bluuebunny/crossref_metadata_2025_split huggingface dataset:
Over-Expression of Flt3 Induces the Expression of NFkB Target Genes, cIAP-1 and IL-6.
Authors: Takahashi Shinichiro , Harigae Hideo , Inomata Mitsue , Ishii Keiko Kumura , Licht Jonathan D. , Sasaki Takeshi , Kaku Mitsuo 
Published: October 2019

Abstract
Nuclear factor kappa B (NFkB) regulates transcription of many genes involved in the immune response, including cytokines (e.g. IL-6) and growth factors. NFkB suppresses apoptosis as well, through various mechanisms including activation of cellular inhibitor of apoptosis-1 (cIAP-1). NFkB can be activated by the activation of Ras/phosphatidylinositol 3′-kinase (PI3K)/protein kinase B pathway in acute myeloblastic leukemia (AML). It was reported that in large proportion of primary AML cells, NFkB is constitutively activated, and the target genes are upregulated. Flt3 is a member of receptor tyrosine kinases. Flt3 receptor phosphorylation/activation results in the activation of downstream kinase pathways, like Ras, PI3K, leading to abnormal cell growth and aberrant gene regulation. Increased levels of Flt3 transcript are observed in a large number of AML specimens, and the over-expression of Flt3 contributes to the phosphorylation of Flt3 and activation of this pathway. In this study, we addressed the role of Flt3 over-expression in the activation of NFkB, which is a target molecule of various kinase pathways. We first tested the effect of Flt3 expression to NFkB responsive reporter by transient transfection and revealed the significant induction of the reporter. Next, we generated Flt3 transgenic BaF3 (BaF3-Flt3) cells that stably over-expressing Flt3 to confirm this effect. The introduction of NFkB-responsive reporter, as well as serum response element (SRE)-luciferase fused reporter into these cells resulted in the activation of the reporters. These results suggest that Flt3 over-expression mediated signal transduction activates NFkB pathways and affects growth factor pathways as well. Real time quantitative PCR demonstrated that in BaF3-Flt3 cells, mRNA expression of IL-6 and cIAP-1, both known target genes of NFkB, were increased. AG1296 or PDTC, potent inhibitors of Flt3 and NFkB respectively, completely abrogated the induction of these genes, suggesting the importance of Flt3-NFkB pathway to these effects. Moreover, we measured the expression level of Flt3 and NFkB target genes in 24 primary AML samples. As a result, there was a tendency of moderate to weak positive correlation between Flt3 and NFkB target genes. (Flt3 vs IL-6 [Spearman’s rank correlation coefficient: r= 0.35], Flt3 vs cIAP-1 [r=0.16]). These facts may also indicate the positive influence of Flt3 over-expression to NFkB target genes. Overall, these suggest a role of Flt3 over-expression in the activation of NFkB pathway in AML.
R399H Mutated ETS Domain Leads to a Functional ETV6 Double-Knock-Out in Acute Leukemia and Completely Alters the Physiological Function of ETV6.
Authors: Hennig Heike , Hillmer Frauke , Becker Annegret , Podleschny Martina , Pukrop Tobias , Truemper Lorenz H. , Griesinger Frank 
Published: October 2019

