bluuebunny/crossref_metadata_2025 huggingface dataset:
The Influence of Human Plateley Antigen Match on the Success of Stem Cell Transplantation after Myeloablative or Reduced-Intensity Conditioning Regimen.
Authors: Leitner Gerda C. , Greinix Hildegard T. , Kalhs Peter , Paul Hoecker , Simon Panzer
Published: October 2019

Background: Mismatches between donor and recipient for human platelet antigens (HPA) may affect the success of transplantation due to: a) serving as minor histocompatibility antigens and therefore render recipients at risk for GvHD, b) inhibition of thrombopoiesis due to platelet antibodies.

Patients and Methods: We evaluated in 54 patients receiving hematopoietic stem cell transplantation (HSC T) from HLA matched siblings after a myeloablative conditioning regimen the occurrence of GvHD, transplant related mortality (TRM), and the need of platelet support by prospective analyzes of donor-recipient pairs for HPA -1, -2, -3, and -5 allotypes. Patients were screened for platelet antibodies prior to transplantation and in weekly intervals until day 100 after transplantation. Forty five patients receiving HSCT from related or unrelated donors after a reduced-intensity conditioning regimen (RIC) were enrolled retrospectively after a median observation time of 372 days.

Results: Neither the incidence of GvHD and TRM, nor the onset of thrombopoiesis, nor the CCI after platelet transfusions, nor the frequency of platelet transfusions were affected by HPA mismatches. TRM among patients receiving a RIC was higher when grafts were from unrelated donors with 2 or more mismatches. Six of 7 patients who died due to TRM had 2 mismatches in the HPA system, although TRM was not significantly associated with mismatched HPA (p = 0.06). However, within donor-recipient pairs with more than one HPA mismatch TRM occurred when grafts were from unrelated donors (p = 0.03).

Conclusion: Thus, the HPA match does not affect the success of transplantation.
Diverse Functional Implications of ADAMTS13 Gene Mutations in Patients with TTP and Congenital Deficiency.
Authors: Donadelli Roberta , Banterla Federica , Capoferri Cristina , Galbusera Miriam , Ruggeri Zaverio M. , Bucchioni Sara , Noris Marina , Remuzzi Giuseppe
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the VWF cleaving protease ADAMTS13, causing life-threatening disseminated microvascular thrombosis. Here we report four missense mutations (V88M, G1239V, R1219W, R1123C) in three patients with congenital ADAMTS13 deficiency. The three subjects carrying the mutations had less than 10% normal ADAMTS13 plasma antigen levels (measured by ELISA), which is consistent with previously reported data showing that most ADAMTS13 mutations in TTP patients result in impaired secretion of the protein. To evaluate whether the small amount of the mutant protease present in patients’ plasma had proteolytic activity, we cloned the ADAMTS13 cDNA in the pMT/Bip/His Drosophila expression vector and introduced the respective mutations by directed mutagenesis technique using wild-type cDNA as a template. These wild-type and mutant constructs were stably transfected into S2 Drosophila cells under the influence of the Drosophila BiP protein signal sequence, which allows the protein to be secreted into the medium and overcomes impaired secretion caused by the mutations. The induction of the histidine-tagged ADAMTS13 recombinants following the addition of copper was analyzed by Western blotting using an anti-hexahistidine monoclonal antibody. Equal amounts of recombinant ADAMTS13 proteins were used to evaluate proteolytic activity of recombinant proteins by cleavage of the rVWF A1-A2-A3 substrate. The proteolytic carboxyl terminal product of about 30 kDa was visualized by Western blot with a mouse monoclonal antibody directed against an epitope contained within the A3 domain of VWF. All the four missense mutations exhibited reduced activity: V88M:40%, G1239V: 66%, R1219W: 62% and R1123C: 64% of wild type activity . The results were also confirmed by collagen binding assay. For the the V88M mutation (located in the metalloprotease domain), decreased activity was confirmed by kinetic studies. In conclusion, mutant ADAMTS13 proteins found in patients with TTP, besides defective secretion, also have reduced protease activity. The mechanisms of the deficiency will be the matter of further studies.
Favorable Outcomes with Acceptable Toxicity Using a Reduced Intensity Conditioning Regimen for Patients Otherwise Ineligible for Related Donor Allogeneic Stem Cell Transplantation.
Authors: Westervelt Peter , Holmes Wendy , Emmons Robert , Becker Pamela , Lowry Phil , Traynor Ann , Ballen Karen
Published: October 2019

