bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Sphingomyelin Synthase Gene, SMS1-Regulated Sphingomyelin/Ceramide-Rich Microdomain Plays a Crucial Role in Fas-Mediated Apoptosis.
Authors: Miyaji Michihiko , Yamaoka Shohei , Xiong Jin Zhe , Amakawa Ryuichi , Mimori Tsuneyo , Fukuhara Shirou , Umehara Hisanori , Okazaki Toshiro 
Published: April 2020

Abstract
Lipid microdomain consisting of sphingolipids, glycosphingolipids and cholesterol plays an important role in mediating cell death signals. Although functional analysis of lipid microdomain is conventionally performed by exclusion of cholesterol tightly binding with sphingomyein (SM), the exact role of SM in microdomain in signal transduction remains to be cleared. Since we for the first time obtained cDNA (SMS1) responsible for SM synthesis, it became possible to investigate the role of SM in the membrane without affecting cholesterol content. We here examine the role of SM/ceramide-rich microdomain in Fas-induced apoptosis using the SM-deficient cells and its revertant cells by transfection with SMS1.
SM synthase-defective WR19L cells transfected with human-Fas gene (WR/Fas-SM(-)), and its functional revertant cells by transfection with SMS1 (WR/Fas-SMS1) were established. Both SMS1 cells and SM(-) cells expressed similar levels of CD44, CD90, CD95 (Human) and LFA-1 by FACS analysis. Although the levels of cholesterol and GM1-ganglioside on the surface of the membrane were similar in both cells, SM was detected only on the surface of SMS1 cells by FACS and confocal microscopy analysis. Fas crosslinking induced not only higher rate of apoptotic cell death, but also translocation of Fas into TritonX-100-insoluble fractions with a significant increase of ceramide in SMS1 cells as compared with SM(-) cells. After increase of SM and ceramide in microdomain, more efficient aggregation of Fas was detected with increase of DISC formation, resulting in activation of caspase-8 and 3 and decrease of mitochondrial membrane potential in SMS-1 cells than SM(-) cells. Our results clearly demonstrate that SM and ceramide in microdomain of plasma membrane plays a crucial role in Fas clustering and aggregation requires for activation of apoptosis-inducing signals.
Casein Kinase Iε Downregulates PI3K/Akt Signaling, Followed by Genotoxic Stress-Induced Apoptosis of Hematopoietic Cells.
Authors: Okamura Atsuo , Yamamoto Katsuya , Yakushijin Kimikazu , Urahama Norinaga , Minagawa Kentaro , Sada Akiko , Hato Akio , Nishikawa Shinichiro , Yokoyama Kazuaki , Ito Mitsuhiro , Matsui Toshimitsu 
Published: April 2020

Abstract
Casein kinase I (CKI) ε is involved in cytokine-induced hematopoietic cell differentiation by directly interacting with the suppressor of cytokine signaling 3 (SOCS3) and/or β-catenin. Here, we show that CKIε plays an important role in hematopoietic cell survival through modifying Phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIε into murine interleukin-3 (IL-3)-dependent myeloid progenitor 32D cells increased the sensitivity to genotoxic stress, such as γ-irradiation, anti-cancer drug and cytokine-deprivation, whereas kinase-negative (KN)-CKIε suppressed it. While IL-3-induced Akt phosphorylation at Ser473 was sustained by KN-CKIε, it was attenuated by WT-CKIε. Similarly, in human promyelocytic leukemia HL-60 cells, the increase of phosphorylated Akt induced by all-trans retinoic acid was accompanied by the decrease of CKIε expression. A specific inhibitor for CKIε, CKI-7 also increased the phospho-Akt as well as phospho-PTEN (Ser380/Thr382/383; an inactive form of PTEN). Functional expression of CKIε activated PTEN by their physical association, following the decrease of phospho-Akt. The present studies suggest that the expression of CKIε downregulates PI3K/Akt signaling through PTEN, increasing the sensitivity to apoptotic signals. Taken together with the fact that CKIε is ubiquitously expressed in normal hematopoietic cells, CKIε might be critical for prevention against genotoxic stress-induced leukomogenesis. In other words, CKIε could be a candidate of the tumor suppressor in hematological malignancies as well as cancer.
Expression of Complex Junction Proteins in Hematopoietic Progenitor Cells.
Authors: Straub Beate K. , Eckstein Volker , Grund Christine , Miesala Katrin , Horsch Kerstin , Wuchter Patrick , Gottschling Sandra , Franke Werner W. , Ho Anthony D. 
Published: April 2020

