bluuebunny/crossref_metadata_2025_split huggingface dataset:
Successful Translation of a GMP-Based, Clinical Scale Selective Allodepletion Approach for Matched Donor-Recipient Pairs from Bench-to-Bedside.
Authors: Mielke Stephan , Nunes Raquel , Rezvani Katayoun , Fellowes Vicki S. , Venne Annie , Solomon Scott R. , Fan Yong , Scotto Christian , Read Elizabeth J. , Barrett A. John 
Published: October 2019

Abstract
Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from hematopoietic stem cell allografts to prevent graft versus host disease (GvHD) while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion (PD) process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the rhodamide-based photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were co-cultured with responder cells from HLA-matched or mismatched donors. Optimal results were achieved when co-cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule/cm2 light energy at 5x106 cells/ml.
Figure Figure
In six mismatched stimulator-responder pairs, the median reduction of allo-reactivity was 474-fold (range, 43–864) when compared to the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9–3.3) reduction. In three matched pairs alloreactive helper T-lymphocyte precursors (HTLps) were reduced below 1:100,000 while third party responses remained above 1:10,000. CMV-specific T cells and responses to SEB were preserved. The intracellular TH9402 amount after extrusion correlated significantly with the depletion efficacy obtained against the original stimulator (r2=0.76; p=0.0235) but not against the 3rd party (r2=0.05; p=0.68). This establishes a clinical scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current good manufacturing practice (cGMP) being reliably capable of generating at least 107/kg SD T cells for transfusion using a single PD session. In establishing this SD approach, we set out to design a technique that could ultimately be adopted in many cell transplant processing centers. Recent review of our Investigational New Drug (IND) application by the Food and Drug Administration (FDA) and review of our clinical protocol by our Institutional Review Board have been successful, and a clinical trial is in progress where recipients with hematological malignancies are given a myeloablative preparative regimen followed by a transplant of selected CD34+ cells together with 5 x 106 SD T cells/kg from HLA-matched sibling donors.
Defective Expression of Neutrophil Serine Proteases in Myeloid Progenitors of Congenital Neutropenia Patients Carrying Either ELA2 or HAX1 Mutations.
Authors: Skokowa Julia , Fobiwe John-Paul , Lan Dan , Germeshausen Manuela , Welte Karl 
Published: October 2019

Abstract
Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis with two major types of inheritance:
autosomal dominant CN defined by mutations in ELA2 gene encoding neutrophil elastase (NE) (Horwitz M., et al., Nat Genet.1999;23:433) and autosomal recessive CN (including Kostmann syndrome) carrying HAX-1 mutations (Klein C., et al., Nat Genet.2007;39:86), both characterized by a maturation arrest of granulopoiesis at the level of promyelocytes.
In the present study we aimed to evaluate the expression profile of genes specifically expressed in the CD33+ bone marrow promyelocytes of both patient groups harbouring ELA2 or HAX1 mutations. In healthy individuals mRNA expression levels of neutrophil serine proteases (neutrophil elastase (ELA2), cathepsin G, cathepsin D, proteinase 3 and azurocidin) as well as of myeloperoxidase (MPO) and defensins reached highest levels in the azurophil granules at the promyelocytic stage of neutrophil differentiation (Borregaard N., et al., Curr Opin Hematol.2001;8:23). We found downregulation of mRNA expression levels of ELA2 (8.9 fold), cathepsin G (7.6 fold), cathepsin D (11.2 fold), proteinase 3 (9.2 fold) and defensin B1 (6.5 fold) in both groups of CN patients (with ELA2 or HAX1 mutations), in comparison to G-CSF-treated patients with idiopathic neutropenia (IN) and G-CSF-treated healthy controls. In contrast, there were no difference in mRNA expression levels of azurocidin and only slight decrease in the expression of MPO mRNA in CN patients. Additionally, we found significantly reduced protein levels of neutrophil elastase (NE) in plasma of CN patients irrespective of “ELA2 or HAX1” inheritance, in comparison to cyclic neutropenia (CyN) patients, IN patients and G-CSF-treated healthy controls. Taken together, both ELA2 and HAX1 mutations are associated with defective expression of neutrophil serine proteases such as NE, cathepsin G, cathepsin D, proteinase 3 as well as of defensin B1 in CD33+ myeloid progenitor cells of CN patients, suggesting a common pathway for both patient groups. Intriguingly, ELA2 expression is directly regulated by LEF-1, suggesting that abrogated LEF-1 expression in CN promyelocytes (Skokowa J., et al., Nat. Med.2006;12:1191) may be responsible for defective serine proteases expression in both groups, since all are regulated by a similar mechanism.
Cost Effectiveness of Rituximab Maintenance (R-Maint) vs Autologous Stem Cell Transplant (ASCT) from a UK National Health Service (NHS) Perspective.
Authors: Millar Douglas R. , Lewis Gavin , Marcus Robert 
Published: October 2019

