bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells
Authors: Piacibello Wanda , Sanavio Fiorella , Severino Antonella , Danè Alessandra , Gammaitoni Loretta , Fagioli Franca , Perissinotto Eliana , Cavalloni Giuliana , Kollet Orit , Lapidot Tsvee , Aglietta Massimo 
Published: October 2019

AbstractUnderstanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.
Endothelial Cells Undergoing Apoptosis Become Proadhesive for Nonactivated Platelets
Authors: Bombeli Thomas , Schwartz Barbara R. , Harlan John M. 
Published: October 2019

AbstractUnder normal conditions, platelets do not adhere to endothelium. However, when platelets or endothelial cells are stimulated by thrombin or cytokines, respectively, platelets bind avidly to endothelium. Because there is accumulating evidence that endothelial cells may become apoptotic under certain proinflammatory or prothrombotic conditions, we investigated whether endothelial cells undergoing apoptosis may become proadhesive for nonactivated platelets. Human umbilical vein endothelial cells (HUVEC) were induced to undergo apoptosis by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum-deprivation. After treatment of HUVEC or platelets with different receptor antagonists, nonactivated, washed human platelets were allowed to adhere to HUVEC for 20 minutes. To exclude matrix involvement, platelet binding was measured in suspension by using flow cytometry. Independent of the method of apoptosis induction, there was a marked increase in platelet binding to apoptotic HUVEC. Although HUVEC exhibited maximal adhesiveness for platelets after 2 to 4 hours, complete DNA fragmentation of HUVEC occurred only several hours later. Adhesion assays after blockade of different platelet receptors showed only involvement of β1-integrins. Platelet binding to apoptotic HUVEC was inhibited by more than 70% when platelets were treated with blocking anti-β1 antibodies. Treatment of apoptotic HUVEC with blocking antibodies to different potential platelet receptors, including known ligands for β1-integrins, did not affect platelet binding. As assessed by determination of β-thromboglobulin and platelet factor 4 in the supernatants, platelets bound to apoptotic HUVEC became slightly activated. However, significant expression of platelet P-selectin (CD62P) was not found. These data provide further evidence that endothelial cells undergoing apoptosis may contribute to thrombotic events.
Characterization of Cell-Associated Plasminogen Activation Catalyzed by Urokinase-Type Plasminogen Activator, but Independent of Urokinase Receptor (uPAR, CD87)
Authors: Longstaff Colin , Merton R. Elizabeth , Fabregas Pere , Felez Jordi 
Published: October 2019

AbstractThe 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 106to 107 cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.
Interleukin-2–Activated Rat Natural Killer Cells Express Inducible Nitric Oxide Synthase That Contributes to Cytotoxic Function and Interferon-γ Production
Authors: Cifone M. Grazia , D’Alò Simona , Parroni Raffaella , Millimaggi Danilo , Biordi Leda , Martinotti Stefano , Santoni Angela 
Published: October 2019

AbstractNatural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2–activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2–activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2–activated NK cells. Moreover, IL-2–induced interferon-γ production appears also to be dependent, at least in part, on iNOS induction.
Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells
Authors: Ozaki Shuji , Kosaka Masaaki , Wakahara Yuji , Ozaki Yasuko , Tsuchiya Masayuki , Koishihara Yasuo , Goto Tetsuya , Matsumoto Toshio 
Published: October 2019

AbstractTo develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1κ), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16+ cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16+ cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.
Somatic Mutations Within the Untranslated Regions of Rearranged Ig Genes in a Case of Classical Hodgkin’s Disease as a Potential Cause for the Absence of Ig in the Lymphoma Cells
Authors: Jox Andrea , Zander Thomas , Küppers Ralf , Irsch Johannes , Kanzler Holger , Kornacker Martin , Bohlen Heribert , Diehl Volker , Wolf Jürgen 
Published: October 2019

AbstractHodgkin–Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin’s disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vκ3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vκ3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vκ3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3′ splice site of the leader intron and adjacent nucleotides in the rearranged Vκ light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.
Red Blood Cells Inhibit Apoptosis of Human Neutrophils
Authors: Aoshiba Kazutetsu , Nakajima Yuri , Yasui Shuji , Tamaoki Jun , Nagai Atsushi 
Published: October 2019

