bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transplantation with Two Units of HLA-Mismatched Unrelated Umbilical Cord Blood in Treatment of an Adult Patient with Acute Myeloid Leukemia.
Authors: Pang Ying , Feng Ying , Ye Xue , Ren Hanyun 
Published: October 2019

Abstract
Objective To explore the feasibility, long-term hematopoiesis and complication of transplantation with two units of HLA-mismatched unrelated umbilical cord blood in treatment of adult acute myeloid leukemia.
Methods A 32 years old, 50kg male with acute myeloid leukemia in complete remission received transplantation with two units of HLA-mismatched unrelated umbilical cord blood.The conditioning regimen were modified(BU/CY)+ATG. The prophylaxis regimen for graft versus-host disease(GVHD) consisted of cyclosporine(CSA) and mycophenolate mofetil(MMF). The umbilical cord blood obtained from two different donors, both with mismatched HLA B/DRB locus from the recipient and mismatched HLA- B locus between the two donors. The umbilical cord blood was preserved in liquid nitrogen, recovered in a 40o C water bath immediately, infused via a catheter from the femoral artery to the arc of aorta, The doses of nucleated cells infusion were 4.4×107/kg (from donor 1) and 2.8×107/kg (from donor 2).
Results An absolute neutrophil count of more than 0.5×109/L at day 26,and a platelet count of more than 20×109/L at day 42. Septicemia with MRSA and pseudomomanas occurred at day 9 and day 14 because of agranulocytosis. The infection was controlled by Vancomycin, Tienam and HD-dose Gama immunoglobulin. Neither acute nor chronic GVHD developed in a follow-up period of 90 days. DNA-STR and HLA distinct analysis assay revealed a complete implant of cells from only one donor(donor2).
Conclusions 1.No donor with matched HLA bone marrow stem cell was available for the adult patient at the time of his relapse. HLA mismatched umbilical cord blood was obtained from two donors. Although cell counts for transplantation are much lower than the requirement of routine bone marrow transplantation, the speed of blood cell regeneration in the recipient is compatible with routine bone marrow transplantation. 2.Furthermore, Although DNA-STR and HLA analysis indicate complete implant of cells from only one donor, the result indicates transplantation with umbilical cord blood cells obtained from two different donors is promising in the situation where the cell number obtained from one donor is not enough.3.HLA- B/DRB locus was mismatched in the two donors. GVHD did not develop even after tapered off immunosuppresents 3 months post transplantation. No cross rejection observed by clinical presentation and blood cell analysis. The results indicate the incidence of GVHD is low after umbilical cord blood transplantation.
Significance of a Single Human Leukocyte Antigen Mismatch on the Outcome of Nonmyeloablative Allogeneic Stem Cell Transplantation from Related Donors Using the Mexican Schedule.
Authors: Ruiz-Arguelles Guillermo J. , López-Martínez Briceida , Manzano Carlos , Gómez-Rangel J. D. 
Published: October 2019

Abstract
The Mexican approach was used to conduct nonmyeloablative stem cell transplantation (SCT) in patients with various malignant and non-malignant hematologic diseases. Patients received a modified, low-intensity conditioning regimen, which included oral busulphan 4 mg/kg on Days −6 and −5, IV cyclophosphamide 350 mg/m2 on Days −4, −3, and −2, IV fludarabine 30 mg/m2 on Days −4, −3, and −2, oral cyclosporine A 5 mg/kg twice daily staring on Day −1 (continuing until Day 180), and IV methotrexate 5 mg/m2 on Days +1, +3, +5, and +11. Allografts were prospectively performed in 58 patients using sibling donors that were either human leukocyte antigen (HLA) identical (6/6) or compatible with 1 mismatch (5/6). In allografts where the donor was an HLA identical sibling (n = 40), the median overall survival was 33 months compared to 8 months when the donor was an HLA compatible sibling (n = 18; P <.01). The 52-month survival was 47% versus 38% in patients receiving allografts from HLA-identical donors and HLA-compatible donors, respectively. The prevalence of acute graft-versus-host-disease (GVHD) was 57% versus 38%, the prevalence of chronic GVHD was 25% versus 11%, and the relapse rate was 45% versus 55% in patients receiving allografts from HLA-identical donors and HLA-compatible donors, respectively. Two patients failed to engraft; both were 5/6 matches. Despite a trend toward less favorable results in patients allografted from HLA compatible (5/6) siblings, most differences in outcome were not significant, likely due to the small number of patients in the study. These data suggest that nonmyeloablative SCT using the Mexican approach may be a valid option for individuals with either an HLA identical or HLA compatible sibling donor.
Crystal Structure of Coproporphyrinogen Oxidase (CPO).
Authors: Phillips John D. , Whitby Frank G. , Hill Christopher P. , Kushner James P. 
Published: October 2019

