , Chernikovsk Naama , Lotan Sharon , Edlitz-Markus Tali , Shalev Hanna , Shaklai Sigal , Fibach Eithan , Yaniv Isaac , Resnitszky Peretz , Dgany Orly 
Published: April 2020

Abstract
Congenital dyserythropoietic anemia (CDA) type I is a rare autosomal recessive macrocytic moderate to severe anemia rarely associated with distal skeletal deformities. Erythroid bone marrow findings are pathognomonic with internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane. Our and others studied suggested erythroid S phase arrest. We recently identified the gene mutated in this disease, CDAN1 and named its ubiquitous encoded protein codanin-1. The function of this protein remains unknown. The aim of the present study was to determine the sub cellular localization of codanin-1. Human erythroid cultures as well as Hela, K562 and osteodarcoma cell line (U2OS) were grown. A polyclonal anti-codnain-1 antibody was generated by immunizaing rabbits with a synthetic 17 aa peptide. The anti-sera was affinity-purified against the immunizing peptide. Western blots, immunoflurescence stains and immunogold electromicroscopic studies were performed. Western blots of human erythroid cultures as well as HeLa, K562 and osteosarcoma cell lines identified one expected band of 130kD. Fluorescent and confocal microscopic studies of osteosarcoma and HeLa cells using anti-codanin-1 antibody showed that codanin-1 was an abundant nuclear protein. Immunogold electron microscopic studies with anti codanin-1 antibodies demonstrated that coodanin-1 is located within the heterochromatin. Future studies will focus on the role of codanin-1 within the heterochromatin.
Hematopoietic Cell Transplantation in Fanconi Anemia Patients with Biallelic BRCA2 Mutations.
Authors: MacMillan Margaret L. , Auerbach Arleen D. , Wagner John E. 
Published: April 2020

Abstract
Fanconi anemia patients with biallelic BRCA2 mutations have an exceptionally high risk of developing hematological malignancies, Wilms tumors and medulloblastoma at a very young age. We report on the hematopoietic cell transplant (HCT) results of 6 patients from 4 kindreds, all of whom have biallelic BRCA2 mutations. Two patients (800/2 and 984/2) were found to have a Wilms tumor at time of work-up for HCT despite normal previous renal ultrasounds. One was a stage 1 favorable histology, the second a stage II poorly differentiated tumor with diffuse anaplasia. In each case, the patient underwent nephrectomy and proceeded to HCT 3 weeks later. All unrelated or mismatched related donor marrow grafts were T cell depleted either by elutriation in patient 632/1, or CD34 selection with Isolex in the remaining cases. Five of 6 patients achieved neutrophil engraftment. Only one patient developed severe regimen related toxicity and one patient GVHD. Two of 6 patients are alive and well after HCT, both whom had T-cell ALL, which is uncommon in FA patients.
Patient and Transplant Characteristics
Kindred, IFAR No. Age/Gender Clinical Hx Donor Preparative Rx Day to ANC&gt;500 Toxicity, GVHD Outcome 1, 632/1 3.7 yr/F AML at 36 mo, chemotherapy refractory 6/6 URD BM x 2 CY, TBI, ATG, CSA, MP; CY, ATG, MP, CSA graft failure; +31 None AML relapse after both HCT, died day +76 after 2nd HCT 1, 632/2 1.9 yr/F AML at 21 mo, chemotherapy refractory 6/6 MSD BM CY, TBI, FLU, ATG, CSA, MP Engrafted None AML relapse, died day +288 2, 800/1 1.8 yr/M AML at 11 mo, chemotherapy refractory 6/6 URD TCD BM CY, TBI, ATG, CSA, MP +11 Grade II aGVHD AML relapse day +110, died day +124 2, 800/2 0.8 yr/M Neutropenia at 5 mo, Wilms tumor at 9 mo 6/6 URD TCD BM CY, TBI, FLU, ATG, CSA, MP +9 Resp and renal failure Died day +60 from pulmonary hemorrhage 3, 900/1 4.9 yr/M T cell ALL at 5.2 yr, CR after chemotherapy 5/6 Mat TCD BM BU, CY, FLU, ATG, CSA, MP +10 None Alive and well 19 mo after HCT 4, 900/2 6.7 yr/F T cell ALL at 4.9 yr, remission with chemotherapy, relapse with AML 17 mo later, Wilms at 6.6 yr 6/6 URD UCB BU, CY, FLU, ATG, CSA, MP +13 None Alive and well 3 mo after HCT
In summary, HCT has limited effectiveness after the onset of leukemia in patients with Fanconi anemia. While these patients have tolerated chemoradiotherapy well in most cases, refractoriness of the leukemia is the principle obstacle to success. Therefore, patients with biallelic BRCA2 mutations should be screened for solid tumors and referred for HCT as soon as possible, prior to the onset of leukemia. In addition, these patients require routine screening for solid tumors after HCT, the incidence of which remains to be determined.
Isolation of Human CD4+CD25+ T Cells with Regulatory Capacity for Clinical Trials.
Authors: Hoffmann Petra , Eder Ruediger , Piseshka Biserka , Havasi Andrea , Orso Evelyn , Dada Ashraf , Andreesen Reinhard , Holler Ernst , Edinger Matthias 
Published: April 2020

