bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting Hematopoietic Cells Using a Blimp-1 Promoter Helper Dependent Adenoviruses.
Authors: Shi Chang-Xin , Wen Xiao-Yan , Wang Ding-Yan , Gao Bo , Sleep Gillian , Zhuang Li-Hua , Li Zhi-Hua , Hitt Mary M. , Stewart A. Keith 
Published: October 2019

Abstract
B lymphocyte-induced maturation protein-1 (Blimp-1) plays a key role in the maturation of B lymphocytes into end-stage, antibody-screting plasma cells. However its promoter region has not been well characterized. As an initiative aimed at identifying plasma cell specific promoters for use in gene therapy of myeloma, we studied the function of the blimp-1 promoter in a helper-dependent adenovirus (HDAd) vector system. The advantages of employing HDAd include: 1) High capacity encompassing transgene (upto 36 kb); 2). Higher and longer transgene expression; 3). Lower cytotoxicity in vivo; 4). Improved specificity for tissue-specific promoters. Blimp-1 promoter fragments of 2.6, 4.7 and 25 kb driving the luciferase gene were cloned into HDAd precursor plasmid and then rescued into HDAd vectors. A panel of B lymphoma and myeloma cell lines were infected with these HDAd vectors and a positive control vector HDMCMV (an HDAd vector expressing luciferase under the control of MCMV promoter) at an MOI of 2,000 particles/cell. Two days after infection, very high luciferase activity was demonstrated in human myeloma cell lines (KMS11, U266, MM1, and MY5 cells), but low or no activity was seen in human B lymphoma cell lines (Namalwa and Raji). In fibroblasts, the Blimp-1 derived promoter activity appeared to be high in COS7 cells but 10 times lower in NIH3T3 cells. All three differently sized promoter fragments of Blimp-1 demonstrated similar activity. Next we further tested these three Blimp1/Luc HDAd vectors in mice using HDMCMV as a positive control. Six week old FVB mice were intravenously injected with 1X10(e11) particles per mouse. Different tissues were assayed for luciferase activity three days after injection. The control HDMCMV virus directed the highest luciferase activity in the liver because of the tropism of adenovirus. However, the Blimp-1/luciferase viruses directed highest luciferase expression in the spleen and bone marrow with lower level relative expression in the liver and trace amount or no expression in other tissues examined. After normalization, the transciptional activity of the Blimp-1 promoter is ~80 fold higher than the CMV promoter in spleen tissue. All three Blimp-1 promoters demonstrated similar activity, indicating that the 2.6 kb Blimp promoter contains the regulation element for spleen and bone marrow-specific gene expression. FACS analysis of spleen cells co-stained with luciferase antibody revealed luciferase expression in both B220+ve B and B220-ve non-B cells. Taking together, we conclude that 2.6kb blimp-1 promoter sequence is enough to direct gene expression to hematopoietic cells but is not B cell specific.
Gene Transfer in Hematopoietic Progenitor Cells Mediated by an SV40 Based Episomal Vector Carrying A S/MAR Element.
Authors: Papapetrou Eirini , Ziros Panos , Mitcheva Ilina , Athanassiadou Aglaia , Zoumbos Nicholas 
Published: October 2019

Abstract
Background/Aims: Replicating episomal vectors present a potential alternative to currently used oncoretroviral vectors for gene transfer in hematopoietic progenitor/stem cells. Their main advantage is that they can persist in the recipient nucleus as independent units, without integrating into the host’s genome, eliminating thus the risk of insertional mutagenesis. In the present study we explored the capacity of a recently developed SV40-based episomal vector, pEPI-eGFP, to stably transfect hematopoietic progenitor cell lines and primary cells, in order to evaluate its potential for therapeutic applications. pEPI-eGFP contains the enhanced green fluorescent protein (eGFP) cDNA and a Scaffold/Matrix Attachment Region (S/MAR) and it does not code for any proteins of viral origin. These unique properties qualify pEPI-eGFP as an attractive vehicle for gene therapy applications.
Results: The vector was maintained as a stable episome in K562 cells for at least 100 generations and supported long-term EGFP expression, even in cells cultured in non-selective medium. Methylation-dependent cleavage assays demonstrated the vector’s ability to self-replicate in K562 cells and MEL cells, whereas its episomal status was confirmed by Southern blotting and plasmid rescue in E. coli. The vector was also maintained in primary human fibroblasts for at least 30 passages with and without selection pressure.