Abstract
Objectives: Both ASCT and R-Maint have been shown to be effective therapies for treating rituximab naïve patients with relapsed/refractory follicular lymphoma. The aim of this research was to assess the cost effectiveness of ASCT compared to R-Maint in this patient population (following a response to chemotherapy induction) from a UK perspective.
Methods: A literature review of randomised control trials (RCTs) and case series studies suitable for comparing the efficacy of the two treatments in relapsed / refractory follicular lymphoma patients was performed. Given the heterogeneity of relevant study outcomes and designs a naïve indirect comparison was performed. Equivalent efficacy between the two therapies was assumed based upon the indirect comparison; hence a cost minimization approach was utilized. The starting time point of the analysis was following 3 cycles of CHOP therapy. In the EORTC 20981 trial both RCHOP and CHOP induction therapies were used; to reduce heterogeneity between the trial populations being compared, only results from patients who received CHOP induction in this trial were considered. Drug, administration, adverse event, and monitoring costs were evaluated from the start of treatment to the point of relapse after which time-point cost equivalence was assumed between the two therapies. Resource use and unit costs were sourced from published NHS reference costs, the EORTC 20981 study, and clinical expert opinion. Uncertainty around the assumption of equal efficacy was explored using threshold analysis based on an assumed incremental cost effectiveness ratio (ICER) threshold of £30,000 and an average utility score of 0.71 for patients with follicular lymphoma (derived from the literature).
Results: One RCT and 6 case series for ASCT and one RCT for R-Maint were identified. The estimated cost of R-Maint and ASCT was £12,275 and £25,563 respectively, resulting in an estimated incremental cost of ASCT over R-Maint of £13,288 per patient. ASCT would have to offer an additional 7.5 months of overall survival to be considered cost effective compared to R-Maint.
Conclusions: There is a high degree of certainty that ASCT produces higher costs compared to R-Maint, however it is uncertain whether ASCT offers an increase in either progression-free or overall survival over R-Maint in rituximab naïve patients. Consequently R-Maint may be considered a cost-saving treatment option for such patients in the treatment of relapsed/refractory follicular lymphoma patients compared to ASCT.
Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.
Authors: Rohrabaugh Sara , Mantel Charlie , Broxmeyer Hal E. 
Published: October 2019

Abstract
Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.
Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.
Authors: Okamura Daisuke , Yagasaki Fumiharu , Maeda Tomoya , Ishikawa Maho , Jinnai Itsuro , Bessho Masami 
Published: October 2019

Abstract
Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.
Pivotal Role of FoxO3 and mTORC1 in the Opposite Effects of SDF-1 and TGF-β on Cell Cycle Progression in CD34+ Stem/Progenitor Cells.
Authors: Chabanon Aurelie , Desterke Christophe , Rodenburger Emilie , Clay Denis , Guerton Bernadette , Boutin Laetitia , Pierre-Louis Olivier , Capron Claude , Bennaceur-Griscelli Annelise , Uzan Georges , Abecassis Lucile , Bourgeade Marie-Francoise , Lataillade Jean-Jacques , Le Bousse-Kerdiles Marie-Caroline 
Published: October 2019

Abstract
Control of cell cycling is a key process in stem cell fate. A balance between cell quiescence and proliferation within the stem cell niche in interaction with microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. Among growth factors regulating haematopoietic stem/progenitor cell (HSC/HP) cell cycling, TGF-β is known to be a major negative regulator by blocking cell cycle progression during the G1 phase. Besides its effect on HSC/PH and lymphocyte trafficking, the SDF-1/CXCL12 chemokine is another key regulator which helps maintaining hematopoiesis homeostasis. We have previous demonstrated that SDF-1 acts as a survival and cell cycle promoting factor for CD34+ HSC/HP by triggering the G0-G1 transition and then regulating early cell cycle phases suggesting that it may act as an antagonist of TGF-β on cell cycle progression (Lataillade et al., Blood 2000 and 2002). The general aim of this study was to investigate the mechanisms of this antagonist effect on cell cycle. This work was performed on CD34+ cells purified from the peripheral blood of healthy un-mobilized donors. Since they are mainly in G0, they allow dissecting the early cell cycle phases including the G0-G1 transition. We demonstrated that SDF-1 and TGF-β exert an opposite effect on the expression of cell cycle key regulators such as cyclins and CDKI. We showed that cross-talk between SDF-1 and TGF-β signaling pathways involving PI3K/Akt phosphorylation participates in the control of CD34+ cell cycling. We demonstrated a pivotal role of FoxO3 and mTOR in the TGF-β/SDF-1 control of the quiescence/cycling of hematopoietic progenitors. A model integrating a pivotal role for the activation of two Akt substrates, FoxO3 and mTORC1, in the control of CD34+ cells quiescence/cycling by TGF-β and SDF-1 is proposed. Altogether, our results shed new light on the intracellular signaling mechanisms of SDF-1 and of its implication, together with TGF-β, in cell cycle promotion in primary CD34+ cells. Regarding their opposite effect on cell cycle and survival, we suggest that SDF-1 and TGF-β can be considered as natural mutual competitors, both acting as organizers of the stem cell niche where a balance between HSC hibernation and activation is an essential part of the stemness control.
Caspase-3 Activation Is Involved in the Early Megakaryocyte Differentiation, but Not in Platelet Production.
Authors: Kozuma Yukinori , Yuki Satoshi , Suzuki Hidenori , Kojima Hiroshi 
Published: October 2019

Abstract
Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.
Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.
Authors: Broxmeyer Hal E. , Srour Edward F. , Cooper Scott , Wallace Carrie T. , Hangoc Giao , Youn Byung 
Published: October 2019

Abstract
Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.