Abstract
The interaction of hematopoietic stem cells with the microenvironment plays a pivotal role in regulating self-renewal and differentiation. N-cadherin-based adherens junctions have been reported to be involved in this process (Puch et al., 2001; Zhang et al., 2003). However, the molecular composition of such junctions in hematopoietic progenitor cells (HPC) remains to be defined as well as their function. CD34+ cells and CD34+/CD38− cells were isolated from human umbilical chord blood (CB). In immunoblotting, CD34+ cells were positive for the transmembrane proteins N- and E-cadherin, cadherin 11 (low amounts) and the cytoplasmic plaque proteins α- and β-catenin, protein p120ctn, vinculin, spectrin/fodrin and actin as markers for adherens junctions as well as for ezrin, moesin and drebrin, actin-binding proteins known to be enriched in cellular protrusions. Other classical cadherins such as VE-, P-, R-cadherin or cadherin-6 were negative. Moreover HPCs were positive for the intermediate filament cytoskeletal protein vimentin, the integral heparan sulphate proteoglycans syndecan-1 (CD138), syndecan-2 (fibroglycan), syndecan-3 (N-syndecan) and syndecan-4 (ryudocan, amphiglycan) and the gap junction protein connexin 43. CD34+/CD38− cells showed positive reactions with antibodies against vimentin, ezrin, vinculin, α- and β-catenin and with a pan cadherin antibody, with no obvious differences to CD34+/CD38+ and Kg1a cells. In parallel, we established single- and double-immunohistochemistry with antigen-retrieval-treated, formalin-fixed, paraffin-embedded bone marrow using peroxidase, alkaline phosphatase and glucose oxidase coupled secondary antibodies. The architecture of human, rat and bovine bone marrow tissue was analyzed with a panel of antibodies against components of adhering, gap and tight junctions. Immunohistochemical sections of bone marrow incubated with the various antibodies remarkably showed the presence of all these antigens in their natural microenvironment. Intercellular junctions of rat and bovine bone marrow were also observed with transmission electron microscopy. Our results show that HPC are equipped with components of cadherin-catenin-based adherens junctions. Immunoprecipitation and immunolocalisation experiments are underway to further elucidate the homo- and heterotypic intercellular junctional complexes between HPC and stromal cells in vitro and in vivo.
Promotion of Chimerism and Tolerance by Flt3 Ligand-Mobilized Facilitating Cells Is Associated with Upregulation of CXCR4 and SDF-1.
Authors: Huang Yiming , Kucia Magda , Rezzoug Francine , Ratajczak Mariusz Z. , Tanner Michael , Schanie Carrie , Xu Hong , Fugier Isabelle , Ildstad Suzanne T. 
Published: April 2020

Abstract
Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient.
Figure Figure
Early Kinetics of Bone Marrow Cell Engraftment in Bone after Transplantation: Insights into the Mechanism of Engraftment.
Authors: Garlits John E. , Dominici Massimo , Hofmann Ted J. , Horwitz Edwin M. 
Published: April 2020

Abstract
Despite their anatomic proximity and recognized physiologic relationship, the interactions between bone and bone marrow (BM) are incompletely understood. Consistent with our data from clinical BMT in children with osteogenesis imperfecta, murine models show significant engraftment of transplanted donor BM cells in the osteogenic compartment early after BMT, which declines to a nearly undetectable level by 9 months post-BMT. To gain insight into the mechanism of donor BM cell engraftment in bone, we studied the kinetics of hematopoietic and osteopoietic engraftment. Lethally irradiated (1100 cGy) FVB/N mice were transplanted with 2x106 BM cells transduced with a retroviral vector encoding the green fluorescent protein (GFP). Transduction efficiency, assessed by flow cytometry, was 53%. Engraftment in the hematopoietic and osteopoietic compartments was analyzed at 2, 4, or 6 weeks post-transplant (wpt). Flow cytometric analyses of blood leukocytes, as a marker of functional hematopoietic marrow, showed 66% ± 2% GFP+ (mean ± SEM) at 2 wpt (n = 8 for each time point), increased at 4 wpt to 73% ± 2% and plateaued at 77% ± 2% by 6 wpt (p &lt; 0.01). Erythrocytes and platelets exhibited a similar profile. BM cellularity, estimated by microscopic evaluation, increased from approximately 50% to 80% while GFP expression was observed in about 50% of marrow cells and remained at that level from 2 to 6 wpt. Analysis of 5 immunostained bone sections per animal revealed 40% ± 4% GFP+ osteoblasts at 2 wpt with a decline to 20% ± 2% by 6 wpt (p &lt; 0.01). Mature osteocytes were 20% ± 3% GFP+ at 2 wpt, then declined to 10% ± 3% by 6 wpt (p &lt; 0.05). Thus, both hematopoietic and osteopoietic engraftment are early events; however, donor-derived hematopoietic BM proliferates over the first 6 weeks post-BMT while the donor-derived osteopoietic cell population declines. Our data suggest that either the transplantable marrow osteopoietic cell is a progenitor capable of a single burst of differentiation or that bone specific mechanisms related to the BMT procedure underlie the capacity of BM cells to engraft in the osteopoietic environment. Elucidating this cellular pathway will be a key element to the development of BM cell therapy for mesenchymal tissues.
G-CSF-Stimulation of Human Marrow Stromal Cells Induces In Vitro Migration of Hematopoietic Progenitor Cells Involving MMP-2 and MMP-9 but Not MMP-1.
Authors: López Adriana , Chabot Valérie , Vaudin Pascal , Clément Nathalie , Hérault Olivier , Charbord Pierre , Domenech Jorge 
Published: April 2020

