bluuebunny/crossref_metadata_2025_split huggingface dataset:
Foamy Viral Vectors Efficiently Transduce Quiescent Hematopoietic Stem/Progenitor Cells (HSC) and Restore the Long Term Repopulating Activity of Fancc −/− Stem Cells.
Authors: Clapp D. Wade 
Published: April 2020

Abstract
Fanconi anemia (FA) is characterized by bone marrow aplasia and myeloid leukemia. The identification of FA genes raises the potential of using gene transfer technology to introduce cDNAs into autologous HSCs. Current strategies using Moloney retroviruses require a 2–4 day ex vivo culture of HSC to facilitate stable integration of the transgene. However, ex vivo culture results in a time-dependent increase in apoptosis of Fancc−/− primitive HSC and mice reconstituted with the surviving cells have an increased risk of acquiring myeloid malignancies. Therefore we examined the potential of a recombinant foamy virus construct (MD9-FANCC-EGFP) to transduce murine Fancc −/− HSC in the absence of prestimulation. Forty-80% of progenitors that were in G0 – G1 at the time of transduction were transduced following a single 10–14 hr transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice or recipients treated with IFN-g only. Four-six months following transplantation, recipient BM cells were isolated and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. Fancc−/− progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wildtype (WT) controls while Fancc−/− progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. The repopulating activity of MD9-FANCC-EGFP-transduced Fancc−/− stem cells was comparable to WT controls 18–24 months following transplantation in primary and secondary recipients. Additionally, while mice reconstituted with Fancc−/− cells transduced with the reporter construct had reduced repopulating ability as compared to the other groups, none of these recipients acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral based vector allows efficient transduction of Fancc−/− HSC, and diminishes the selection pressure that occurs during ex vivo culture of Fancc−/− HSCs.
Secondary or Concomitant Neoplasms among Adults Diagnosed with Acute Lymphoblastic Leukemia (ALL) and Treated According to the LALA-87 and LALA-94 Trials.
Authors: Tavernier Emmanuelle , de Botton Stephane , Dhedin Nathalie , Bulabois Claude-Eric , Reman Oumedaly , Vey Norbert , Garban Frederic , Boiron Jean-Michel , Gardin Claude , Fegueux Nathalie , Witz Francis , Lioure Bruno , Gratecos Nicole , Isnard Francoise , Delannoy Andre , Vernant Jean-Paul , Dombret Herve , Lheritier Veronique , Fiere Denis , Thomas Xavier 
Published: April 2020

Abstract
Second malignant neoplasms are a serious complication after successful treatment of childhood ALL. Although treatment intensity and outcome were not comparable, with improvements in survival, it is important to evaluate the rate and the type of second neoplasms in adults with ALL. We analyzed the data from the GET-LALA group. A cohort of 1493 patients, aged 15 to 60 years and enrolled on two successive multicenter protocols between 1987 and 2002, was observed to determine the incidence of second neoplasms and associated risk factors. The median follow-up time from diagnosis was 6 years. By February 2005, secondary or concomitant neoplasms were documented in 23 patients (median age: 36 years, range:18–57) including 9 acute myeloid leukemias, 4 non Hodgkin lymphomas, 5 skin tumors, and 5 other solid tumors (1 lung cancer, 1 tongue carcinoma, 1 thymoma, 1 condrosarcoma, 1 histiocytosis). Neoplasms developed 0.5 to 13.8 years (median, 4.5 years) after the diagnosis of ALL. 22 patients were in first remission, one was in second remission. The overall cumulative risk of secondary neoplasms was 2.1% at 5 years, 4.9% at 10 years, 9.4% at 15 years. The cumulative risk of developing a second hematologic malignancy was 1.8% at 5 years, 2.2% at 10 years, 3.3% at 18 years; that of developing a solid tumor was 0.2% at 5 years, 2.8% at 10 years, 6.2% at 15 years. The development of secondary neoplasm was not associated with the use of any specific cytotoxic agent. However, risk of skin tumor increased with radiation dose and transplantation (p = 0.01). Overall survival after the diagnosis of a second malignant neoplasm was 55% at 10 years. However, the median overall survival in patients developing acute myeloid leukemia was of 5.7 months. Considering the low-survival rate of this large unselected adult ALL cohort (32% at 10 years), considering the poorer results comparing to childhood ALL treatment, the risk of secondary or concomitant neoplasm remains probably under estimated. Larger series with long-term follow-up are, however, mandatory.