Abstract
ETV6 is the only known transcriptional repressor which plays an important role in the malignant transformation of haematopoietic cells in different leukemias.
In most cases of ETV6-AML1 + leukemias, the second ETV6 allele is knocked out by deletions. Other mechanisms of knocking out the second ETV6 allele in acute leukemias carrying ETV6 fusion genes, have not been described as of yet. Recently, we have published that the MT-ALL cell line with multilineage differentiation potential carries an ETV6-ARG fusion gene. The second ETV6 allele carries a point mutation in the highly conserved ETS-domain (G to A, bp 1220), leading to the replacement of arginine, normally directly binding to DNA, by histidine (R399H). Therefore, we investigated whether the R399H point mutation leads to a functional double knock-out and will reduce the DNA binding affinity of ETV6.
For the ETV6 binding studies the single point mutated and wild type ETS-domains were amplified and the proteins were expressed. Electro mobility shift assays (EMSA) were performed with the EBS (ETS binding site) carrying the specific GGAA core motif. The wild type ETS domain (ETSwt) incubated with the EBS showed a clear shift of the protein DNA complex loaded on a gel, but not the point mutated ETS domain (ETSmt). In order to quantify the binding of the ETSwt and mt-domains to EBS we established a transient transfection reporter gene assay. For this assay we constructed vectors which contain the EBS fused to the TK promoter, firefly luciferase serves as reporter gene. After transient cotransfections of HeLa cells with plasmids which expressed the ETSwt- or ETSmt-domain together with the vector containing the EBS(wt or mt), the activities of luciferase were measured in a tube luminometer. As expected, cotransfection of ETSwt and EBSwt reduced luciferase activity up to 75% compared with control vector and EBSwt cotransfection. Cotransfection of ETSmt with EBSwt did not lead to a reduction of luciferase activity. The specificity of the reporter gene analysis was controlled by using EBSmt.
These results show that the transcriptional repressor function of ETV6 is dependent on binding of ETV6-ETS domain to EBS and that the point mutation completely alters the physiological function of ETV6. Further studies would explain whether the multilineage differentiation potential of MT-ALL is dependent on ETV6 double-knock-out.
Randomized Clinical Trial Comparing Melphalan-Prednisone (MP), MP-Thalidomide (MP-THAL) and High-Dose Therapy Using Melphalan 100 MG/M2 (MEL100) for Newly Diagnosed Myeloma Patients Aged 65–75 Years. Interim Analysis of the IFM 99-06 Trial on 350 Patients.
Authors: Facon Thierry , Mary Jean Yves , Hulin Cyril , Benboubker Lofti , Attal Michel , Harousseau Jean Luc , Pegourie Brigitte , Renaud Marc , Guillerm Gaelle , Voillat Laurent , Chaleteix Carine , Troncy Jacques , Monconduit Mathieu , Casassus Philippe , Maisonneuve Herve , Dorvaux Veronique , Jaubert Jerome , Dib Mamoun , Doyen Chantal 
Published: October 2019

Abstract
The results of the IFM 95-01 trial comparing MP to Dexamethasone-based regimens were consistent with the fact that the standard MP remained the reference treatment for patients (pts) older than 65 years with multiple myeloma (MM) (Blood2003;102,147a). In May 2000, the IFM designed a new trial, IFM 99-06, for pts aged 65–75 years, comparing standard MP (12 courses at 6 weeks intervals) to MP-THAL (same MP, THAL at the maximum tolerated dose but ≤400 mg/day, stopped at the end of MP) and a MEL100-based treatment (intermediate-dose MEL procedure developped by the Italian MM study group); VADx2, cyclophosphamide 3g/m2 for stem cells collection, 2 courses of MEL100. IFM99-06 is planned to randomize 500 pts, according to a 3 (MP) - 2 (MP-THAL) - 2 (MEL100) allocation, and schedules two main comparisons ; MP vs MP-THAL and MP vs MEL100. The primary end point is overall survival. In the absence of clear toxicity data for MP-THAL and MEL100 in elderly MM pts, in the setting of a large randomized study, two interim analyses were planned, after the enrollment of 200 (40%) and 350 (70%) pts (followed at least 4 months), to be reviewed by an independent expert review committee (R.Schots / B.Van Camp, Brussels, Belgium ; M. Boccadoro, Torino, Italy ; S.Chevret, Paris-St-Louis, France). The first interim analysis was performed during the first months of 2003 (reference date for analysis January 2, 2003 ; 200 pts enrolled between May, 2000 and June, 2002). The experts considered that (i) the adverse events were as expected with the treatments used (ii) no clear-cut advantage or disadvantage of either MP-THAL or MEL100 over MP was observed according to the statistical rule of the first interim analysis (iii) the trial should pursue as planned. Following a request of the french regulatory agency AFSSAPS, we were asked to look rapidly at some thalidomide toxicities and at the feasability of MEL100. The incidence of deep veinous thrombosis (DVT) was 6%, 9% and 3.5% for MP, MP-THAL and MEL100, respectively (no toxic death due to DVT). Peripheral neuropathy was observed in 25% of MP-THAL pts (14/57 pts, at a median time of 12 months). MEL100 was feasible ; approximately 80% of the pts received the cyclophosphamide and 65% received the 2 MEL100 treatments. No toxic death was noted among 74 courses of MEL100 (39 pts). The second interim analysis, performed on 350 pts (382 pts enrolled on August 1, 2004) is in progress during July/August 2004 (date of point June 1, 2004). Results of the second interim analysis will be presented at the meeting.
Maternal Anti-HPA1a Antibody Level as Predictive Value in Neonatal Alloimmune Trombocytopenic Purpura (NAITP).
Authors: Killie Mette K. , Kjeldsen-Kragh Jens , Skogen Bjørn , Husebekk Anne 
Published: October 2019