Abstract
The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.
The Role of CXCR4 Inhibitors as Novel Antiangiogenesis Agents in Cancer Therapy.
Authors: Ghobrial Irene M. , Timm Michael M. , Hedin Karen E. , Witzig Thomas E. 
Published: April 2020

Abstract
Angiogenesis plays an important role in tumor growth. Endothelial cells express the chemokine receptor CXCR4, and interact with its ligand SDF-1/CXCL12. Previous studies have demonstrated that the ERK MAP kinase and the PI3Kinase pathways are activated in response to SDF-1 stimulation. In this study, we investigate the effect of the role of inhibitors of CXCR4, ERK MAP kinase and PI3Kinase on angiogenesis. The AngioKit (TCS Cellworks, U.K) is a 24 well plate in which human endothelial cells are co-cultured with other human myoblasts and fibroblasts in a specially designed medium. Control wells in the kit include media alone, VEGF (+control) and suramin (-control). Test samples were added on the day the kits arrive, then changed on days 4, 7, and 9, and stained on day 11 with CD31 (PECAM). The wells are then photographed and subjected to image analysis. The software measures angiogenesis as total tubule length per well in microns. Test samples can then be compared to the control wells to determine the drugs affect on angiogenesis in vitro. The following drugs were tested in this angiogenesis model system, a human CXCR4 neutralizing antibody (MAB 171, R&D systems, MN), SDF-1, the MAP kinase inhibitor PD098059, and the PI3Kinase inhibitor LY294002. Treatment with the CXCR4 inhibitory antibody, PD098059, and LY294002 caused marked decrease in angiogenesis (below the level of the negative control suramin). Inhibition of angiogenesis below the level of suramin was first detected at 1mg/ml CXCR4 antibody, and10mg/ml CXCR4 antibody resulted in complete inhibition of angiogenesis. The effect of PD098059 on angiogenesis was dependent on its concentration; 20mM PD098059 inhibited angiogenesis while lower concentrations did not. These results are consistent with the drug’s known concentration-dependent inhibition of MEK-1 and indicate that the MEK-1 inhibitor leads to angiostasis secondary to its specific inhibitory effect on MEK-1. The lowest level tested of 1μM LY294002 led to inhibition of angiogenesis, and 50μM of LY294002 led to complete abrogation of angiogenesis. SDF-1 has been reported to be angiogenic. In this model system, the effect of SDF-1 alone on angiogenesis was subtle. However, the endothelial cells used in this model system may be secreting endogenous SDF-1 leading to the saturation of the CXCR4 receptor and minimal effects of exogenous SDF-1 stimulation. This was demonstrated by the significant effect of the CXCR4 inhibitor on angiogenesis without the addition of exogenous SDF-1. These results indicate that CXCR4 inhibition and its downstream pathways PI3K and ERK MAP kinase lead to significant inhibition of angiogenesis, and suggest that selective inhibitors of CXCR4 may be useful agents to inhibit angiogenesis.
Reduction of Combined Modality Treatment Intensity in Early Stage Hodgkin’s Lymphoma: Interim Analysis of the HD 10 Trial of the GHSG.
Authors: Diehl Volker , Brillant C. , Engert A. , Wolf J. , Nogova L. , Mueller R.-P. , Eich H. , Mueller-Hermelink H.K. , Herrmann R. , Doerken B. , Kanz L. , Greil R. , Pfistner B. ,   for the German Hodgkin′s lymphoma study group (GHSG),   the Competence Network Malignant Lymphoma.
Published: April 2020