Allogeneic stem cell transplantation represents a potentially curative treatment modality for patients with advanced hematologic malignancies. However, the toxicity associated with fully myeloablative conditioning regimens has historically limited its use to younger, otherwise healthy patients. To investigate the feasibility of allogeneic stem cell transplantation in older (over 55 years) or otherwise higher risk patients (eg, prior transplant) with fully HLA-matched sibling donors, a reduced intensity conditioning regimen consisting of fludarabine (30 mg/M2 IV qd X 6, day −8 to −3), ATGAM 10 mg/kg IV qod X 4, day −7/−5/−3/−1) and melphalan (100 mg/M2 IV X 1, day −2) was employed in a single institution setting. GVHD prophylaxis consisted of cyclosporine (2 mg/kg IV bid, adjusted, beginning day −1) and methotrexate (10 mg/m2 IV, day +1/+3/+6).

RESULTS: 21 patients with a variety of hematologic malignancies (AML=4, MDS=5, NHL=5, CML/MPD=3, HD=2, MM=1, CLL=1) were enrolled. Mean age was 54 years (range 28–66). 7 patients had undergone prior autologous transplants. Median followup among surviving patients was 42 months (range 3–58 months). Peripheral blood chimerism at day +100 was >95% donor in 16/18 patients, 80% (with subsequent conversion to 95%) in one patient, and >95% recipient (with documented relapse) in one patient. Acute GVHD grade 1–2 was seen in 52% and grade 3–4 in 10% of patients. Chronic GVHD was observed in 9/20 evaluable patients (45%). Infectious complications included documented bacteremia (6 cases), candida albicans fungemia (1), aspergillus pneumonitis (3), nocardia pneumonitis (2), CMV viremia (6), CMV colitis (1). Veno-occlusive disease (self-limited, grade 1–2) was observed in 4/21 patients (19%). Relapse was observed in 5/21 patients (24%) at a median 249 days post-transplant (range 97–328 days). Event-free survival was 90% (19/21) at 100 days, 58% (11/19) at 1 year and 47% (8/17) at 3 years. Overall survival was 95% (20/21) at 100 days, 68% (13/19) at 1 year and 47% (8/17) at 3 years. Primary causes of death were relapse (4/9), infection (4/9), and idiopathic pneumonitis/multi-organ failure (1/9). Chronic GVHD was a significant contributing factor in 3/4 fatal infections. PTLD was observed in one patient, who subsequently died of CMV infection/sepsis syndrome.

CONCLUSIONS. Allogeneic BM/SCT using a reduced intensity conditioning regimen is feasible among older patients, and those who are otherwise poor candidates for myeloablative BM/SCT regimens; however, GVHD, infection, and relapse remain formidable obstacles to achieving successful outcomes.
Impact of Allogeneic Stem Cell Transplantation (ASCT) on Outcome of Biphenotypic Acute Leukemia (BAL):.
Authors: Krishnan Amrita Y. , Chang Karen L. , Fung Henry C. , O’Donnell Margaret , Bhatia Ravi , Spielberger Ricardo , Pullarkat Vinod , Stein Anthony , Kogut Neil , Kirschbaum Mark , Bolotin Ellen , SarkodeeAdoo Clarence , Dagis Andrew , Cheng Nigel , Forman Stephen J.
Published: October 2019

