bluuebunny/crossref_metadata_2025_split huggingface dataset:
Umbilical Cord Blood Derived CD133+ Cells: Homing Capability in Vasculogenesis and Immunogenicity.
Authors: Fanning L. R. , Finney M. R. , Winter D. G. , Kadereit S. , Banks J. , Greco N. , Kozik M. , Pompili V. , Laughlin M. J. 
Published: April 2020

Abstract
Human CD133+ cells constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal angiogenesis. CD133+ homing to stimuli, IL-8 production and immunogenic properties are anticipated important characteristics to consider in potential use of allogeneic UCB CD133+ as a therapeutic in arterial ischemia. CD133+ cells were isolated from UCB mononuclear cells (MNC) by magnetic bead selection (AutoMACS, Miltenyi Biotech) according to manufacturer’s protocol and analyzed by flow cytometry. Yields per cord blood unit averaged 1.05 x 106 cells (+/− 0.7 SEM n=30). Surface phenotyping of UCB CD133+ showed co-expression of VEGFR2 (3.5%), CD105 (22.7%) and CXCR4 (8.7%) ligand for SDF-1. Both HLA-DR (57.6%) and HLA-ABC (66.5%) were expressed on CD133+ cells suggesting that CD133+ cells may be capable of presenting immunostimulatory antigens and eliciting an allogeneic reaction. To test this, we performed 96 hour mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated (30Gy) CD133+ from UCB (ratio 3:1). Proliferation was measured by 3H-thymidine incorporation. CD133+ and MNC from UCB induced proliferation from allogeneic healthy adult MNC in vitro (46939 +/− 2764 and 49548 +/− 2018 cpm respectively, n=2). MLR studies with CSFE-stained responder cells revealed equivalent rates of lymphocyte cell division comparing selected UCB CD133+ and MNC cells used as stimulators. Comparison studies of responding lymphocyte cytokine production including pro-inflammatory protein assays are ongoing. Initial angiogenic protein assays of CD133+ cells demonstrated elevated levels of IL-8 production as compared to MNC (103+/−380 pg/mL greater in CD133+ than MNC from the same UCB unit) when cultured for 24h in basal media. Transwell migration assays of CD133+ cells to SDF-1 (100ng/mL) demonstrated a 1.8 ± 0.7 fold increase in homing compared to a negative control, coinciding with the CXCR4 expression observed on these cells. In summary, UCB derived CD133+ cells demonstrate homing capability as well as potential for cellular recruitment (IL-8 production) for angiogenesis and cellular therapeutics. CD133+ cells selected from UCB maintain immunostimulatory capacity and initiate proliferation of adult MNC. Further studies of UCB derived CD133+ pro-inflammatory potential; cell recruitment and homing to ischemic signals are warranted.
Gene Transfer of Cytidine Deaminase (CDD) Protects from Cytidine Analogs in an In Vivo Murine Transplant Model.
Authors: Rattmann Ina , Sorg Ursula R. , Brueckner Annette , Opalka Bertram , Flasshove Michael , Moritz Thomas 
Published: April 2020

