bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutropenic Mice Can Be Protected from Fungus Infection by Ex Vivo Expanded Allogeneic Myeloid Progenitors.
Authors: Domen Jos , Wahedi Mastura , Danenberg Esther , Smith-Berdan Stephanie , Christensen Julie , Fong Tim , Brown Janice (Wes) 
Published: April 2020

Abstract
Neutropenia induced by chemotherapy, hematopoietic cell transplantation, as well as radiation-induced neutropenia is accompanied by a significant morbidity and mortality due to increased susceptibility to a variety of pathogens. Despite clinical advances, such as the availability of growth factors that can stimulate granulopoiesis, like G-CSF, and new generations of antibiotics and antifungal agents, the problem persists. Direct cellular therapy with mature granulocytes has met with limited success, and would be difficult to implement due to the large number of cells needed, their very short lifespan and the inability to store the cells between harvesting and use. Our long-term goal is to develop a non-HLA-restricted cell-based short-term bridging therapy to reduce susceptibility to infection and enhance recovery from infections in the setting of neutropenia. The recent characterization of surface marker profiles of early myeloid progenitors has allowed the prospective testing of these cells. It has been clearly shown that the use of limited numbers of a combination of Common Myeloid Progenitors (CMP) and Granulocyte-Macrophage-Progenitors (GMP), purified from bone marrow, can protect mice from a normally lethal challenge with fungus or bacteria [Blood100:4660-7(2002)]. We have extended these observations and tested whether myeloid progenitors (MP) with similar properties can be obtained in large numbers from hematopoietic stem cells (HSC) in vitro. Here we report that a one week, serum-free culture can result in a 200-fold expansion of cells with myeloid progenitor activity with respect to the starting CD117+, CD90.1low, Linneg/low and Sca-1+ KTLS-HSC. Furthermore, these myeloid progenitor cells are as effective as Myeloid Progenitors sorted directly from bone marrow in preventing death due to invasive aspergillosis, even when the cells are fully allogeneic and have been cryopreserved prior to use. These results indicate a way in which myeloid progenitor cells, which can be stored frozen prior to use, can be obtained in large numbers. This process, once translated to human cells, would also open the way for the stockpiling of MP for use in nuclear disasters.
Outcomes after Unrelated Cord Blood Transplantation in Children with Acute Lymphoblastic Leukemia. An Eurocord-Netcord Survey.
Authors: Rocha Vanderson , Michel Gerard , Kabbara Nabil , Arcese William , Ortega Juan , Iori Anna Paola , Madero Luis , Chan Ka-Wah , Locatelli Franco , Garnier Federico , Ionescu Irina , Wernet Peter , Gluckman Eliane 
Published: April 2020

Abstract
Unrelated cord blood transplantation (UCBT) is an alternative option to treat children with haematological diseases without an HLA-identical donor. We have analyzed a total of 323 children with ALL receiving an UCBT, from 1994 to 2004 in 99 transplant centres in 24 countries, mostly in Europe. Cumulative incidence with competing risk and KM estimates were used to calculate outcomes. Seventy six children were transplanted in CR1, 136 in CR2 and 111 in more advanced phase of the disease. Among those children poor cytogenetics were observed in 89% of children in 1CR, 33% in 2CR and 42% in advanced phase. Twenty percent of children transplanted in advanced phase had been previously autografted. The median age was 6.5 years at UCBT, median cell dose infused was 4.1x107/kg and the median follow time was 22 months (3–96). The cord blood was HLA identical (6/6) in 12% of the cases, 5/6 in 46%, 4/6 in 39% and 3/6 in 3%. All children received myeloablative conditioning regimen (TBI in 66%) and the majority (67%) received CsA+corticoids as GVHD prophylaxis. Cumulative incidence of neutrophil recovery at day 60, platelets recovery (&gt;20.000) at day 180, acute (grade II–IV) and chronic GVHD were 76±5%, 54±5%, 42±3%, 14±2%, respectively. Overall 2 year-LFS was 36±3%. In a multivariate analysis, only CR1 or CR2 were associated with better LFS (HR=1.8; p&lt;0.0001).
Outcomes CR1 (n=76) CR2 (n=136) Advanced (n=111) TRM at day 100 22+/−5% 25+/−4% 34+/−5% Relapse at 2 years 34+/−8% 37+/−5% 48+/−7% LFS at 2 years 42+/−6% 41+/−4% 24+/−4%
For those patients transplanted with poor cytogenetics, LFS at 2 years was 32±6% and it was 37% for CR1, 43% for CR2 and 0% for advanced phase of the disease. In conclusion, in these large series of high risk ALL patients, these results show that UCBT should be proposed as alternative source of allogeneic transplantation for children lacking an HLA identical donor, in earlier status of the disease.
Functional Correction of Lymphocytes and Dendritic Cells of Patients with WAS or XLT, Using Lentiviral Vectors Targeting the Expression of WAS Protein to Hematopoietic Cells.
Authors: Charrier Sabine , Dupre Loic , Blundell Mike , Scaramuzza Samantha , Jeanson Laurence , Vainchenker William , Danos Olivier , Roncarolo Maria-Grazia , Thrasher Adrian , Galy Anne 
Published: April 2020