Figure Figure
SNX5, a Novel Gene Linked to Fanconi Anemia Causes Hematopoietic Failure When Knocked down in Zebrafish.
Authors: Flynn Catherine M. , Mendenhall Eric M. , Eckfeldt Craig E. , Wagner John E. , Verfaillie Catherine M. 
Published: April 2020

Abstract
Sorting nexin 5 (SNX 5) is a member of the sorting nexin family, a diverse group of cellular trafficking proteins unified by the presence of a phospholipid binding domain (PHOX) which is involved in protein-protein interactions. While the function of SNX5 is unknown, there is evidence from yeast two hybrid studies that SNX5 binds to FANCA, and SNX5 is one of the 20 genes located on a region on mouse chromosome 2 associated with accelerated stem cell aging. We have recently shown that SNX5 is significantly higher expressed in CD34+CD33-CD38-Rho+ (Rhohi) cells from umbilical cord blood (UCB) and bone marrow (BM), depleted of SCID repopulating cells (SRCs) than in CD34+CD33-CD38-cKIT+ Rho- cells (Rholo), containing all SRCs. A large scale high throughput functional analysis was carried out in zebrafish and genes differentially expressed between Rholo and Rhohi cells were knocked down using morpholino (MO) antisense oligonucleotides. Morpholinos are a useful tool which can specifically inhibit the translation of target mRNA. 16 out of 70 genes knocked down, including snx5, demonstrated decreased circulating blood in the zebrafish injected. Further analysis has shown that MO knock down of snx5 in zebrafish (n=152) results in hematopoietic failure with normal vasculature, with normal expression of scl and gata-1 by in situ hybridization, indicating a defect at or beyond the hematopoietic stem cell stage. Quantitative RT-PCR was used to further confirm the phenotype. Levels of hemoglobin, l-plastin and myeloperoxidase mRNA in snx5 morphants compared to uninjected controls, were significantly lower, consistent with a multi-lineage hematopoietic differentiation defect. The decrease in circulating blood could be partially rescued by overexpressing human SNX5 cDNA, confirming specificity of the morphant phenotype. To determine whether SNX5 might be linked to Fanconi anemia (FA), we measured levels of SNX5 mRNA and protein in EBV transformed cell lines from patients with Fanconi-complementation group A (FANCA), -complementation group C (FANCC) or unknown complementation group (FANC-NX). We have found no significant difference between SNX5 mRNA expression in 11 cell lines compared to Jurkat controls. However, levels of SNX5 protein appeared lower in FANCA cells suggesting a possible role of SNX5 in FANCA, which is being evaluated further. Ongoing studies evaluating the role of SNX5 in normal human hematopoiesis will also be presented.
Update of the Modified Hyper-CVAD Regimen with or without Rituximab in Newly Diagnosed Adult Acute Lymphocytic Leukemia (ALL).