Abstract
CPO, an essential enzyme in the heme biosynthetic pathway, catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III to form vinyl groups in the product protoporphyrinogen IX. CPO mutations are responsible for the disease hereditary coproporphyria. All eukaryotes express a highly conserved, oxygen-dependant form of CPO. Yeast and human CPO have a sequence identity of 52% at the amino acid level. To probe the biochemical basis of catalytic activity and to determine the deleterious effects of clinically identified mutations, we determined the crystal structure of CPO from S. cerevisiae. This is the first reported structure of a eukaryotic CPO. A cDNA encoding CPO (Hem13p) was expressed in E. coli as a histidine tagged protein. Hem13p was purified and concentrated to 25 mg/ml and crystals were grown in the presence of 18% PEG 8000, 0.1 M HEPES, pH 7.5, 2% isopropanol, 0.2 M Na-acetate. The crystal structure was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein folds into a novel structure featuring a central flat seven-stranded antiparallel beta-sheet flanked on each side with helices. The homodimeric structure of CPO is formed by a short isolated strand that forms a beta-ladder between the two monomers. Each monomer contains an active site formed between the flat beta-sheet and adjacent helices near the dimer interface. Many of the conserved residues are located at this interface. The deep active site cleft is lined by conserved residues and has been captured in an open and a closed conformation in two different crystal forms. The substrate cavity is completely buried in the closed conformation by an approximate 8 Å movement of a helix that forms a lid over the active site. The volume of the enclosed cavity precisely accommodates a modeled molecule of coproporphyrinogen III. The model indicates binding of two propionate side chains of coproporphyrinogen with two invariant arginines. The pyrrole nitrogens of the substrate are positioned to be coordinated by an invariant asparagine in a fashion similar to that described for uroporphyrinogen decarboxylase (EMBO J, 2003, 22: 6225–33). Nineteen point mutations have been described in association with hereditary coproporphyria and we have mapped these to the crystal structure. Only two mapped to the active site. Fourteen mutations were predicted to destabilize the protein and 4 of these occur at the dimer interface. Three mutations mapped to the solvent exposed surface of CPO. The crystal structure supports a model in which the enzymatic reaction occurs in an isolated cavity formed by a conformational change induced by substrate binding. The conformational change could create a binding site for molecular oxygen, the cofactor, and provide a mechanism to protect coproporphyrinogen III from inappropriate oxidation and the subsequent peroxidation of protoporphyrinogen IX.
Analysis of Beam (Carmustine, Etoposide, Cytosine Arabinoside, Melphalan) Versus High Dose Melphalan (HDM) with Autologous Rescue in Multiple Myeloma(MM).
Authors: Ramasamy Karthikeyan A. , Branford Ruth , Selvaratnam Radha , Ho Aloysius Y.L. , Duarte Rafael , Devereux Stephen , Pagliuca Antonio , Mufti Ghulam 
Published: October 2019