Biphenotypic acute leukemia is a rare disease accounting for approximately 10% of acute leukemias using the new scoring system. No standard therapeutic approach exists for this entity. In addition, the prognosis for these patients (pts) is poor and thought to be worse than for acute myelogenous leukemia (AML). ASCT may provide improved disease free survival for patients with high risk AML, but its role in BAL is unknown. We undertook a retrospective study of pts with BAL to determine if outcome was similar following ASCT to pts with AML in first remission. Between 7/93 and 2/04, nine patients with BAL who underwent ASCT were identified. Criteria for the diagnosis of BAL was based on the scoring system adapted from the European Group of Immunological Classification of Leukemia. Pts with Philadelphia positive acute lymphoblastic leukemia (ALL) were excluded. Cytogenetics at presentation included 1 pt with deletion7 and trisomy 8, 1 with 11q23, 2 with complex karytoype, 4 with normal cytogenetics ,1 unknown. Six pts were treated with an AML induction of Idarubicin/Cytarabine, two with an ALL regimen Daunorubicin/Vincristine/Prednisone and one with an Idarubicin- AML based first induction, and then a combination of an ALL/AML reinduction at first relapse. Eight pts were in first complete remission (CR) and one pt in second CR at ASCT. Median age at ASCT was 34 years (range 11–60). Median time from diagnosis to ASCT was 3.4 mos (range 1.5–5.2). Seven pts received sibling donor (SIB) and two received unrelated donor (URD) stem cells. Two pts received marrow and seven received peripheral stem cells. The conditioning regimen consisted of 1320cGy total body irradiation (TBI)+VP16 60mg/kg in 6 pts, 1320cGy TBI+Cyclophosphamide 120mg/kg in 1 pt, Fludarabine125mg/m2+Melphalan 140mg/m2 in 2 pts. Seven received a Cyclosporin- based graft versus host disease prophylaxis regimen (GVHD) and two a Tacrolimus- based regimen. Six pts developed grade III GVHD. Two patients died of pulmonary toxicity, one of GVHD, one from infection and one from disease relapse. One year overall survival (OS) is 39%( 95% CI 13–73). The relapse rate at one year was 20%. In conclusion, despite unfavorable cytogenetics in many patients, relapse rates are low following ASCT for BAL and comparable to pts undergoing ASCT for AML. Future approaches include methods to reduce transplant related mortality which was the major cause of reduced survival in this series.
Improved Disease Survival without Increased GVHD Using Low Dose Cyclosporine and Prednisone in Allogeneic Stem Cell Transplantation - a Case Control Study.
Authors: Moore John J. , Ma David D. , Dodds Tony , Milliken Sam , Fay Keith , Concannon Alan , Smith Ian Nivison
Published: October 2019

Allogeneic stem cell transplantation (HSCT) can cure numerous malignant and non-malignant diseases but this is offset by significant morbidity and mortality from graft versus host disease (GVHD). The standard prophylactic regimen contains cyclosporine, methotrexate and/or prednisone however doses and the timing of tapering remain unclear. Recently Ruutu et al demonstrated reduced GVHD using a prednisone based prophylactic regimen without a survival benefit whereas Baclgalupo et al demonstrated an increased survival benefit with 1mg/kg cyclosporine over 3mg/kg which strongly correlated with cyclosporine levels. Since early 2002 we have adopted both these strategies in an attempt to reduce GVHD in myeloblative HSCT but still maintain disease free survival. Consecutive myeloblative allogeneic HSCT patients (n=47, median age 41 yrs, 28 sibling or family, 19 unrelated) underwent HSCT using cyclosporine 1mg/kg, standard dose methotrexate and prednisone commencing at D14 0.5mg/kg, according to Ruutu et al. All sibling allograft patients underwent conditioning with Bu/Cy whereas unrelated recipients received Cy/TBI. This group was compared with 94 historical controls for age and disease status (median age 36 yrs, 63 sibling or family, 31 unrelated). At a median of 1 year follow up (range 100–600 days), overall and disease free survival is significantly increased in the low dose cyclosporine/prednisone arm (OS: 75% vs 50% at 1 yr, p=0.04). Acute GVHD (II–IV) was similar in both arms at D100 with a trend to less GVHD in the low dose cyclosporine/prednisone arm (20% vs 30%, p=0.1). These results suggest that the low dose cyclosporine/prednisone regimen has controlled GVHD whilst maintaining an adequate GVL effect. Randomised trials using this regimen may be warranted to fully determine its place in allogeneic HSCT prophylaxis.
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