Transfection of CD34+ cells from umbilical cord blood with pEPI-eGFP was feasible by electroporation with an efficiency of up to 30%, as estimated by flow cytometric evaluation of eGFP expression 24–48 h post-transfection. Cytokine prestimulation of CD34+ cells did not enhance transfection efficiency, whereas transfection of post-mitotic cells, such as dendritic cells was also feasible. This suggests that efficient transfection with the vector does not require cell cycling. PCR with eGFP-specific primers in single CFU colonies derived from CD34+/eGFP+/PI− FACS-sorted cells demonstrated the presence of the vector in ~20% of the colonies.
Conclusion: Our results demonstrate that pEPI-2 exhibits a considerable potential as a gene transfer vector for hematopoietic cells, as it confers long-term transgene expression in hematopoietic progenitor cell lines as a stable episome and can efficiently transfect unstimulated CD34+ cells. Further studies, both in vitro and in vivo, are required to assess the long-term maintenance in hematopoietic progenitor/stem cells and their progeny as well as other features of the vector, in order to evaluate its overall efficacy and possibly improve its performance.
Lentiviral Delivery of Suicide Gene and Selection Molecules to Non-Dividng T Cells.
Authors: Qasim Waseem , Chatziandreou Ilenia , Thrasher Adrian , Gaspar Hubert 
Published: October 2019

Abstract
T cell suicide gene therapy is designed to permit the use of donor T cells for their powerful anti-leukaemic and anti-viral effects following haematopoietic stem cell transplantation. In the event of graft versus host disease (GVHD) such cells can be eliminated through the administration of suicide-gene dependent prodrugs. Though the feasibility of the strategy has been demonstrated in clinical trials, there has been a lower incidence of GVHD than anticipated and evidence of impaired anti-viral immunity. Current vectors under clinical investigation are based on retroviral delivery systems and require T cells to be actively dividing at the time of vector exposure to for successful transduction. The process of ex-vivo activation to induce mitogenesis has been shown to impair subsequent functional potential, and even under optimized conditions using combinations of anti-CD3 and anti-CD28 antibodies, T cells become prone to exhaustion. It has been previously reported that lentiviral vectors permit T cell transduction without the need for mitogenic stimulation if used in combination with certain cytokines such as Interleukin-2 (IL-2) or Interleukin-7 (IL-7). Thus we have adapted a self-inactivating lentiviral vector system based on HIV-1 and encoding strong promoter elements derived from the long terminal repeat (LTR) regions of Spleen Focus forming virus (SFFV) for the delivery of suicide genes to T cells. The Woodchuck hepatitis virus post-transcriptional regulatory element and the central polypurine tract element of HIV-1 have been incorporated to enhance transduction efficiency and increase transgene expression. T cells cultured in the presence of either IL-2 or IL-7 alone, or in combination could be transduced at between 20–30% efficiency following a single round of exposure to virus pseudotyped with the vesicular stomatitis virus envelope or the feline endogenous leukaemia virus envelope. By the end of the procedure there were minimal changes in T cell surface phenotype, preservation of niave/memory subsets and retention of virus specific populations as quantified by tetramer staining of Cytomegalovirus (CMV) specific T cells. Functional analysis indicated preservation of proliferative responses to autologous dendritic cells pulsed with CMV antigen. In addition, strong responses in mixed lymphocyte culture indicated intact allo-reactive responses. Cells transduced to express a fusion construct encoding a variant herpes simplex thymidine kinase fused to the truncated CD34 selection marker could be enriched to >95% purity by a single round of anti-CD34 magnetic bead selection. The functional integrity of the suicide gene was confirmed by elimination of enriched cells following exposure to the prodrugs Gancilcovir and Aciclovir. In conclusion, non-dividing T cells transduced with suicide gene/selection transgenes using lentiviral constructs, retain phenotypic characteristics and functional responsiveness.
IL-2/B7.1 Fusagene Transduction of AML Blasts by a Self-Inactivating Lentiviral Vector Stimulates T Cell Responses in Vitro: A Strategy To Generate Whole Cell Vaccines for AML.