Abstract
The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.
Regulation of CXCR1, CXCR2 and CXCR4 in Human Neutrophils: Potential Role in the Release from the Bone Marrow, Clearance of Senescent Cells, and Cell Function at Sites of Inflammation.
Authors: Yildirim Sedat , Bautz Frank , Boehmler Andreas M. , Kanz Lothar , Möhle Robert 
Published: April 2020

Abstract
In the mouse model, it has been shown that the interleukin-8 (IL-8) receptor CXCR2 is involved in the release of mature neutrophils from the bone marrow into the circulation. When neutrophils age, upregulation of CXCR4 and downmodulation of CXCR2 result in homing and subsequent sequestration of senescent cells in the bone marrow. In our study, we observed a similar time-dependent (starting at 3 hrs., maximum at 12–18 hrs.) downregulation of CXCR2 in human neutrophils during aging in ex vivo culture, while expression of the second IL-8 receptor CXCR1, which is mainly responsible for the IL-8-induced chemotaxis, was unchanged. Furthermore, strong upregulation of CXCR4 was noted on the cell surface which could not solely be attributed to re-expression of internalized, intracellular receptors, as an increased amount of CXCR4 mRNA was detected by Northern blot analysis in these cells. The increase in CXCR4 expression was not influenced by inflammatory cytokines such as TNF and IL-1, as well as by IL-8 or G-CSF. Accordingly, SDF-1-induced transendothelial migration of aged neutrophils was 6-fold increased and even exceeded migration in response to IL-8. We conclude that also in human neutrophils, loss of CXCR2 and gain of CXCR4 expression on the cell surface may favor homing and sequestration of senescent cells in the bone marrow. At sites of inflammation however, retained expression of CXCR1 and increased expression of CXCR4 still allow a response of aged, pre-apoptotic neutrophils to the chemotactic mediators IL-8 and SDF-1, as the latter is not only released in the bone marrow, but also at sites of tissue damage and necrosis.
Rb Plays an Essential Role in Erythroid Differentiation through Inhibition of Apoptosis Mediated by NFKB.
Authors: Matsuoka Sahoko , Hirao Atsushi , Arai Fumio , Takubo Keiyo , Miyamoto Kana , Hosokawa Kentaro , Suda Toshio 
Published: April 2020

Abstract
Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day14. However, molecular mechanisms of the function of Rb in erythroid differentiation have been unclear. Recent studies have suggested that Rb has both intrinsic and extrinsic roles on erythroid differentiation. Using Rb-deficient (Rb−/−) embryos(E12), we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis mediated by NFKB. Enucleation of erythroblasts was impaired in semisolid culture of Rb−/− hematopoietic progenitors in fetal liver. The lethally-irradiated recipient mice transplanted with Rb−/− hematopoietic stem cells (HSCs) showed severe anemia with splenomegaly, whereas the number of leukocytes and platelets were normal. In Rb−/− recipient mice, the nucleated erythrocytes and reticulocytes were significantly increased in the peripheral blood. We analyzed cell surface markers for erythroid lineage (TER119 and CD71) in the enlarged spleen. A block of erythroid differentiation at the early erythroblast stage (TER119high CD71high), accompanied with increased apoptosis, was observed in the recipient mice with Rb−/− HSCs. We speculated that the defect in the erythroid differentiation of Rb−/− HSCs might be caused by inappropriate cell death. Thus, we examined expression of apoptosis-related genes in early erythroblasts (CD71high Ter119high) and observed decrease of Bcl-XL expression. To clarify the function of Bcl-XL, we introduced exogenous cDNA of mouse Bcl-XL with GFP (Bcl-XL ires GFP) or GFP alone as control into HSCs and then transplanted them to lethally irradiated mice. From the point of CD71 and Ter119 expression pattern in GFP positive cells, Rb−/− erythoblasts still showed the block in differentiation. In contrast, overexpression of Bcl-XL in Rb−/− erythoblasts inhibited inappropriated apoptosis and restore the differentiation capacity. Further, we found that inactivation of NFKB, but not STAT5 in Rb−/− erythroblasts. Treatment of NFKB inhibitor suppressed erythroid differentiation, accompanied by enucleation, and also inhibited upregulation of Bcl-XL. These data demonstrates that Rb is essential for erythroid differentiation through inhibition of apoptosis mediated by NFKB.
The Role of the Small GTPase Rab27a in the Regulated Secretion of Granulocytes.
Authors: Catz Sergio D. , Munafo Daniela B. , Ellis Beverly A. , Johnson Jennifer L. 
Published: April 2020