Abstract
NAITP is suspected if a neonate unexpectedly presents with petecchiae and is severely trombocytopenic. The thrombocytopenia is caused by maternal alloantibodies to platelet-specific antigens, most often anti-HPA 1a. A mother who has previously given birth to an affected child is usually considered at high risk for giving birth to a trombocytopenic child also in the subsequent pregnancy. The aim of the present study was to compare to what extent quantification of anti-HPA 1a versus obstetric history can predict the severity of thrombocytopenia in new-borns. Anti-HPA 1a was repeatedly quantified in 152 immunized pregnant women who were included in a large Norwegian prospective study. Sixty-eight of the women gave birth to thrombocytopenic children of which 42 suffered severe thrombocytopenia (plt. count <50x109/l). Ten women had previously given birth to children with symptomatic NAITP. Five of these did not give birth to children with severe thrombocytopenia in their subsequent pregnancy. Positive predictive values for thrombocytopenia (<150x109/l) were 0.75 using obstetric history and 0.80 for anti-HPA 1a antibody level above 100 Arbitrary Units (AU). Positive predictive values for severe NAITP (<50x109/l) were hence 0.50 and 0.51 for obstetric history and antibody level. However, the diagnostic sensitivity for obstetric history was only 0.12, whereas anti-HPA 1a level above 100 AU had a diagnostic sensitivity of 0.88. In addition, we found that the level of anti-HPA1a tend to increase in first pregnancy and decrease in subsequent non-compatible pregnancies. This observation is in accordance with the fact that half of the women, who gave birth to NAITP children in their first pregnancy, did not get an affected child in their next pregnancy. In conclusion, obstetric history only identifies a few of the affected cases since all first time cases of sever NAITP are missed. Both obstetric history and antibody level has comparable positive predictive values for severe NAITP. However, the level of anti-HPA 1a antibody would be the best marker to identify severe NAITP cases because of independence of parity and obstetric history.
Can IVIg Preparations with Specificity for Soluble Antigens Mimic the Therapeutic Effects of IVIg in the Treatment of Immune Thrombocytopenia?.
Authors: Siragam Vinayakumar , Brinc Davor , Crow Andrew R. , Song Seng , Freedman John , Lazarus Alan H. 
Published: October 2019

Abstract
Intravenous immunoglobulin (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP). In addition, an IVIg specific for a cell-associated antigen (anti-D) is also able to increase platelet counts in D positive subjects with ITP. Whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP is, however, unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenia. Ovalbumin (OVA) was selected as the primary target antigen because it can be used in its soluble form or can be coupled to syngeneic red blood cells (OVA-RBC), and the same anti-OVA antibody could be used with both OVA and OVA-RBC. Mice were injected with either preformed soluble OVA + anti-OVA, OVA-RBC’s presensitised with anti-OVA, or appropriate control preparations, followed by an anti-platelet antibody to induce thrombocytopenia. Both experimental regimes, but none of the control preparations, protected mice from ITP. Similar to IVIg, soluble OVA + anti-OVA did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcγRIIB (FcγRIIB−/−). In contrast, injection of anti-OVA sensitised OVA-RBC’s did ameliorate thrombocytopenia in FcγRIIB−/− mice. Finally, mice injected with IgG specific for the endogenous soluble antigens, albumin and transferrin, also inhibited ITP in an FcγRIIB-dependent manner. We conclude that IgG antibodies directed to soluble antigens can inhibit or reverse immune thrombocytopenia in an FcγRIIB-dependent manner.
Gene Transfer to Repopulating Human CD34+ Cells Using Amphotropic, GALV or RD114 Pseudotyped HIV-1 Based Vectors from Stable Producer Cells.
Authors: Richter Johan , Johansson Maria , Relander Thomas , Olsson Karin , Ikeda Yasuhiro , Takeuchi Yasuhiro , Collins Mary 
Published: October 2019