Abstract
Autologous stem cell transplantation is an established treatment that leads to prolongation in overall and disease free survival in MM. BEAM and M have been widely used as conditioning regimen for autologous stem cell transplant (ASCT) in patients with MM. At our centre MM patients with low creatinine clearance (Crcl) and a low performance status were offered Melphalan only as conditioning regime for ASCT. We report on our experience of HDM in comparison with standard BEAM. Records of 53 consecutive patients who had ASCT for myeloma at our centre between 2000 and 2003 were examined. Patients were staged as per Salmon and Durie (SD) criteria and EBMT response criteria were used for assessment post transplant .35 patients had BEAM conditioning (SD criteria 31-IIIA, 3-IA, 1-IIA) and 18(SD criteria 14- IIIA, 4-IIIB) had M.).Patients who received BEAM had a median age of 55 (range 38–66), median follow up of 19 months and those who received HDM had a median age of 60 (range 45 –69), follow up of 20.5 months. Stem cell source was peripheral blood (PBSC) in 50 patients, 1 Bone marrow (BM), and 2 BM and PBSC. HDM at a Dosage of 140 –200 mg/m2was administered in all HDM cases except for 1 patient who had 70mg/m2 because of Crcl of 17.4 ml/min. BEAM group had Carmustine 300mg/m2 (−6), Ara- C 800mg/m2 (−5 to −2), Etoposide 800mg/m2 (−5 to −2), M 140mg/m2 (−1). Patients in the M group had significantly lower haemoglobin and higher serum creatinine levels in comparison with the BEAM group. The 100-day treatment related mortality (TRM) was 5.5 % in HDM group, which was comparable to 2.2 % in the BEAM group. 9 out of 18 patients who had HDM relapsed and 9 out of 35 patients who had BEAM relapsed at follow up with Median relapse free survival (RFS) being 575 days in HDM group and has not achieved yet in BEAM group (p= .017). This data confirm that BEAM ASCT lead to significant prolongation of disease free survival with low TRM.Our data also suggest the effectiveness of HDM in patients who because of poor performance status and/or abnormal renal function may not tolerate BEAM ASCT
Race Has No Effect on Survival in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation.
Authors: Buadi Francis , McClune Brian , Moore Yoriann , Womeodu Robin , Yunus Furhan , Walsh William , Minhas Sohail , Przepiorka Donna 
Published: October 2019

Abstract
Multiple myeloma (MM) is more common in African Americans (AA) compared to White Americans (WA) with an incidence ratio of 2:1. The incidence of MM as well as mortality from MM has been on the rise in AA. SEER data shows a higher death rate in AA than WA diagnosed with multiple myeloma. The benefit of autologous stem cell trasnplantation (ASCT) for multiple myeloma has been clearly demonstrated in randomized trials. However the influence of race on survival in patients receiving ASCT for multiple myeloma has not been well studied. Between January 1996 and December 2003, 60 patients with multiple myeloma underwent ASCT in our program. We evaluated the influence of race on survival and also compared the survival of all transplanted patients to 74 myeloma patients from the hospital tumor registry who were diagnosed with multiple myeloma in the same time period, who received standard chemotherapy without ASCT, and who are in the same age range at diagnosis as the transplant patients. The transplant group included 32 AA and 28 WA, while the registry group had 42 AA and 32 WA. There was no difference between groups in the distribution by gender, but the transplant patients were significantly younger (median age 56.5 years, range 37–72 years) than the registry patients (median age 65 years, range 39–72 years). At analysis, median time from diagnosis was 4.6 years (range 1.2–10.3 years) for the transplant group and 7.7 years (range 3.7–10.6 years) for the registry group. For AA patients, median survival was 2.1 years in the registry group and 5.9 years in the transplant group (p=0.003). For WA patients, median survival was 2.0 years in the registry group and 5.8 years in the transplant group (p<0.0001). Median survivals did not differ in the transplant group when compared by race (5.9 years vs 5.8 years). The benefit of ASCT was seen in all age groups, so the difference in survival was not due to the younger age of the transplant patients. In a multivariate analysis of all 134 patients, ASCT was predictive of improved survival, but gender, race and quartile of age at diagnosis had no significant association with survival. We conclude that the improvement in survival with ASCT is independent of race.
Recombination Occurs in Lentiviral Vectors Designed To Express Both EGFP and EYFP in Human Hematopoietic Cells.
Authors: Dijon Marilyne , Torne-Celer Caroline , Tonnelle Cecile , Chabannon Christian 
Published: October 2019