AbstractOxidative stress has been implicated in the triggering of apoptosis in neutrophils. Because red blood cells (RBCs) are well known to scavenge oxidants including H2O2, we tested the hypothesis that RBCs inhibit apoptosis of neutrophils by reducing intracellular oxidative stress. Apoptosis of neutrophils was evaluated by light microscopy and DNA gel electrophoresis. We found that coculture with RBCs protected against neutrophil apoptosis. Neither physical contact between RBCs and neutrophils nor the cellular integrity of RBCs was required to protect against neutrophil apoptosis. Neutrophil apoptosis was promoted by exogenous H2O2 but suppressed by catalase, indicating a role for H2O2 as a mediator of apoptosis. The protective effect of RBCs against apoptosis was due to catalase and glutathione metabolism because blocking of these antioxidant systems in RBCs attenuated the protective effect of RBCs. These results suggest that neutrophils are protected against apoptosis in the circulation.
Lipopolysaccharide Regulates Macrophage Fluid Phase Pinocytosis Via CD14-Dependent and CD14-Independent Pathways
Authors: Peppelenbosch Maikel P. , DeSmedt Marjory , ten Hove Tessa , van Deventer Sander J.H. , Grooten Johan 
Published: October 2019

AbstractLipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the biological effects of LPS in these cell types are only partially understood. We decided, therefore, to investigate the influence of LPS on macrophage pinocytosis and Fc receptor–mediated endocytosis, two prominent and related macrophage effector functions. We observed that LPS did not greatly influence endocytosis in either macrophages or monocytes, but did exert a dual action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS caused a decrease in pinocytosis in both macrophages and monocytes, whereas at higher LPS concentrations, enhanced pinocytosis in macrophages was observed. Detoxified LPS was two orders of magnitude less potent in producing these effects. After inhibition of the LPS receptor CD14, the LPS-induced decrease in pinocytosis was absent, and stimulation of pinocytosis at lower LPS concentrations was unmasked. We conclude that LPS can influence pinocytosis via CD14-dependent and CD14-independent signaling pathways. Furthermore, as addition of LPS to macrophages effected pinocytosis but not Fc receptor–mediated endocytosis, these two processes are independently regulated in macrophages.
Granulocyte Colony-Stimulating Factor–Mobilized Allogeneic Stem Cell Transplantation Maintains Graft-Versus-Leukemia Effects Through a Perforin-Dependent Pathway While Preventing Graft-Versus-Host Disease
Authors: Pan Luying , Teshima Takanori , Hill Geoffrey R. , Bungard David , Brinson Yani S. , Reddy Vijay S. , Cooke Kenneth R. , Ferrara James L.M. 
Published: October 2019

AbstractMinimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)–mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF–mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 → B6D2F1). B6 mice (H-2b) were injected subcutaneously with human G-CSF (100 μg/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v0%, P &lt; .001). Systemic levels of lipopolysaccharide and tumor necrosis factor- were markedly reduced in recipients of allogeneic G-CSF–mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2d) at the time of transplantation, all surviving recipients of G-CSF–mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell–depleted (TCD) splenocytes from G-CSF–mobilized B6 donors died of leukemia. When splenocytes from G-CSF–mobilized perforin-deficient (pfp−/−) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF–mobilized PBSCT. These data illustrate that G-CSF–mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.
Cladribine Activity in Adult Langerhans-Cell Histiocytosis
Authors: Saven Alan , Burian Carol 
Published: October 2019