Abstract
Background: Combined modality treatment of chemotherapy (usually ABVD) followed by involved field irradiation (IF) is today a standard treatment regimen for early stage Hodgkin′s lymphoma (HL)in stages I-II without risk factors in most study groups. The complete remission rates achieved with this treatment strategy are more than 90%. However, the question arises, whether treatment intensity of chemotherapy, radiotherapy or both may be further reduced in these patients.
Aim: In the HD10 trial of the GHSG, reduction of ABVD cycles as well as reduction of IF- RT dose were investigated.
Methods: Patients were randomized between 4 cycles of ABVD followed by 30 Gy IF radiotherapy as standard treatment (arm A), 4 cycles ABVD + 20 Gy IF (arm B), 2 cycles ABVD + 30 Gy IF (arm C) and 2 cycles ABVD + 20 Gy IF (arm D).
Results: Between May 1998 and May 2002, 1131 HL patients with early stage disease (CS I,II without risk factors) were randomized. 847 patients (75%) were evaluable for this second interim analysis performed in August 2003. Patient characteristics were well balanced between the treatment arms. In total, 98.4% of the patients reached complete remission. Progressive disease or no change were observed in 0.9% of patients, relapse rate was 2.5%. In total, thirteen patients died during the study (1,5%). During chemotherapy there were 19% leukopenia as the most common WHO grade III/IV toxicity (4xABVD: 22 %, 2xABVD: 15%). Radiotherapy was well tolerated with dysphagia in 2.3%. Altogether ten secondary neoplasias were seen: 1AML, 4 NHL and 5 solid tumors.
After a median observation time of two years, overall survival (OS) and freedom from treatment failure (FFTF) rates were 98.5% and 96.6%, respectively. No statistical differences in FFTF and OS were detected between 4xABVD and 2xABVD. In addition, there was no statistical difference between the different doses of radiotherapy (30Gy vs. 20Gy).
Conclusions: Since only few events were observed, the results of this interim analysis can be interpreted as a trend that therapy intensity in combined modality treatment of early stage might be further reduced in terms of the number of chemotherapy cycles as well as the radiotherapy dose, even in IF-RT setting.
Intensification of Chemotherapy and Concomitant Reduction of Radiotherapy Dose in Intermediate Stage Hodgkin′s Lymphoma: Results of the Fourth Interim Analysis of the HD 11 Trial of the GHSG.
Authors: Diehl Volker , Brillant C. , Engert A. , Wolf J. , Nogova L. , Mueller R.-P. , Eich H. , Hermelink H.K. Mueller , Herrman R. , Markova J. , Ho A. , Hiddemann W. , Doerken B. , Greil R. , Pfistner B. ,   for the German Hodgkin′s Lymphoma Study Group (GHSG),   the Competence Network Malignant Lymphoma.
Published: April 2020

Abstract
Background: Combined modality treatment consisting of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for intermediate stage (early unfavourable) Hodgkin′s lymphoma (HL). Despite high complete remission (CR) rates comparable to early favorable stage HL, failure rates are remarkably higher in this group of patients.
Aim: In the HD 11 multicenter trial of the GHSG, introduction of the BEACOPP regimen for intensification of chemotherapy in combination with dose reduction of IF-RT on 20 Gy was tested in an attempt to improve therapy outcome.
Methods: Between May 1998 and July 2002, 1363 patients aged 16–75 suffering from intermediate stage HL (CS I, IIA with risk factors or IIB with elevated ESR and/or 3 3 nodal areas) were randomised according to a factorial design between: 4 cycles of ABVD followed by 30 Gy IF-RT (arm A - standard treatment), 4 ABVD + 20 Gy IF-RT (arm B), 4 BEACOPP basis + 30 Gy IF-RT (arm C) and 4 BEACOPP basis + 20 Gy IF-RT (arm D).
Results: In the fourth interim analysis in August 2003, 1047 (77%) patients were evaluable for chemotherapy and 982 (72%) for radiotherapy evaluation. Patient characteristics were well balanced between the treatment arms in regard to age, gender, clinical stage, histopathology and performance status. 95.1% patients reached CR, 2.0% suffered progression, the relapse rate was 5.9%, the mortality rate 3.2%. The most frequent haematological toxicity was leucopenia observed in 33% of patients (ABVD: 27%, BEACOPP: 40%). Infection rate was 5% (ABVD: 4%, BEACOPP:7%). The most frequent toxicity during radiotherapy was dysphagia in 5.6%. Nine secondary neoplasias were observed: 2 AML, 3NHL, 4 solid tumors.
After a median observation time of two years overall survival (OS) was 97.4% (95%-CI: 96-98) and freedom from treatment failure (FFTF) was 89.9% (95%-CI: 88-92). Both for FFTF and OS, there was no sequential significant difference either between ABVD and BEACOPP arms nor between 30 Gy and 20 Gy IF-RT arms.
Conclusions: At two years of median observation time, no differences in treatment outcome were detected either between chemotherapy regimes nor between the different doses of radiotherapy. The low FFTF (89,9 % at two years) led us to further intensification of treatment by the introduction of 2 cycles of escalated BEACOPP + 2 cycles ABVD +30 Gy IF-RT in the next study generation (HD14 study).