Abstract
Recombinant lentiviruses are widely used vectors for in vitro and in vivo long-term gene transfer. Many of the initial gene transfer studies were conducted using single reporter genes. However, for many gene delivery applications, expression and detection of two genes are useful. The goal of this study was to explore the feasibility of directing the independent expression of two marker genes from a single HIV-1 derived lentiviral vector. We used the enhanced green fluorescent protein (EGFP) and the enhanced yellow fluorescent protein (EYFP) as markers. Because of spectral overlap of these fluorescent proteins, we modified the optical bench of a FACScalibur flow cytometer (BDIS), with insertion of Omega Optical filters, in order to detect the two emitted fluorescences. To independently co-express the marker genes, we tested two different promoter associations in the same lentiviral backbone. In the first case, the EF1α promoter drove EGFP transcription, and EYFP was translated from spliced RNA transcribed from the LTR promoter. In the second case, two internal promoters : EF1α and CMV were inserted in a SIN (Self Inactivating) vector. Different vectors were generated from the pTRIP deltaU3 EF1α EGFP and pTRIP EF1α EGFP lentiviral vectors (gifts from P. Charneau), with transcriptional units and viral elements in variable positions. Following transduction of hematopoietic and non-hematopoietic cell lines, FACS and PCR analyses allowed to quantify transduction and integration efficiencies, respectively. Addition of EYFP in the original single gene lentiviral vector resulted in a decrease in the overall percentage of fluorescent cells. Moreover, vectors using splice mechanism produced weak co-expression of both genes (less than 4% of double positive cells). However, one of the SIN vector that contains two internal promoters transduced 32 to 100% of Nalm6 human pre-B cells, depending on the MOI; of these fluorescent cells, 12 to 78% were double positive. The size of the provirus was assessed in transduced cells, using southern blotting. Cells transduced with vectors coding for the two marker genes contained DNA sequences with sizes below the expected values; these observations suggest that recombination occurred within the vector sequence during or after transduction. This was further confirmed by southern blotting of sorted single and double positive cells; again, bands with lower than expected sizes were detected. We conclude that two highly homologous sequences can lead to recombination when inserted in close positions into a lentiviral backbone. This conclusion was reinforced by the observation that substitution of the DsRed2 coding sequence to EYFP in the same lentiviral vector that also encodes EGFP produced 100% double positive cells following transduction.
Generation of Transgenic Mice Expressing High Levels of Activated Murine Coagulation Factor VII.
Authors: Aljamali Majed N. , Margaritis Paris , Schlachterman Alexander , High Katherine A. 
Published: October 2019

Abstract
Treatment of acute bleeding episodes in hemophilic patients with inhibitors can be successfully managed by the infusion of recombinant human factor VIIa (rhFVII, NovoSeven“). We have recently shown the efficacy of a gene transfer approach to treat hemophilia B (HB) mice by adeno-associated virus (AAV) expressing activated murine FVII (mFVIIa) (J Clin Invest. 2004 Apr; 113(7): 1025–31). To assess the consequences of long-term expression of different levels of mFVIIa, we generated transgenic mice expressing mFVIIa driven by a liver-restricted (transthyretin) promoter. Results from four founders have been analyzed. The levels of mFVII antigen in both founders and their offspring were 3.5–7.5 microgram/ml, about 2.5–5 fold the baseline compared to their non-transgenic littermates. Moreover, the expressed protein retains its coagulation activity in the extrinsic pathway as demonstrated by shortening of the prothrombin time (PT) from 22.3±0.6 sec in the non-transgenic mice to 12.3±1.6 sec in the transgenic littermates. We found two male HB mice that were also transgenic for mFVIIa, resulting from the breeding of one male founder and an HB heterozygote female. The high levels of mFVII antigen (7.5 and 5.5 microgram/ml) were accompanied by significantly shorter PTs (9.8 and 12.3, respectively) compared to wild-type baseline of 22 sec, and most importantly, by a shorter activated partial thromboblastin time (aPTT) compared to their two HB littermates (36.3 and 28.8 versus 61.3 and 61.8 sec, respectively), i.e., mice transgenic for mFVIIa show aPTT similar to their wild type littermates. Additionally, kinetics and general characteristics of in vivo clot formation after laser-induced injuries to the arteries of the cremaster muscle were similar in an HB-mFVIIa transgenic mouse (the only one tested) and in normal mice, while clots were absent in HB control mice. On the other hand, thrombin anti-thrombin (TAT) levels in the transgenic mice were comparable to their HB and wild type littermates. These findings support the efficacy and safety of a gene therapy approach for the expression of mFVIIa and should further allow us to assess the risk of continuous expression of elevated levels of mFVIIa in mouse plasma.
Efficient Non-Viral Transfection of Mouse and Human Embryonic Stem Cells.
Authors: Berneman Zwi N. , Brown Jeremy P. , Van der Sar Sjaak , Van den Plas Dave , Van den Eeden Lena , Van Bockstaele Dirk R. , Jorens Philippe G. , Van Tendeloo Viggo F. , Merregaert Joseph , Singh Prim B. , Ponsaerts Peter 
Published: October 2019