Authors: Thomas Deborah A. , Cortes Jorge , O’Brien Susan , Faderl Stefan , Ravandi Farhad , Garcia-Manero Gullermo , Giles Francis , Beran Miloslav , Koller Charles , Wierda William , Verstovsek Srdan , Ferrajoli Alessandra , Kornblau Steven , Keating Michael , Andreeff Michael , Kantarjian Hagop 
Published: April 2020

Abstract
Hyper-CVAD is effective therapy for adult ALL [Kantarjian et al, JCO18:547, 2000; Kantarjian et al, Cancer101:2788, 2004]. Intensive chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine [VCR], doxorubicin and dexamethasone) was alternated with high dose methotrexate and cytarabine every 21 days for 8 courses with G-CSF and prophylactic antibiotics, followed by maintenance with POMP (6-MP, methotrexate, VCR, prednisone). Complete response (CR) rate was 92% with 3-year disease-free survival (DFS) rates 38% overall. A modified hyper-CVAD regimen was developed to address the following: (1) higher induction mortality in patients (pts) aged 60 or older (17% versus 3%); (2) longer DFS reported with early anthracycline intensification; (3) worse survival with CD20 expression (excluding Burkitt’s [BL] and lymphoblastic lymphoma [LL] subtypes); (4) CNS relapse rate of 6% and 1% in low and high risk pts, respectively and (5) late relapses after completion of therapy.
Modifications to Hyper-CVAD Parameter Hyper-CVAD Modified Hyper-CVAD Laminar air flow rooms No For age ≥ 60 yrs or poor PS Dose-intensive anthracycline No C2 Liposomal DNR & cytarabine Rituximab No For CD20 ≥ 20 Intrathecal treatments 4–16 6–8 Maintenance (POMP) 2 years 3 years Intensifications (MTX, asparaginase) Months 7 & 11 Months 6,7 & 18,19 with hyper-CVAD
Newly diagnosed or primary refractory (1 course only) pts were eligible. BL and Ph+ ALL pts were excluded. From May 2000 to December 2001, 77 pts were treated with the modified regimen detailed above (9 courses of intensive chemotherapy). The program was then modified further with elimination of course 2 anthracycline intensification. An additional 80 pts were treated with hyper-CVAD with or without rituximab (8 courses of intensive chemotherapy). The median age for these 157 pts was 40 yrs (range, 15–83) with 20% aged ≥ 60 yrs; 57% were males. Overall response rate was 94% in the evaluable pts (8 too early) with no difference by CD20 expression (49% were CD20+). No induction deaths were observed in the elderly subgroup (2 younger pts with induction deaths). Outcome with anthracycline intensification appeared worse, particularly in the CD20 negative group. The addition of rituximab appeared to improve DFS in CD20 positive group (with or without anthracycline intensification) compared with hyper-CVAD alone (2 yr DFS 90% versus 65%, p=.03); however, overall survival in the CD20 positive group was influenced by deaths in CR (10%) in elderly patients related to GNR sepsis or pneumonia during the intensive phase. Additional accrual and follow-up is needed to further define the role of rituximab in non-Burkitt’s adult ALL.
The Role of CD4+ T Cells in a Murine Model of Acute Lymphoblastic Leukaemia.
Authors: Hegazy Ahmed N. , Wolenski Mathias , Welte Karl , Klein Christoph 
Published: April 2020

Abstract
To assess CD4-mediated anti-tumor immunity in a murine acute lymphoblastic leukaemia model system, we have generated a series of BCR-ABL positive pre-B cell lines expressing the surrogate tumor antigen ovalbumine. Upon intravenous injection, PKH-26-labeled leukaemia cells were taken up by splenic CD8+ but not by CD8− dendritic cells (DC). In comparison to PBS-injected DCs, CD8+ DCs also showed increased expression of CD40, CD80, and CD86. Purified DCs from leukemic mice stimulated transgenic DO11.10 T cells recognizing OVA323–339 in the context of I-Ad, suggesting efficient presentation of the surrogate tumor antigen. Next, we utilized adoptive transfer of DO11.10 T cells to measure tumor-specific T cell responses in vivo. OVA-expressing BM185 cells were unable to directly stimulate DO11.10 T cells, as shown by 3H-thymidine incorporation. In contrast, DO11.10 T cells were activated in vivo in spleen and lymph nodes, as shown by upregulation of CD44 and CFSE staining, suggesting that DC effectively present tumor antigens to DO11.10 T cells in vivo. However, despite of detectable T cell activation and proliferative T cell responses, all animals succumbed to progressive leukemia. Furthermore, adoptive transfer of naïve DO11.10 cells did not induce protective anti-leukemia immunity. Interestingly, in vivo primed DO11.10 T cells did not express interferon-γ. We therefore hypothesized that inefficient in vivo priming of TH1 cells may contribute to immune evasion of ALL cells. To address this question, we primed DO11.10 T cells in vitro prior to adoptive transfer. In this setting, DO11.10 T cells expressed interferon-γ and induced regression of preestablished leukaemia. This effect was dependent on CD8 cells, as shown by in vivo depletion experiments. Our experimental system supports the concept of CD4-dependent antitumor immunity and provides a platform to assess immunological mechanisms of novel strategies to therapeutically enhance antileukaemic immune responses.