Authors: Chan Lucas , Hardwick Nicola , Darling Dave , Galea-Lauri Joanna , Gäken Joop , Devereux Stephen , Kemeny Mike , Mufti Ghulam , Farzaneh Farzin 
Published: October 2019

Abstract
Despite recent treatment advances prognosis for the majority of AML patients remains bleak. AML patients over 60 years of age treated with intensive chemotherapy have an estimated 2-year survival of 20% and only 10% at 4–5 years. This is attributed to unfavourable cytogenetics, preceding myelodysplastic syndrome and limited abilities to tolerate intensive chemotherapy. We and others have previously demonstrated that tumour cells engineered to express B7.1 and IL-2 can stimulate T cell and NK cell responses against the unmodified tumour cells and prolong the survival of vaccinated animals in mouse models of leukaemia. However, development of whole cell vaccines for AML have been severely hampered by the fact that primary AML cells are poorly infectable with RV, AdV, AAV and HSV. Therefore we have constructed self-inactivating lentiviral vectors incorporating enhancer elements and a myeloid efficient promoter to express B7.1 and IL-2 in a monocistronic “fusagene” configuration as a single fusion protein. This polyprotein is post-synthetically cleaved to generate biologically active membrane anchored B7.1 and secreted IL-2. This provides transduction efficiencies of between 40%–99% for B7.1 and 2-15ng/106/24hrs of IL-2 secretion in both established and primary AML cells, following a single round of infection at an MOI of 10. The combined expression of IL-2 and B7.1 in AML blasts enables increased in vitro stimulation of allogeneic T cells and autologous T cells obtained from both non-remission and remission patients (post-BMT or non-BMT recipients). In addition, IL-2/B7.1 AML cells stimulated an increase in lymphocyte numbers, expansion of CD8+ cells and increased expression of T cell activation markers. Furthermore, the stimulated lymphocytes secrete greater levels of Th1 cytokines and show evidence of specificity, as indicated by their increased proliferation in the presence of unmodified AML compared to remission bone marrow cells. This data supports the notion that IL-2/B7.1 whole cell vaccination could stimulate therapeutically useful immune responses in AML patients.
Long-Term Follow-Up of 98 Cases of Recombinant Human Erythropoietin (epoetin) Induced Pure Red Cell Aplasia (PRCA): Outcomes and Treatment Data from the Research on Adverse Drug Reactions and Reports (RADAR) Program.
Authors: Carson Kenneth R. , Klinge Stephen A. , Belknap Steven M. , McKoy June M. , Raisch Dennis W. , Tallman Martin S. , Kuzel Timothy M. , Evans Andrew M. , Kim Benjamin , Yarnold Paul R. , Lyons Elizabeth A. , Bennett Charles L. 
Published: October 2019

Abstract
Background: With the recent reporting of 191 cases of epoetin induced PRCA (Bennett CL, NEJM 2004), information is needed about the long-term follow-up of individuals with this diagnosis. While 47 cases from France, Germany, and England have been reported recently (Verhelst D, Lancet 2004), these data are limited by short follow-up and absence of information from other regions.
Methods: RADAR investigators identified cases of epoetin-associated PRCA from the Food and Drug Administration MEDWatch database and proprietary reports from both the manufacturers epoetin alpha (Epogen from AMGEN, and Eprex from Johnson and Johnson) and epoetin beta (Neorecormon from Roche). Patients exposed to more than one epoetin form were excluded. PRCA recovery was operationally defined as transfusion independence, clearance of anti-erythropoietin antibody, and/or normalization of reticulocyte count.
Results: See table.
Conclusions: The overall recovery rate of 82% following transplantation versus 40% following immunosuppression suggests that transplantation might be the optimal therapy for epoetin-associated PRCA. Almost half of the patients rechallenged with epoetin developed recurrent PRCA, raising concern over the benefits of rechallenge.
Eprex (N=82) Neorecormon (N=11) Epogen (N=5) Origin: US 60% 64% 100% Europe 60% 64% – Pacific Rim/Asia 21% 36% – Canada 19% – – Subcutaneous delivery 99% 100% 60% Recovery with Immunosuppression 37% 73% 0% Recovery with renal Transplant 11% – – No recovery with renal transplant 1% – 20% No Recovery 41% 18% 40% Unknown Recovery 10% 9% 40% Male gender 68% 73% 40% Rechallenged with epoetin N=12 – – Response rate 50% Median age 68 years 47 years 51 years
Establishment of a Retroviral Mouse Model for inv(16)-Mediated Acute Myeloid Leukemia Suggests That the p14ARF Tumor Suppressor Is a Transcriptional Target for Repression by the inv(16) Fusion Protein.