Abstract
A novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation to NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used; the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+) and a modified feline endogenous virus RD114 envelope (RDpro). Titration of vectors harvested from the stable packaging cells gave the following results: MLV-A: 1–3x106 inf.u/ml, GALV+: 1x106 inf.u/ml and RDpro: 3–10x106 inf.u/ml CB CD34+ cells were transduced on Retronectin® preloaded with vector either in fresh medium (PL alone) or in vector containing medium (PL+VCM). Gene transfer to freshly thawed CD34+ in the absence of cytokines was very low. Addition of cytokines (CK; SCF, TPO and FL) increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 hours wit the same cytokines. Concentration of the vectors (15-fold) by low-speed ultracentrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 70% of the cells under the same conditions. Detailed in vitro transduction data is shown in table as percentage GFP positive cells:
RDpro MLV-A GALV+ PL alone PL+VCM PL alone PL+VCM PL alone PL+VCM No CK 9.2±2 0.9±0.8 12.5±3.5 6.6±0.1 0.9±0.4 1.4±0.5 With CK 22.4±8.3 3.3±5.3 18.2±3.8 10.8±3.8 4.8±1.7 16.7±9.0 CK prestim f 24 hours 57.6±14.9 35.3±32.1 45.4±14.9 36.3±14.8 3.4±1.7 19.5±12.2 Concentrated vector CK prestim f 24 h 93.4±3.3 93.3±3.7 73.3±7.6 40.3±20.4 66.1±7.5 77.8±10.9
Transplantation of CD34+ cells prestimulated for 24 hours and then transduced with a single hit of concentrated vector to irradiated NOD/SCID mice revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells which was significantly higher than the MLV-A (12.6%) or GALV+ (25.1%) pseudotyped vectors. Thus, the STAR packaging system, especially with the modified RD114 envelope, appears to be a new valuable tool for the genetic modification of primitive human progenitor cells.
The Human Xist Gene Promoter Prevents Silencing of an Integrated Reporter Gene.
Authors: Greene Michael R. , Lowrey Christopher H. 
Published: October 2019