Abstract
In vitro gene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control).
Protection from araC was investigated in three experiments. Gene transfer efficiency was analysed by EGFP expression in the peripheral blood after hematopoietic reconstitution. In these experiments 42±4%, 31±1% and 69±2% EGFP+ granulocytes and 9±1%, 10±1% and 36±3% EGFP+ lymphocytes were detected following SF91-CDD-IRES-EGFP transduction (n=5–6 for each experiment). After araC application (500mg/kg; 1xdaily; d1-4; i.p.) substantial protection of granulocytes and thrombocytes was observed. Following the first treatment cycle nadir counts [x103/μl] were 2.9±0.6 vs. 0.7±0.1, 1.1±0.2 vs. 0.5±0.1 and 2.3±0.3 vs. 0.7±0.2 for granulocytes and 510±148 vs. 80±9, 165±48 vs. 70±42 and 728±133 vs. 85±22 for thrombocytes, respectively (SF91-CDD-IRES-EGFP vs. control; all differences significant at p&lt;0.01 for granulocytes and p&lt;0.05 for thrombocytes). Similar protection was noted during repetitive araC treatment cycles. However, no significant enrichment of CDD transduced cells following araC application was seen and lack of selection was confirmed in two additional experiments utilizing low efficiency gene transfer (&lt;5% and &lt;12% EGFP+ granulocytes or lymphocytes throughout the experiment). No major toxicity of CDD-expression in hematopoietic cells was noted and steady levels of gene marked cells were seen for up to six months in primary and at 5 weeks in secondary recipients. Substantial protection of hematopoiesis by CDD overexpression also was observed for gemcitabine. Mice receiving CDD transduced cells and exposed to 250mg/kg gemcitabine (1xdaily i.p., d1-2), showed granulocyte and thrombocyte nadirs [x103/μl] of 2.4±0.6 vs. 1.4±0.2 and 416±62 vs. 277±97 (SF91-CDD-IRES-EGFP vs. control). When a third day of gemcitabine treatment was added, all mice in the control group died within 4 days while animals receiving CDD-transduced bone marrow survived with nadir counts [x103/μl] of 0.8±0.1 granulocytes and 1130±292 thrombocytes [n=3]. Again, no enrichment of transduced cells was observed.
In summary, our data demonstrate efficient protection from araC and gemcitabine hematotoxicity by CDD overexpression. This protective effect was on progenitor rather than stem cells, as no selection of CDD-expressing cells was demonstrated. Thus, a promising approach to utilize CDD clinically may be the combination with a selectable drug resistance gene such as MGMT or MDR-1 to allow enrichment of transduced cells while at the same time protecting against additional drugs.
Cross Talk between IRAK4 and the NADPH Oxidase.
Authors: Catz Sergio D. , Pacquelet Sandrine , Ellis Beverly A. , Johnson Jennifer L. 
Published: April 2020

Abstract
The ability of human neutrophils to combat bacterial and fungal infections depends on their production of reactive oxygen species. The enzymatic complex NADPH oxidase which is responsible for the generation of superoxide anion, is essential for the microbicidal activity of neutrophils, since patients with chronic granulomatous disease, whose NADPH oxidase is inactive, suffer recurrent infections. The exposure of neutrophils to lipopolysaccharide (LPS) amplifies their oxidative response to formylated-peptides (priming). However, the relationship between the signaling downstream of the toll-like receptor 4 after LPS stimulation and the activation of the oxidase has not been elucidated. The phosphorylation of the NADPH oxidase cytosolic factor p47phox is an essential step during the oxidase activation. Here, we test the hypothesis that Interleukin-1 receptor-associated kinase-4 (IRAK4) regulates the NADPH oxidase through phosphorylation of p47phox. We first show that p47phox is a substrate for IRAK4. We showed that IRAK4-phosphorylated p47phox could be subsequently phosphorylated by PKC suggesting that they phosphorylate p47phox at different residues. IRAK4 phosphorylated p47phox to a similar extent as PKC, however, while p47phox was phosphorylated by PKC only in serines, IRAK4 phosphorylated p47phox also in threonines as determined by two-dimensional electrophoresis. Importantly, IRAK4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system (significantly different from the unphosphorylated control, p&lt;0.05). Furthermore, cophosphorylation of p47phox by IRAK4 and PKC significantly potentiated the activity of the NADPH oxidase when compared to PKC phosphorylation alone (p&lt;0.01). Finally, we identified, by mass spectrometry, the residues in p47phox that are targets for IRAK4 phosphorylation. We found that IRAK4 phosphorylates p47phox at a threonine rich domain which possibly constitutes a novel regulatory domain.
The Direct Association of GATA-1 to Rb Is Necessary for Definitive Erythropoiesis.
Authors: Kadri Zahra , Shimizu Ritsuko , Gisselbrecht Sylvie , Ohneda Osamu , Yamamoto Masayuki , Romeo Paul Henri , Chretien Stany 
Published: April 2020

Abstract
GATA-1, the founding member of GATA family, is a master gene for erythropoiesis. It binds to the consensus sequence WGATAR and is involved in the activation of most of the erythroid specific genes. GATA-1 can also repress the transcription of genes coding for proteins involved in the control of cell proliferation. Here we describe a novel function of GATA-1 i.e. the control of the cell proliferation through a direct association to the retinoblastoma protein (pRb). This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA and, using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb-), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb- did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb- in a GATA-1 deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb- with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb- rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1/pRB interaction is necessary for erythropoiesis enlighting the mechanism of the autonomous defect of erythropoiesis observed in the Rb−/− mice.
Phenotypic and Genotypic Characteristics of Mastocytosis Differ According to the Age of Disease’s Onset.
Authors: Lanternier Fanny , Cohen-Akenine Annick , Palmerini Fabienne , Feger Frederic , Nasca Laurent , Barete Stephane , Guez David , Casassus Philippe , Bodemer Christine , Arock Michel , Dubreuil Patrice , Hermine Olivier , Lortholary Olivier 
Published: April 2020