Abstract
Donor derived CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but actively suppress GVHD induced by CD25− conventional T (Tconv) cells when co-transplanted at a 1:1 ratio in murine model systems. Purified human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as their murine counterparts, i.e. they constitutively express high levels of intracellular CTLA-4, preferentially express the forkhead/winged helix transcription factor Foxp3, they are anergic to T cell receptor-mediated stimulation, and suppress the proliferation of Tconv cells after polyclonal or allogeneic activation in vitro. Thus, their adoptive transfer is a promising strategy for the prevention of GVHD in patients after allogeneic BMT. For potential clinical applications, efficient protocols for the isolation of CD4+CD25+ Treg cells from peripheral blood are required. In laboratory-scale experiments we determined that depletion of B cells (that partially express CD25), followed by repetitive positive selection of the remaining CD25+ cells with paramagnetic beads, yields a more than 90% pure population of CD4+CD25+ T cells. We now applied this protocol for the large-scale isolation of human CD4+CD25+ T cells from leukapheresis products of healthy volunteers under GMP conditions using the CliniMACS® device. B cell depletion with CD19-beads, followed by three cycles of CD25-enrichment with CD25-coupled microbeads resulted in an average 90% purity of CD4+CD25+ T cells (range 86% to 94%). Positively selected cells constituted on average 1.56% of MNC (range 0.84% to 2.0%), resulting in absolute numbers of 63 to 261 x 106 cells, depending on the initial size of the leukapheresis product. Since T cells with regulatory capacity predominantly reside within the CD4+CD25high subpopulation, we further determined the relative proportion of CD25high to CD25intermediate CD4+ T cells within the cell product. CD4+CD25high T cells represented on average 42% of the purified CD4+CD25+ T cell population (range 26% to 60%) as compared to 10.6% (range 8.5% to 13%) of the CD4+CD25+ T cells within the starting population. CD4+CD25+ T cells isolated according to this GMP protocol expressed higher levels of Foxp3 mRNA and Foxp3 protein as well as higher levels of intracellular CTLA-4 as compared to CD4+CD25− T cells. In addition, they showed functional characteristics of Treg cells as they were hypoproliferative to polyclonal stimulation in vitro and inhibited the proliferation of co-cultured CD4+CD25− T cells in a standard suppression assay. The ability to isolate highly enriched Treg cell populations in sufficient numbers under GMP conditions will accelerate their clinical application in allogeneic BMT.
Isolation and Expansion of Functional Differentiating Mesenchymal Stem Cells with a Serum Deprived Medium.
Authors: Dominici Massimo , Mueller Ingo , Cafarelli Luigi , Kordowich Sandra , Sternieri Rita , Spano Carlotta , Chiodino Caterina , Mariotti Ilaria , Petak Istvan , Horwitz Edwin , Paolucci Paolo , Conte Pierfranco 
Published: April 2020

Abstract
The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy.
We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/− 1,3%) and 10% (+/− 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the “classical” MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications.
Ex-Vivo Expansion of Megakaryocyte Progenitors.
Authors: Giulivi Antonio , Halpenny Mike , Birch Paul , Yang Lin , Martin Lisa 
Published: April 2020

Abstract
Following Bone Marrow Transplant, thrombocytopenia and neutropenia always occur and patients require additional post transplant support in the form of platelet transfusions.
Megakaryocytes (Mk), the precursors of platelets, are contained in hematopoietic progenitor cell products but their number is variable and relatively low. The infusion of ex vivo expanded Mk precursors could be beneficial by shortening the time to platelet engraftment and therefore reducing the amount of platelet transfusion support required by bone marrow transplant patients. The objective of this project was to investigate the expansion of Mk progenitors from peripheral blood stem cell (PBSC) harvests from patients with haematological malignancies. Briefly, CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) and Interleukin 1 (IL-1) then incubated at 37°C /5% CO2 for 8 – 12 days. Megakaryocyte progenitor analysis was accomplished using flow cytometry analysis (CD34+/41+, CD41+, CD61+) and Mk culture analysis (CFU-Mk) (Stem Cell Technologies). Mk progenitor expansion efficiency was determined as “fold expansion” of Mk progenitors produced over input levels.
After 8 days of culture, a mean expansion of 46 fold (range 1.2 – 327.0, n=10) in megakaryocytic cells (CD61+) and a 15 fold expansion (1.2 – 41.7, n=10) in megakaryocyte progenitor cells (CD34+/41+) was observed. After 12 days, a 116 fold expansion (1.5 – 286, n=7) in megakaryocytic cells (CD61+) and a 19 fold expansion (2.4 – 40, n=7) in Mk progenitors (CD34+/CD41+) was observed.
This study demonstrates that CD34+ cells can be used to effectively expand megakaryocytic cells using just two cytokines for an incubation period of 8 – 12 days. This data could be used to develop future protocols for use in clinical applications.