Abstract
Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.
Prospective Evaluation of Graft-Versus-Host Disease-Specific Proteomics Pattern in Patients after Allogeneic Stem Cell Transplantation.
Authors: Weissinger Eva M. , Hillmann Meike , Kolb Hans-Jochem , Zander Axel R. , Holler Ernst , Mischak Harald , Ganser Arnold , Hertenstein Bernd 
Published: April 2020

Abstract
We have recently described and published a proteomic pattern specific for the early diagnosis of acute graft versus host disease (aGvHD), based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of proteome based GvHD-patterns to prospectively collected samples from 86 patients (45 AML, 14 ALL, 8 MM, 5 CLL, 5 SAA, 3 MDS, 3CML, 3 NHL). Fifty three patients were transplanted from matched unrelated donors (MUD), 29 received stem cells from matched related donors (MRD), 3 from haplo-identical donors and 1 was transplanted from a syngeneic sibling. GvHD prophylaxis was methotrexat or mycophenolate and cyclosporin A in the majority of the patients. The follow up is currently between 60 and 549 days after allogeneic HSCT. Urine samples were collected on ice prior conditioning, once a week and monthly after day +60. To avoid degradation of the proteins/peptides the samples were frozen as soon as possible. After thawing and removal of confounding substances, like salts, lipids and of all molecules larger than 30 kDa, the samples were loaded onto the CE, separated according to their charge and, after ionization, directly analyzed in an electrospray ionization time of flight (ESI-TOF) -MS. Between 500 and 2500 peptides and proteins were detected in individual samples. All data generated are stored in a Microsoft MS database. Reproducibility of these analyses is greater 98%. The polypeptide patterns specific for the early detection of acute GvHD were applied to the data generated prospectively and the outcome of these analyses was compared to the clinical diagnosis of aGvHD, sepsis and CMV-reactivation. Forty patients were diagnosed with aGvHD in the current evaluation period, 18 had aGvHD ≥ II. From 461 samples, 68 gave a score with the aGvHD pattern, 15 were false positive, whereas 5 scored false negative. The sensitivity of the aGvHD pattern is more than 90%, the specificity is about 96%. Thus the application of the aGvHD pattern for early recognition of acute GvHD is very useful for predicting the development of aGvHD. In addition, we are currently evaluating samples of patients with chronic GvHD (cGvHD). Seven patients in the prospective cohort have developed cGvHD so far. First results show that in the majority of the patients the cGVHD markers are different from those forming the aGvHD pattern. The polypeptide patterns for aGvHD and cGvHD will be discussed together with those of other complications, like CMV-reactivation or sepsis.
Taken together our results demonstrate that the proteome analysis of body fluids collected from patients after HSCT maybe extremely useful for diagnosis of complications.
Suicide Gene Therapy of Graft-Versus-Host Disease Induced by Central Memory Human T Lymphocytes.
Authors: Bondanza Attilio , Valtolina Veronica , Magnani Zulma , Kaneko Shin , Ponzoni Maurilio , Sanvito Francesca , Bonyhadi Mark , Traversari Catia , Toma Salvatore , Radrizzani Marina , La Seta-Catamancio Simona , Fleischhauer Katharina , Ciceri Fabio , Bordignon Claudio , Bonini Chiara 
Published: April 2020

Abstract
Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.
Nucleophosmin Regulates Differentiation, Cell Cycle Progression, and Stress Response in Hematopoietic Progenitor Cells.
Authors: Li June , Sejas Daniel P. , Pang Qishen 
Published: April 2020

Abstract
Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Strong evidence indicates that NPM is involved in hematopoiesis and leukemic development. Here we report that NPM enhances the proliferative potential of hematopoietic stem/progenitor cells and increases cell survival upon stress challenge. Specifically, lin-Sca1+c-kit+ bone marrow cells transduced with retroviral vector expressing NPM exhibited higher proliferative rates in both short-term liquid culture and clonogenic progenitor cell assays, compared to the cells transduced with empty vector. Interestingly, NPM overexpression appears to inhibit differentiation of myeloid progenitors. Hematopoietic stem/progenitor cells infected with the NPM retrovirus expressed significantly lower levels of mature cell markers Gr-1 and Mac-1 compared to empty vector transduced cells, and majority of the NPM-overexpressing cells remained Sca1+C-Kit+ during the 5-day culture. Bone marrow transplantation experiments demonstrated that NPM overexpression increases long-term multi-lineage repopulating capacity of hematopoietic progenitors. We have not observed any evidence of proliferative disorders or leukemia in recipients transplanted with NPM-expressing progenitors thus far (4 months posttransplantation). Through cell-cycle profile analysis and single-cell division experiments, we showed that NPM overexpression induces rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G0/G1 to S transition. Furthermore, immunocytochemical and Western-blot analyses demonstrated that NPM-transduced cells expressed higher level of cyclin A compared to vector-transduced cells. Finally, overexpression of NPM significantly increased the survival of hematopoietic progenitors exposed to mitomycin C or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of progenitor differentiation, cell cycle progression, and stress response.