Authors: Moreno Isabel , Pan Ling , Keates Jennifer , Kim Hyung , Klug Christopher , Hiebert Scott 
Published: October 2019

Abstract
The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia. The chimeric gene encodes a fusion protein consisting of most of the RUNX1 co-factor core binding factor beta (CBFβ) fused to a smooth muscle myosin heavy chain, SMMHC. Given that RUNX1 and RUNX1-ETO regulate the p14ARF promoter, we tested whether the inv(16) fusion protein (IFP) can also repress p14ARF expression. In reporter assays, the IFP cooperated with RUNX1 to repress the p14ARF promoter. To investigate whether the IFP can regulate endogenous ARF expression, we triggered the oncogene checkpoint and the expression of p19ARF (the murine homologue) by expressing c-Myc in primary myeloid progenitor cells. After induction of c-Myc activity, p19ARF levels increase 20–50 fold; however, the co-expression of the IFP and c-Myc effectively blunted the induction of p19ARF. Next, we sought to take advantage of this information to establish a murine model of the inv(16). We reasoned that the IFP provides a growth advantage and perhaps allows mutations to accumulate by repressing p19ARF. If so, then removing p19ARF during the rapid growth of hematopoietic stem cells and progenitors during embryogenesis might allow the formation of the same mutations that would occur after expression of the IFP, but accelerate leukemogenesis upon IFP expression. Therefore, we used p19ARF-null mice as bone marrow donors and used recombinant retroviruses to express the IFP together with a green fluorescent protein (GFP). Given that repression of the p19ARF promoter is likely to be incomplete, we also tested whether removing one allele of p19ARF might enhance the ability of the IFP to induce leukemia by further reducing the expression of p19ARF. We found that expression of the IFP induced a myelomonocytic AML in 10 to 12 weeks when expressed in p19ARF-null cells. In addition, the IFP cooperated with the removal of only a single allele of p19ARF and the complete deletion of p19ARF only modestly shortened the latency period. The leukemic blasts in all the cases showed large nuclei and many of them contained granules and/or vacuoles in the cytoplasm. In addition, the GFP+ blast cells that express the IFP did not express any of the standard lineage markers and the majority of these cells were ckit+/Sca1−. All these findings are consistent with a myeloblastic acute leukemia. We conclude that repression of p19ARF by the IFP may stimulate formation of AML and that the IFP sets the stage for the rapid development of AML.
Hematopoietic Stem Cells Overexpressing Smad7 Exhibit Increased Self-Renewal and Regeneration Capacity in Vivo.
Authors: Blank Ulrika , Larsson Jonas , Utsugisawa Taiju , Magnusson Mattias , Klintman Jenny , Karlsson Stefan 
Published: October 2019

Abstract
The hematopoietic stem cell (HSC) resides in the bone marrow (BM) and can self-renew to generate more stem cells as well as differentiate into all hematopoietic lineages. The precise molecular mechanisms, which govern HSC fate decisions are poorly understood. The Transforming Growth Factor-β (TGF-β) superfamily of ligands, including the TGF-βs, Activins and Bone Morphogenetic Proteins (BMPs), encompasses an important group of growth factors, many of which have been shown to modulate and regulate hematopoiesis. Smad7 is known to block the phosphorylation event of receptor-activated Smads, thus creating a block in the entire signaling cascade downstream of TGF-β and related factors. To assess the effect of blocking the entire Smad signaling pathway downstream of TGF-β/Activin and BMP in HSCs in vivo, we have overexpressed the inhibitory Smad7 by a retroviral gene transfer approach. Both control and Smad7 vectors were MSCV based and expression was driven by the LTR promoter. The Smad7 vector contained the cDNA sequence for murine Smad7 and an internal ribosomal entry site (IRES) followed by GFP, whereas the control vector contained IRES and GFP only. In these experiments BM from wild type C57/B6 mice could efficiently be transduced with Smad7 or control vectors respectively. Upon transduction, cells (Ly5.2) were transplanted in a competitive fashion into lethally irradiated recipients (Ly5.1) and transduced cells were monitored by GFP fluorescence. Smad7 