Abstract
Mastocytosis is a heterogeneous group of disease with respect to clinical, biological and genotypic features, which may either occur during childhood or at the adult’s age. Adult’s mastocytosis is usually associated with persistent systemic involvement and c-kit 816 mutations, while pediatric’s disease is mostly limited to the skin and often resolves spontaneously during adolescence. No study has attended to compare characteristics of adult’s mastocytosis according to the age of disease’s onset. Among 155 adult patients with histologically proven cutaneous mastocytosis and complete phenotypic and genotypic data available recruited through the French mastocytosis network (AFIRMM), disease started before 15 years in 23 patients (15%) and later than 18 years in 132 patients (85%). We compared phenotypic and genotypic features of patients whose disease started during childhood (Group 1, n = 23) with those of 31 randomly allocated patients whose disease started at adult’s age (Group 2). Genotypic analysis was performed on skin biopsy by direct sequencing of c-kit exons 17 and 8 to 13 in which most of activating mutations are found.
According to the WHO classification, the percentage of systemic disease was similar in both groups (61 vs. 79%) in groups 1 and 2, respectively. Bone pain (57% vs. 23%) and flush (83% vs. 39%) differ between groups (p &lt; 0.01). Bullous lesions were only observed in group 1 (14%) while telangiectasia macularis eruptiva perstans was only observed in group 2 (16%). Major asthenia, syncope, mood disturbances, anaphylactoid reactions, gastrointestinal and respiratory disturbances and blood cell count, serum tryptase and liver enzymes levels did not differ between groups. C-kit 816 mutations was found in 33% and 77% of patients in groups 1 and 2, respectively (p &lt; 0.001). 45% of patients whose disease’s onset occurred before the age of 2 years vs. 7% of those whose disease’s onset occurred later during childhood presented with C-kit 816 mutation (p &lt; 0.001). Other c-kit activating mutations were found in 29% and 3% (p &lt; 0.001) and lack of c-kit activating mutation in 38% and 20% (p &lt; 0.001) in groups 1 and 2, respectively. In conclusion, pathogenesis of mastocytosis significantly differs according to the age of disease’s onset. Our data may have major therapeutic relevance when considering c-kit-targeted therapy.
Extracorporeal Photopheresis Induces the Generation of Regulatory T Cells.
Authors: Strobl Frank , Campbell Kim , Krutsick Amy , Huber Janine , Peritt David 
Published: April 2020

Abstract
Extracorporeal photopheresis (ECP) is an immunomodulatory cell therapy being investigated as a treatment for various immune-mediated inflammatory disorders. Clinically, ECP involves the intravenous reinfusion of autologous, apoptotic peripheral blood leukocytes. The exact mechanism of action behind ECP, however, remains unclear. In animal models, delivery of apoptotic cells has been shown to regulate immune responses through the downregulation of antigen-presenting cell (APC) function, the modulation of cytokines, and the generation of regulatory T cells. We and others, have shown that activation of naive T cells in the presence of APCs that have engulfed ECP-treated apoptotic cells leads to the generation of a T cell population that can suppress syngeneic T cell proliferation and function. In the present study, we demonstrate that the direct interaction of ECP-treated peripheral blood mononuclear cells (PBMCs) with naive human CD4+ T cells in vitro also promotes a T cell phenotype with regulatory activity. Transfer of ECP-derived regulatory T cells to a secondary mixed lymphocyte reaction results in a greater than 50% inhibition of syngeneic responder T cell proliferation and IFNg production at a Treg:responder ratio of 1:10. In addition, transwell assays demonstrate that inhibition of responder T cells by ECP-derived regulatory T cells is contact-dependent. The generation of human regulatory T cells by ECP is dependent on the presence of ECP-treated monocytes and can be reversed by the addition of IL-2. These regulatory T cells also proliferate in response to concanavalin-A, but do not express increased levels of Foxp3 or IL-10, nor do they secrete significant levels of the pro-inflammatory cytokines IL-2, IFNg, IL-4, and TNFa. Additional studies are underway to further characterize the phenotype of these regulatory cells and to determine their suppressive capacity in vivo. It is hoped that a better understanding of the mechanism of action behind ECP will help to define more efficacious ECP treatment regimens.
African American Patients with Multiple Myeloma Have Prolonged Responses after Autologous Stem Cell Transplantation.
Authors: Saraf Santosh , Chen Yi-Hsiang , Dobogai Lisa C. , Saunthararajah Yogen , Peace David , Hoffman Ronald , Mahmud Nadim , Chunduri Sandeep , Rondelli Damiano 
Published: April 2020