AbstractLangerhans-cell histiocytosis (LCH) results from the accumulation of tissue histiocytes derived from the same progenitor cells as monocytes. Because cladribine is potently toxic to monocytes, we conducted a phase II trial of cladribine. Cladribine was administered to 13 LCH patients at 0.14 mg/kg per day by 2-hour intravenous infusion for 5 consecutive days, every 4 weeks for a maximum of six courses. Median age was 42 years (range, 19 to 72) and median pretreatment disease duration was 99 months (range, 6 to 252). One patient was untreated, one had received prior prednisone only, one prior radiation only, six prior radiation and chemotherapy, and four prior surgery, radiation, and chemotherapy. Seven patients had cutaneous involvement, six multifocal osseous, six pulmonary, two each with soft tissue and nodal involvement, and four had diabetes insipidus. Of 13 patients, 12 were evaluable for response and all for toxicity. After a median of three courses (range, 1 to 6), seven (58%) patients achieved complete responses (two pathologic and five clinical) and two (17%) patients achieved partial responses; overall response rate, 75%. Median response follow-up duration was 33 months (range, 1 to 65). Seven patients experienced grade 3 to 4 neutropenia. Only one patient had a documented infection, dermatomal herpes zoster. At a median follow-up of 42 months (range, 5 to 76), 12 patients remain alive and one patient has died. Thus, cladribine has major activity in adult LCH and warrants further investigation in both pediatric and adult LCH as a single agent and in combination with other drugs.
Presenting White Blood Cell Count and Kinetics of Molecular Remission Predict Prognosis in Acute Promyelocytic Leukemia Treated With All-Trans Retinoic Acid: Result of the Randomized MRC Trial
Authors: Burnett Alan K. , Grimwade David , Solomon Ellen , Wheatley Keith , Goldstone Anthony H. 
Published: October 2019

AbstractAll-trans retinoic acid (ATRA) is an essential component of the treatment of acute promyelocytic leukemia (APL), but the optimal timing and duration remain to be determined. Molecular characterization of this disease can refine the diagnosis and could be potentially useful in monitoring response to treatment. Patients defined morphologically to have APL were randomized to receive a 5-day course of ATRA before commencing chemotherapy or to receive daily ATRA commencing with chemotherapy and continuing until complete remission (CR). The chemotherapy was that used in current MRC Leukaemia Trials. Outcome comparisons were by intention to treat with additional analysis for relevant risk factors. Patients were characterized by molecular techniques for the fusion products of the t(15;17) and monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) during and after treatment. Two hundred thirty-nine patients were randomized. Treatment with extended ATRA resulted in a superior remission rate (87% v 70%, P &lt; .001), due to fewer early and induction deaths (12% v 23%, P = .02), and less resistant disease (2% v 7%, P = .03), which was associated with a significantly more rapid recovery of neutrophils and platelets. Extended ATRA reduced relapse risk (20%v 36% at 4 years, P = .04) and resulted in superior survival (71% v 52% at 4 years, P = .005). Presenting white blood cell count (WBC) was a key determinant of outcome. The 70% of patients who presented with a WBC less than 10 × 109/L had a better CR (85% v62%, P = .0001) and reduced relapse risk (22% v42%, P = .002) and superior survival (69%v 43%, P &lt; .0001). Within the low count group, extended ATRA resulted in a better CR (94% v 76%, P= .001), reduced relapse risk (13% v 35%, P = .04), and improved survival (80% v 57%, P = .0009). There was no evidence of benefit in patients presenting with a higher WBC (&gt;10 × 109/L). Molecular monitoring after the third chemotherapy course had a correlation with risk of relapse. The relapse risk was 57% if the RT-PCR was positive versus 27% if the RT-PCR was negative (P = .006). APL patients who present with a low WBC derive substantial benefit from combining ATRA with induction chemotherapy until remission is achieved, whereas patients with a higher WBC did not benefit. Molecular characterization of disease can improve diagnostic precision and a positive RT-PCR after consolidation identifies patients at a higher risk of relapse.
The Mouse GATA-2 Gene is Expressed in the Para-Aortic Splanchnopleura and Aorta-Gonads and Mesonephros Region
Authors: Minegishi Naoko , Ohta Jun , Yamagiwa Hironori , Suzuki Norio , Kawauchi Shimako , Zhou Yinghui , Takahashi Satoru , Hayashi Norio , Engel James Douglas , Yamamoto Masayuki 
Published: October 2019

AbstractWe previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2–expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5′ end of the IS first exon direct expression of β-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5′ end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5′ to the IS exon.