Abstract
BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells.
METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimized on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analyzed by FACS for EGFP expression, viability and phenotype. Also, stably-transfected mouse ES cell lines containing Lox-P or FRT-flanked reporter genes were electroporated with mRNA encoding Cre- or FLPe-recombinase proteins. Monitoring recombination efficiency was done based on the appearance and/or disappearance of fluorescent reporter genes, as determined by FACS analysis. ES cells that underwent recombination were further analyzed for potential to differentiate towards the neural lineage and differentiated cells were analyzed by FACS for expression of neural markers.
RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not influence ES cell phenotype and further cell culture of undifferentiated ES cell populations. Electroporation of mRNA encoding Cre- or FLPe-recombinase proteins in stably-transfected mouse ES cell lines containing LoxP- or FRT-flanked reporter genes resulted in a recombination efficiency of respectively 75% and 90%. Moreover, these recombination events did not have influence on ES cell phenotype, viability, growth potential, and their ability to differentiate towards neural cell types upon retinoic acid stimulation. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation.
CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cell-fate within in vitro cultured embryoid bodies.
RNA Electroporation as a New Gene Transfer Method in Hematopoietic Progenitor Cells, Mesenchymal Cells and Activated T-Cells.
Authors: Berneman Zwi N. , Smits Evelien , Ponsaerts Peter , Lenjou Marc , Nijs Griet , Van Bockstaele Dirk R. , Van Tendeloo Viggo F. 
Published: October 2019

Abstract
Electroporation of messenger RNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. When electroporating dendritic cells, this method combines high transient gene expression with low cell mortality without the risk for insertional mutagenesis. Here, we have investigated the potential of mRNA electroporation to induce short-term transgene expression in other cell types such as bone marrow CD34+ progenitor cells, in vitro cultured bone marrow mesenchymal cells and phytohemagglutinin A (PHA)-stimulated peripheral blood T-cells. Transgene expression after electroporation with mRNA encoding the enhanced green fluorescent protein (EGFP) was evaluated by flow cytometry. Flow cytometric analysis 24h after EGFP mRNA electroporation revealed that 35% of fresh uncultured bone marrow-derived CD34+ hematopoietic progenitor cells was efficiently transfected. In the population of in vitro cultured mesenchymal cells, 90% of the viable CD45-CD13+ cells showed detectable EGFP expression. The level of transient EGFP expression in the PHA-stimulated T-cell population was 47% at 24h after electroporation. Importantly, no significant difference of cell mortality between electroporated and non-electroporated cells was observed, due to the mild electroporation procedure ensuring high cell viability. When looking at the kinetics of EGFP expression, we saw that EGFP was still expressed at day 7 after electroporation with highest expression at day 4. Short-term gene introduction by mRNA electroporation in hematopoietic progenitor cells, mesenchymal cells and T-cells could be used to direct the differentiation and/or to modulate the function of these cells. We conclude that mRNA electroporation is an efficient method for short-term gene transfer in CD34+ cells, mesenchymal cells and activated T-cells with potential applications in gene therapy procedures.