Two Distinct Precursors of Hematopoietic Stem Cells and Endothelial Progenitors Characterized by the PCLP1 Expression.
Authors: Onitsuka Izumi , Takeuchi Masaki , Okabe Tomoya , Kamiya Yoshiko , Hirata Ayami , Yahara Ichiro , Miyajima Atsushi 
Published: April 2020

Abstract
Blood cells and endothelia are believed to arise from their common progenitor hemangioblast. However, it still remains unknown how these lineages develop. Here we report the existence of two distinct precursors for hematopoietic stem cells (HSCs) and endothelial progenitors in murine fetal liver (FL). Podocalyxin-like protein 1 (PCLP1) is a member of the sialomucin family and was shown to be expressed in hemangioblasts in the aorta-gonad-mesonephros region in murine embryo. To further analyze the fates of hematopoietic/endothelial cells, we focused on embryonic day 14.5 (E14.5) FL, since it is a major hematopoietic organ during embryonic period. Based on the PCLP1 expression levels, E14.5 FL cells could be fractionated into four distinct populations. In vitro colony-forming assay and in vivo transplantation analysis revealed that lineage-committed progenitors with colony-forming activities and long-term repopulating hematopoietic stem cells (LTR-HSCs) were in PCLP1neg cells. PCLP1dull cells contained erythroid lineage-committed cells. Interestingly, while PCLP1med cells lacked colony-forming activities, they showed LTR-HSC activity in vivo. To further characterize these cell populations, we cultured them with OP9 stromal cells, since OP9 cells have been used to induce hematopoietic and endothelial lineages from embryonic stem cells. In co-culture with OP9 cells, PCLP1neg cells immediately generated blood cells with colony-forming activity but lacking in vivo hematopoietic activity, indicating that OP9 cells failed to support hematopoietic progenitor/HSCs. However, PCLP1med generated colony-forming hematopoietic progenitors with LTR-HSC activities in the presence of OP9 cells. These results indicated that PCLP1med cells contained stromal cell-dependent immature precursors for HSCs. PCLP1high cells did not express the hematopoietic markers or endothelial cell markers such as PECAM1 and VE-cadherin. However, they formed endothelia-like cell colonies which were highly proliferative and serially transferable in OP9 co-culture. Interestingly, the addition of vascular endothelial growth factor (VEGF) to the culture strongly induced the expression of PECAM1 and VE-cadherin in these colonies. PCLP1high cells contributed to PECAM1+ endothelium in several organs in vivo when transplanted to conditioned neonatal liver. Therefore, PCLP1high cells contained immature precursors for endothelial progenitors. These results indicate that PCLP1 expression levels distinguish previously unrecognized early precursors for HSCs and endothelial progenitors, which are distinct from hemangioblasts.
Regulation of Hematopoiesis In Vitro and In Vivo by Invariant NKT Cells.