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

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

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

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

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

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

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

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

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.
Adenovirus Vector E4 Gene Promotes Angiogenesis through Modulation of Junctional Connexin 40 and 43 Expression.
Authors: Cheng Joseph , Zhang Fan , Lam George , Hackett Neil R. , Wang Shiyang , Amano Hideki , Shmelkov Sergey V. , Young Lauren M. , Hempstead Barbara , Crystal Ronald G. , Rafii Shahin
Published: October 2019

Gap junction channels formed of connexin (Cx) proteins provide a means for intercellular communication via direct linkage of the cytoplasm of adjacent cells and play important roles in the pathophysiology of vascular and cardiac homeostasis and disease. We have previously reported that introduction of E1/E3-deleted adenovirus (AdE4+) to endothelial cells (EC) selectively activates angiogenesis and increases cell survival via activation of the junctional adhesion molecule vascular endothelial-cadherin which targets the PI3K/Akt pathway. We now show that AdE4+ also potentiates Cx expression in EC in vitro and mouse heart tissue in vivo. Infection of EC with AdE4+, but not AdE4−, vectors resulted in time- and dose-dependent induction of Cx40 and suppression of Cx43 protein and mRNA as determined by immunoblot and Northern blot respectively. Maximal induction of Cx40 and suppression of Cx43 protein and mRNA were observed by 48 hours after infection, and between multiplicity-of-infection of 100 and 200. Immunohistochemical analysis of infected EC revealed that AdE4+ induced Cx40 expression localized to the plasma membrane at intercellular junctions, and attenuated total Cx43 expression. Cx40 expression was also markedly increased and Cx43 significantly decreased in the heart tissue of mice treated with AdE4+ via intra-tracheal administration compared to control mice. Pretreatment of EC with either PKA inhibitor (H89) or PI3K inhibitor (LY294002) abrogated the effects of AdE4+ in regulating Cx40 and Cx43, and was associated with diminished AdE4+-enhanced EC survival. Furthermore, neither PKG inhibitor (KT5823) nor guanylyl cyclase inhibitor (NS2028) affected AdE4+-modulation of Cx. It is of interest to note that cAMP has been shown to trigger Cx trafficking by way of PKA activation, as we have also observed. However, neither forskolin- nor cholera toxin-induced cAMP affected Cx expression in AdE4+-infected EC, indicating that PKA activation by AdE4+ may be independent of cAMP. Taken together, alteration of the Cx expression profile, as mediated through PKA and PI3K signaling pathways, may play a role in AdE4+-associated EC survival. These findings of Cx modulation by AdE4+ may not only explain why E4+ adenoviral vectors may induce increased survival of endothelial cells in vivo, but also may help us to understand the mechanisms of the pathophysiology of vascular and heart disorders by regulation of Cx.
Risk Factors for Bleeding in Patients with Nonvalvular Atrial Fibrillation Who Are Receiving Anticoagulant Therapy with Ximelagatran or Warfarin: Findings from the SPORTIF III and V Trials.
Authors: Douketis James D. , Arneklev Karin , Goldhaber Samuel , Spandorfer John , Halperin Frank , Horrow Jay
Published: October 2019

Background: Ximelagatran is a novel oral direct thrombin inhibitor that is as effective as warfarin in preventing stroke and other thromboembolic complications in patients with nonvalvular atrial fibrillation (AF). Risk factors for bleeding with warfarin are known, but risk factors for bleeding with ximelagatran have not been described. Unlike warfarin, ximelagatran has a predictable anticoagulant effect, does not require anticoagulation monitoring, has a low potential for interactions with drugs, food, or alcohol, and is not affected by genetic polymorphisms. We undertook an exploratory analysis of a large patient database to identify conventional and novel risk factors for bleeding in ximelagatran-treated patients, in warfarin-treated patients, and in all patients, irrespective of treatment.