Abstract
The incidence of multiple myeloma (MM) in African American (AA) patients is higher than in other racial groups. In this retrospective study we analyzed the outcome of 70 consecutive patients (AA n=38, non-AA n=32) with MM receiving an autologous stem cell transplantat (ASCT) at the University of Illinois at Chicago. No significant differences, including age, gender, stage of disease, Hgb and b2 microglobulin levels, skeletal involvement, plasma cell marrow infiltration, were observed between AA and non-AA patients at diagnosis. Chemotherapy prior to ASCT was also comparable in the two groups. Conditioning regimen with high dose melphalan was used in 84% AA and 72% non-AA, while the remaining patients received BCNU/melphalan. After induction chemotherapy no difference occurred in complete remission (CR) and partial remission (PR) rates in AA (9% and 42%) as compared to non-AA (13% and 33%). Two months after transplantation CR and PR rates were 31% and 25% in AA and 30% and 20% in non-AA. However, at 6 months after ASCT 25%AA vs 60% non-AA patients showed a progression of the disease (p=0.009). At a median follow-up of 26 months (range:1 to 89), 79% AA and 69% non-AA patients are alive. The estimated median Event-Free-Survival in the two groups is 21 vs 12 months (p=0.02), while the estimated median Overall Survival is 64 vs 39 months (p=0.19) (Figure 1). In conclusion, AA and non-AA patients have similar CR and PR rates after ASCT. However, AA patients seem to have more prolonged clinical responses. In a setting where equal access to modern modalities of treatment is available, race does not appear to affect responses to therapy in multiple myeloma.
Figure 1 Figure 1.
Expression of C/EBPε Restores G-CSF-Induced Granulocytic Differentiation of Myeloid Cells Expressing the Carboxy Truncated G-CSF Receptor Associated with AML/SCN.
Authors: Dong Fan , Zhuang Dazhong , Qiu Yaling 
Published: April 2020

Abstract
Mutations in the G-CSF receptor gene causing carboxy terminal truncation of the G-CSF receptor have been associated with the development of acute myelogenous leukemia (AML) in patients with severe congenital neutropenia (SCN). The truncated G-CSF receptors mediate augmented cell proliferation and survival, but are defective in inducing granulocytic differentiation in myeloid cell lines and transgenic mice. Little is known about the mechanism by which the carboxy terminus of the G-CSF receptor regulates granulocytic differentiation. It has been shown that myeloid transcription factors including C/EBPα, C/EBPε and PU.1 are critically involved in granulopoiesis. Of the three transcription factors, expression of C/EBPε was dramatically upregulated upon G-CSF treatment of myeloid 32D cells expressing the wild type G-CSF receptor (32D/WT). C/EBPε upregulation by G-CSF was abolished in 32D cells expressing the G-CSF receptor mutant D715 (32D/D715), which lacked a region of 98 amino acids in the carboxy terminus. Forced expression of C/EBPε in 32D/D715 cells at levels comparable to those in G-CSF-treated 32D/WT cells resulted in markedly attenuated cell proliferation and survival, and rapid terminal granulocytic differentiation in response to G-CSF. These data indicated that the functional defect of the D715 mutant in mediating granulocytic differentiation may arise from its inability to upregulate C/EBPε expression. Notably, forced expression of C/EBPε failed to induce terminal granulocytic differentiation of 32D/D715 cells cultured in IL-3, suggesting that the D715 receptor acted in collaboration with C/EBPε to drive granulocytic differentiation. Of the four cytoplasmic tyrosine (Tyr) residues of the G-CSF receptor, the D715 mutant contains only Tyr 704 that is involved in Stat3 activation. Because Stat3 is an important regulator of granulopoiesis, experiments are under way to address whether Tyr 704 in the D715 mutant is required for granulocytic differentiation driven by C/EBPε.
Distinctive Roles of Stem Cell Factor and IL-7 in T Cell Development.
Authors: Wang Hongfang , Pierce L. Jeanne , Spangrude Gerald J. 
Published: April 2020