overexpressing cells were able to long-term reconstitute as well as give rise to both lymphoid and myeloid compartments at normal distributions. When self-renewal was assessed by secondary transplantations, Smad7 overexpressing cells showed significantly increased reconstitution ability compared to control transduced cells (blood samples at 12 weeks post transplant: 37.3 ± 5,9 for Smad7 vs. 5.06 ± 1,7 for control. Data represent % GFP positive cells ± SEM). Furthermore, Western blot analysis showed efficient expression of Smad7 protein in BM cells originating from transduced cells of transplanted mice. In addition, Smad2 and Smad1 phosphorylation was blocked upon TGF-β, Activin or BMP stimulation in BM cells, suggesting that Smad7 was functionally active in BM cells in vivo. However, when cultured under serum-free conditions in vitro, Smad7 overexpressing cells exhibited reduced proliferative capacity as compared to control transduced cells (3.5 times fewer cells by day 12 post transduction), suggesting that the in vivo phenotype was dependent on the BM microenvironment. Taken together, our data indicate that blocking of several TGF-β pathways simultaneously increases the self-renewal ability of HSCs in vivo, but not in vitro.
Mutations of the SHP-2 Proteine Tyrosine Phosphatase in Adult Patients with Acute Myeloid Leukemia.
Authors: Thiede Christian , Steudel Christine , Schäkel Ulrike , Ehninger Gerhard 
Published: October 2019

Abstract
Acute myeloid leukaemia (AML) represents a heterogeneous group of early stem cell malignancies characterized by an uncontrolled expansion of malignant cells blocked at certain stages of myeloid differentiation. Several genetic abnormalities have been described, including translocations involving genes of the CBF family, as well as activating mutations of signal transduction proteins, e.g. N-RAS, K-RAS or the Flt3- receptor tyrosine kinase (RTK). More recently, mutations in the SHP-2 protein, a tyrosine phosphatase involved in RTK signal transduction, have been described in patients with juvenile myelomonocytic leukaemia (JMML) as well as paediatric patients with AM. Only limited data on this aberration are available in older patients. In order to investigate the prevalence and the prognostic relevance of this gene in adults, we analyzed the mutational status of SHP-2 in a cohort of patients with AML.
Patients and Methods: DNA samples prepared from bone marrow or peripheral blood samples taken at diagnosis from 977 patients treated in the AML96 protocol of the SHG Dresden were analyzed. Screening for mutations in the mutational hot spot in exon 3 of the PTPN11 gene coding for SHP-2 was performed on a dHPLC system (Transgenomic). Samples with aberrant dHPLC chromatogram were sequenced.
Results: In the 977 patients investigated so far, 29 PTPN11 exon 3 mutations were found (3%). All mutations consisted of single base pair exchanges in codons 60–76, mostly affecting codons 69 (n=6), 72 (n=7) and 76 (n=6). Patients with PTPN11 mutations were more likely to have FAB M0 (p= .038) or M5b (p=.045) morphology than negative cases and had a significantly lower prevalence of FLT3-ITD mutations (p= .017). PTPN11 mutations were associated with a normal karyotype in 15/28 evaluable patients, whereas 13 had cytogenetic abnormalities, all of which were either high risk (−7/7q-, n=4; inv3, n=2, complex, n=2, −5, n=1) or intermediate risk (n=4) aberrations. No significant differences were found in the complete remission rate as well as the overall and event free survival, but a trend for a shorter disease free survival was observed in patients with PTPN11 mutations (median 7.8 vs 14.4; p= 0.06) associated with a significantly increased rate of relapse (p= .02). Although not significant yet, a similar trend was seen for patients with normal karyotype.
Conclusion: PTPN11 mutations can be found in a small but considerable number of adult patients with AML. Consistent with the data in children, this mutation seems to more prevalent in patients with monosomy 7 and leukemias with monocytic differentiation. Although very preliminary, the data point to a worse prognosis of patients with SHP-2 mutations. We are currently further extending the number of patients and analyze additional exons to delineate the potential prognostic role of this aberration.