Methods: We undertook a pooled analysis of the SPORTIF III and V trials trials, which included 7329 patients with nonvalvular AF who received oral ximelagatran, 36 mg twice daily, or warfarin, administered to achieve a target international normalized ratio of 2.0–3.0. Patients had nonvalvular AF and 1 or more risk factors for stroke: hypertension; age ≥75 years; previous stroke, transient ischemic attack (TIA) or systemic embolism; left ventricular dysfunction; age ≥65 years and coronary artery disease; or age ≥65 years and diabetes mellitus. Major exclusion criteria were: mitral stenosis; previous heart valve surgery; transient AF; increased risk for bleeding. Multivariate logistic regression analysis was used to identify independent risk factors for major bleeding. The hazard ratio (HR) for major bleeding, and corresponding 95% confidence interval (CI), was calculated for each variable in the regression model.

Results: The Table presents risk factors in which there was a significant or a non-significant (NS) association with major bleeding in ximelagatran-treated or warfarin-treated patients, and in the combined patient population.

Risk factor Ximelagatran-treated patients, HR (95% CI) Warfarin-treated patients, HR (95% CI) Combined patient population, HR (95% CI) Aspirin use 1.65 (1.07, 2.55) 2.40 (1.69, 3.42) 1.96 (1.49, 2.58) Increasing age 1.03 (1.01, 1.05) 1.06 (1.03, 1.08) 1.04 (1.03, 1.06) Prior liver disease NS 4.96 (1.57, 15.62) NS Prior stroke or TIA 1.78 (1.16, 2.73) NS NS Diabetes mellitus 1.80 (1.18, 2.75) NS 1.39 (1.05, 1.86) Asian race NS NS 1.99 (1.16, 3.42) Statin use 0.62 (0.39, 0.97) 0.61 (0.42, 0.88) 0.62 (0.39, 0.97)

Conclusions: Overall, the bleeding risk was lower with ximelagatran compared with warfarin. Aspirin use and increasing age were associated with an increased risk of bleeding in both ximelagatran- and warfarin-treated patients. Statin use was associated with a decreased risk for bleeding in both groups.
Early Results from a Choice Based Conjoint Analysis Assessing Patient Preferences for Selected Features of Erythropoietic Agents.
Authors: Bolge Susan C. , Fastenau John M. , Howell Jamie B. , Piech Catherine T. , Donohue Jane A.
Published: October 2019

Background: Understanding patients’ preferences regarding treatment features is important, as they may not be aligned with those of providers. Epoetin alfa (EPO) and darbepoetin alfa (DARB) are two erythropoietic agents indicated for the treatment of chemotherapy-related anemia (CRA) to increase hemoglobin (Hb) levels and reduce transfusion requirements. These outcomes are associated with fatigue reduction and improved quality of life (QoL), but the degree of improvement and current dosing schedules for both agents may vary. Preliminary results from a comparative trial suggest EPO provides a greater early and overall Hb rise compared to DARB at the commonly prescribed doses (EPO 40,000 units weekly vs. DARB 200 mcg every two weeks) (Proc Am Soc Clin Oncol.2004;23:763 (8153)).

Objective: To assess patient preferences for two attributes, effectiveness and convenience, associated with the use of erythropoietic agents.