Abstract
Notch signaling plays a critical role in T lineage commitment during lymphoid differentiation. However, Notch signaling alone is not sufficient to support T cell development through the CD4/CD8 double positive (DP) stage in vitro. We here report distinct effects of several cytokines on T cell differentiation in the OP9-DL1 cell culture model. Our studies show that Flt3 ligand enhances the proliferation of progenitors but has no obvious effect on differentiation. In contrast, stem cell factor (SCF) favors the proliferation of CD4/CD8 double negative (DN) lymphoid progenitors and inhibits differentiation to the DP stage in a dose-dependent manner. Differentiation of the NK lineage is promoted under these conditions. Conversely, blocking the function of SCF that is expressed endogenously by OP9-DL1 cells inhibits proliferation of lymphoid progenitors and accelerates T lineage differentiation. IL-7 is necessary for differentiation from the DP to the CD8 single positive (SP) stage, and is also required for γδ T lineage development. We also find a dosage effect of IL-7 during T cell development. OP9 and OP9-DL1 stromal cells produce endogenous levels of IL-7 that are sufficient to support B and DP T cell differentiation. However, the amount of endogenous IL-7 is not sufficient to support T cell differentiation from the DP to the SP stage. Addition of exogenous IL-7 (1–10 ng/ml) to the cultures promotes SP differentiation, while blocking endogenous IL-7 with anti-IL-7 antibody inhibits both B and T cell development. We conclude that activation through the Notch pathway is sufficient to suppress B lineage differentiation and thereby promote T lineage commitment, but is not sufficient to promote the subsequent stages of T cell development. SCF promotes expansion and directs NK lineage differentiation at the expense of T cell development, while IL-7 provides both proliferation as well as T lineage differentiation signals. T cell development from the DN to the DP stage requires a low amount of IL-7, while differentiation from the DP to the SP stage requires a higher level of IL-7. The balance between the effects mediated by these cytokines, along with Notch signaling, plays a critical role in regulating development of the T and NK lineages.
Induction of Transient Cytopenia To Analyze Hematopoiesis in Mutant Mice.
Authors: Stanford William L. , Anderson Nicole M. , Minden Mark D. , Barber Dwayne L. 
Published: April 2020

Abstract
The mouse plays an indispensable role in developing our current understanding of mammalian hematopoiesis. Most hematopoietic phenotyping assays in the mouse are non-viable techniques designed to evaluate homeostatic populations, enumerate progenitor populations, and perform functional analysis. The worldwide effort to generate mouse models of human disease and functionally annotate the mammalian genome using mouse mutagenesis (including dominant ENU screens) requires the development of robust standardized viable phenotyping tools. We have developed a phenotyping assay that induces transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea), the responses to which are monitored by tracing changes in peripheral blood levels of red blood cells, white blood cells and platelets. We have performed detailed analysis of lineage recovery kinetics, developing lineage recovery curves for various strains of mice for both males and females, which allowed us to identify appropriate testing days to identify phenodeviants. We have compared the recovery data with conventional progenitor assays and analyzed a cohort of well-studied naturally occurring and targeted hematopoietic mutants using the transient anemia assays that has yielded novel phenotypes of hemizygous mutant animals. For example, erythropoietin receptor null embryos die of severe anemia at mid-gestation; however, no defect in erythropoiesis has been reported in EpoR +/− mice. We have found that 5-fluorouracil and phenylhydrazine elicit delayed RBC recovery in EpoR +/− mice, demonstrating a critical dose-dependent role for the erythropoietin receptor in stress erythropoiesis. In addition, Stat5 has been shown to play an important role in erythropoiesis and in the regulation of steady state hematopoiesis. We have found that Stat5a/b+/− mice treated with 5-fluorouracil show altered recovery kinetics in RBC, WBC and platelets. Finally, we have adapted the transient cytopenia assay to develop a sensitized dominant ENU screen, enabling us to identify hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. Thus, these standardized cytopenia response assays function as surrogate viable assays to analyze progenitor populations.