Authors: Kotsianidis Ioannis , Silk Jonathan D. , Patterson Scott , Almeida Antonio , Tsatalas Costas , Bourikas George , Cerundolo Vincenzo , Roberts Irene A.G. , Karadimitris Anastasios 
Published: April 2020

Abstract
Invariant NKT cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. Upon activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete Th1/2 cytokines but also cytokines with hematopoietic potential such as IL-3 and GM-CSF. In mice, NKT cells activated by alpha-galactosylceramide (alphaGC), a potent glycolipid ligand, cause an in increase in extramedullary hematopoietic committed progenitor activity through secretion of these cytokines.
We tested the role of iNKT cells in regulating hematopoiesis under conditions of activation and in steady state hematopoiesis. We found that GM-CSF-secreting alphaGC -activated iNKT cells enhanced (by 64%, n=5, p&lt;0.05) the myeloid clonogenic potential of human cord blood hematopoietic progenitors; conversely, in the absence of NKT cells short- and long-term progenitor activity is decreased: 48% reduction in GFU-GM frequency, n=6; p&lt;0.05 and a reduction of 18–60% in LTC-IC frequency in 4 independent experiments. These findings suggest that NKT cells are implicated in the regulation of hematopoiesis both in the presence and absence of immune activation. In accordance with these findings, iNKT cell-deficient mice compared to wild type animals display impaired hematopoiesis characterized by peripheral blood cytopenia (43% and 32% reduction in leucocyte and platelet counts respectively, n=10, p&lt;0.001), reduced marrow cellularity (reduced by 35%, n=13, p&lt;0.001), lower frequency and absolute numbers per hind limb of hematopoietic stem cells (HSC) as assessed by the cKit+Lin-Sca-1+ phenotype (reduced by 40% n=8, p&lt;0.05, and 61% n=8, p&lt;0.001, respectively), and reduced early (50% and 68% reduction in frequency and absolute numbers of LTC-IC respectively, n=4, p&lt;0.05) and late (40% and 57% reduction in frequency and total number of CFU-GM per hind limb respectively, n=8, p&lt;0.05) hematopoietic progenitors. We also show that CD1d is expressed on human HSC and CD1d-expressing HSC display short- and long-term clonogenic potential and can present the glycolipid a-galactosylceramide to iNKT cells suggesting that the iNKT cell-mediated regulation of hematopoiesis might operate through cell contact of NKT cells with HSC and interaction of the invariant TCR with CD1d on HSC.
In conclusion, iNKT cells when activated enhance the clonogenic capacity of myeloid progenitors and can thus modulate innate immune responses. Additionally, they are required for maintenance of normal hematopoiesis in the absence of immune activation. Our findings offer further evidence for a concerted regulation of the immune and hematopoietic systems and the potential for new therapeutic approaches for the manipulation of hematopoiesis.
FLT3 Mutant Activation Is Still Dependent on FLT3 Ligand.
Authors: Zheng Rui , Piloto Obdulio , Small Donald 
Published: April 2020

Abstract
FLT3 is a member of the class III receptor tyrosine kinase family and is primarily expressed on hematopoietic stem/progenitor cells. FLT3 ligand (FL) interacts with wild-type FLT3 (wtFLT3) receptors leading to the activation of FLT3 signaling. Constitutive activation of wt FLT3 through autocrine activation by FL is also observed in a number of cell lines and primary AML samples. Incubation of leukemic blasts expressing FLT3 with FL results in enhanced proliferation and decreased spontaneous apoptosis in many cases of acute leukemia. Somatic mutations of FLT3 involving internal tandem duplication (ITD) of the juxtamembrane domain or point mutations in the tyrosine kinase domain (TKD) have been identified in approximately 17–34% and 7–9% of acute myeloid leukemia (AML) patients, respectively. The ITD and TKD mutations appear to activate the tyrosine kinase domain of FLT3 in a FL-independent manner. However, these data were all obtained in human and murine cell lines that express FL. Thus, it is not clear whether FL also plays a role in the activation of FLT3 mutants. In order to determine whether or not FLT3 mutants are completely or somewhat dependent on FL, a FL deficient murine embryo fibroblast cell line (MEF) was derived from FL deficient (FL−/−) mice by SV40 large T antigen transformation. This eliminates the possibility of autocrine stimulation of transfected FLT3 receptor by FL. Expression of FLT3/ITD and FLT3/TKD mutations in FL−/−MEF cells resulted in some constitutive phosphorylation of FLT3 receptor. However, a more than 4 fold increase of FLT3 activation was induced by addition of FL. Retroviral introduction of FL in FL−/− MEF cells expressing FLT3 mutants led to more than 3 fold and 2 fold increase of tyrosine phosphorylation of FLT3 and AKT, respectively. In addition, FL expression resulted in a 3 fold increase of phosphorylation of STAT5 in FL−/−MEF cells expressing FLT3/ITD. In these FL expressing cells, addition of exogenous FL showed no further stimulation of FLT3, AKT and STAT5. These data strongly suggest that the level of FLT3 activation mediated by FLT3 mutants is still largely dependent on FL.