Abstract
Epigenetic silencing and position dependent expression are long-standing problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small number of genes which are expressed from the inactive X chromosome. The product of this gene is an untranslated structural RNA which coats the X chromosome destined for inactivation prior to H3 Lys 9 hypoacetylation, H3 Lys 9 methylation, CpG island methylation and the subsequent silencing of most of the genes on the chromosome. Continued expression of the XIST gene in this highly repressive environment is required to maintain the chromosome in an inactive state. The region of the proximal promoter of the XIST gene on the inactive X chromosome has been shown to retain an active chromatin structure. Based on these findings we hypothesized that the XIST gene promoter would be able to resist the epigenetic changes which lead to transgene silencing. To test this idea, we subcloned a minimal XIST promoter upstream of an enhanced GFP reporter gene in a pUC-based plasmid which also contained a neomycin resistance gene. The same plasmid, with a CMV promoter, served as a control vector. The plasmids were electroporated into mouse erythroleukemia (MEL) cells and then grown in media containing G418. The MEL cell line was chosen because genes transferred into these cells are frequently silenced and because it is often used as a first screen for vectors with potential for use in therapeutic gene transfer to erythroid cells. Individual colonies were selected and G418 removed. After expansion of the clones, flow cytometry was used to determine the percentage of cells in each clonal population which were expressing GFP as determined by comparison to the untransduced MEL cell line. Silencing typically involves a gradual decrease in the proportion of cells expressing the integrated transgene. Statistical analyses of results were performed using the t-test. 16 XIST and 13 CMV clones were available for analysis at the start of the experiment (time 0). 11/13 CMV clones and 12/16 XIST clones initially expressed GFP. Of the clones which were expressing GFP, the average percentages of positive cells were higher for those with the XIST promoter (63% vs. 41%, p= 0.015). Expression was reanalyzed after 6 weeks of culture in the absence of G418 selection. At this time point, the average percentage of GFP expressing cells was much higher for the XIST clones (57% vs. 19%, p=0.00008) and when analyzed for silencing, XIST clones were expressing at an average of 90% of their time 0 levels vs. 46% for the CMV clones (p=.0008). These results indicate that the XIST promoter is resistant to silencing in our model system and is a candidate for further development and mechanistic studies.
Clinical Relevance of Recipient Leukocyte Infusion (RLI) Therapy.
Authors: Saito Toshiki I. , Rubio Marie T. , Sykes Megan 
Published: October 2019

Abstract
BACKGROUND
Surprisingly, anti-tumor responses can occur in patients who reject donor grafts following nonmyeloablative hematopoietic cell transplantation (Dey et al., Biol Blood Marrow Transplant 7:604). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced anti-tumor responses against host-type tumors (Rubio et al. Blood 102:2300).
To further investigate the clinical relevance of this RLI model, we:
Evaluated the effect of RLI from tumor-bearing mice Compared RLI with allogeneic lymphocyte infusion in untreated mice
METHODS
Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day-5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. Some RLI donor mice received BALB/c A20 B cell lymphoma cells (1x105) two weeks before RLI. Some groups received RLI depleted of B cells using B220 negative selection by MACS column. A20 cells (5x105) were given i.v. one week after RLI in chimeras or after allogeneic lymphocyte infusion (3x107 B10.BR spleen cells) to untreated BALB/c mice.
RESULTS
In the clinical setting, RLI would be obtained from tumor-bearing hosts. We therefore examined whether RLI is still effective when the lymphocytes are obtained from the tumor-bearing mice. Contamination of B220+ cells in the RLI was less than 0.03%. RLI recipients from the tumor-bearing mice (n=10) showed similar tumor survival compared to recipients of RLI from naïve donors (n=10) (median survival time [MST] 53 versus 50 days respectively, p=0.48) (Figure 1). Thus, with a purging procedure, the same anti-tumor effect was achieved with RLI from tumor-bearing hosts as from non-tumor-bearing hosts.
Allogeneic lymphocyte injection is a potentially feasible anti-tumor therapy. We therefore compared anti-tumor effects of allogeneic lymphocyte infusion into naïve mice with that of RLI given to mixed chimeras. Chimeric RLI recipients mice (n=13) had longer survival (MST 61 days) than naïve mice receiving allogeneic lymphocytes (n=10) (MST 39.5 days, P<0.01) (Figure 2). This result suggests not only that the anti-tumor effect of RLI therapy is stronger than allogeneic lymphocyte infusion therapy but also suggests that rejection of allogeneic cells is insufficient and mixed chimerism is required prior to the induced rejection to achieve maximum anti-tumor effects.
CONCLUSION
Together, these data reinforce the potential of RLI therapy to be a new HSCT strategy which does not have the risk of graft-versus-host disease.
Cord Blood CD4+CD25+ Derived T Regulatory Cell Lines Express FoxP3 Protein and Manifest Potent Suppressor Function.
Authors: Godfrey Wayne R. , Spoden Darrin J. , Ge Ying G. , Baker Seth R. , Liu Baoling , Levine Bruce L. , June Carl H. , Blazar Bruce R. , Porter Stephen B. 
Published: October 2019

Abstract
CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with >95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production.
Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines.
Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.