Methods: A cross-sectional study of adults (age 18+) with solid tumor cancers and anemia is on going. 500 planned participants are being recruited through 50 oncologists’ offices across the U.S. Information is collected via a self-administered questionnaire that is divided into two parts. The first, completed by the provider, captures clinical information; the second, completed by patients records their knowledge, experience and preferences regarding anemia and its treatments, including resource use, QoL and productivity. This preliminary analysis focuses on the choice based conjoint (CBC) section of the questionnaire that assesses effectiveness, defined as time to a noticeable relief of fatigue, and convenience, defined as number of provider visits during an 8-week period. Each attribute has three levels (4, 6, and 8 weeks or visits).

Results: 276 provider/patient-matched questionnaires have been received. 264 providers and 237 patients have completed the CBC section (96% and 86% response rates, respectively). 80% of providers view time to a noticeable relief of fatigue as being more important to patients than frequency of visits when choosing a medication to treat anemia. Similarly, patients also view time to a noticeable relief of fatigue as being the more important characteristic of anemia medications (1.8 times more important than frequency of visits). For time to a noticeable relief of fatigue, patients prefer an early onset (4 over 6 over 8 weeks). For convenience, patients prefer 8 over 6 over 4 visits during an 8-week period.

Conclusion: Providers and patients both view effectiveness as a more important attribute of anemia treatment over convenience. While patients prefer relief of fatigue as soon as possible, interestingly, their preference to see providers more frequently rather than less may be contrary to some providers’ beliefs.
Topical Progesterone Cream Does Not Increase Thrombotic and Inflammatory Factors in Postmenopausal Women.
Authors: Stephenson Kenna , Price Carol , Kurdowska Anna , Neuenschwander Pierre , Stephenson John , Pinson Barbara , Stephenson Douglas , Alfred Denita , Krupa Agnieszka , Mahoney Debra , Zava David , Bevan Mary
Published: October 2019

Postmenopausal women have an increased risk of cardiovascular disease, and heart disease is the leading cause of death in postmenopausal American women. Conventional hormone replacement therapy has been shown to result in an increase in thrombotic events in large prospective clinical trials including HERS I, and the recently halted Women’s Health Initiative. One possible mechanism for this observed increase is the unfavorable net effects of conjugated equine estrogens and medroxyprogesterone acetate on the hemostatic balance and inflammatory factors. An estimated 50 million American women are peri or postmenopausal and clinical therapies for menopausal symptoms remain a significant challenge in light of the known thrombotic risks. In this prospective blinded study, we examined the short-term effect of topical progesterone cream on menopausal symptom relief in 30 healthy postmenopausal women. Potential adverse effects of topical progesterone on hemostatic and inflammatory factors and cortisol levels were also examined. Subjects were randomized to first receive either 20 mg of topical progesterone cream or placebo cream for 4 weeks. Following a subsequent 4-week washout period, subjects were crossed over to either placebo cream or active drug for an additional 4-week period. In each case, progesterone and cortisol levels were monitored by salivary sampling. Baseline values, 4-week follow-up values and end-of-study values were also obtained for the Greene Climacteric Scale, total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, PAI-1, CRP, TNFα, and IL-6. For subjects receiving 20 mg of topical progesterone cream for 4 weeks, Greene Climacteric Scale scores were consistently and significantly improved (decreased) over baseline, demonstrating significant relief from menopausal symptoms. In addition, in a subpopulation of hypercortisolemic women, topical progesterone was associated with a favorable decrease in nocturnal cortisol. Surprisingly, and in sharp contrast to earlier studies with conventional hormone replacement therapy, topical progesterone had no effect on any of the hemostatic components examined: total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, and PAI-1 levels were all unchanged. Levels of CRP, TNFα and IL-6 also remained unchanged. From this study we conclude that administration of topical progesterone cream at a daily dose of 20 mg significantly relieves menopausal symptoms in postmenopausal women without adversely altering prothrombotic potential. Since the thrombotic complications that are typically observed with conventional hormone replacement therapy do not seem to occur with topical progesterone, this treatment should be seriously considered as an effective and safe alternative clinical therapy for women suffering from menopausal symptoms.