Over-Expression of Stromal Cell-Derived Factor-1 in Non-Specialized Endothelium Enhances the Endothelium-Supported Transmigration, Maintenance, and Proliferation of Hematopoietic Progenitor Cells.
Authors: Hwang Jin-Hee , Kim Seong-Woo , Yun Hwan-Jung , Lee Younghee , Kim Samyong , Jo Deog-Yeon 
Published: April 2020

Abstract
Stromal cell-derived factor-1 (SDF-1) plays a central role in the homing of hematopoietic stem cells and progenitor cells (HPCs) to bone marrow (BM). Tissue damage, most notably ischemia, induces the recruitment of bone marrow (BM)-derived stem/progenitor cells, which can lead to tissue repair. Moreover, damaged tissues and endothelium express and produce SDF-1. However, the effects of aberrant expression and production of SDF-1 in human endothelium on the biology of BM-derived stem/progenitor cells are not fully understood. To clarify this, we over-expressed the SDF-1 gene in human umbilical vein endothelial cells using an adenoviral vector (HUVEC/AdeSDF-1) and examined the transmigration, maintenance, and proliferation of HPCs supported by the endothelium. HUVEC/AdeSDF-1 monolayers induced the migration of MO7e cells and mobilized peripheral blood (mPB) CD34+ cells underneath the endothelium within a few hours, while HUVEC monolayers transfected with adenoviral vectors expressing LacZ (HUVEC/AdeLacZ) did not. A transmigration assay revealed that HUVEC/AdeSDF-1 monolayers supported the spontaneous transmigration of CD34+ cells to a greater degree than did HUVEC/LacZ monolayers (15 ± 4% vs. 5 ± 3%) and that transmigration was blocked with pretreatment of the input cells with pertussis toxin (PTX). The co-culturing of mPB CD34+ cells with HUVEC/AdeSDF-1 for up to 2 weeks led to a greater expansion of CD45+ cells and colony-forming cells, and reduced cell apoptosis. Furthermore, the co-culturing of mPB CD34+ cells with HUVEC/AdeSDF-1 led to the formation of numerous cobblestone areas, while mPB CD34+ cells plus HUVEC/AdeLacZ supported the formation of only a few cobblestone areas (253 ± 28 vs. 10 ± 3 per 2 x 104 mPB CD34+ cells at week-2), which were further enhanced by adding a combination of early-acting hematopoietic growth factors at low concentrations. To further elucidate whether SDF-1 is directly involved in the formation of cobblestone areas, mPB CD34+ cells were co-cultured with murine BM MS-5 stromal cells. Weekly PTX treatment, beginning 24 hours after start of co-culturing, markedly decreased the numbers of cobblestone areas by weeks 2 and 5, which indicates that SDF-1 is involved not only in the localization of primitive HPCs underneath the endothelium or BM stroma but also in the maintenance and proliferation of the cells. These results indicate that aberrant SDF-1 over-expression in any non-specialized endothelium induces BM-derived progenitor cell trafficking into extravasculr sites and promotes the maintenance and proliferation of the migrated cells.