bluuebunny/crossref_metadata_2025_split huggingface dataset:
Knockdown of Peroxiredoxin VI in K562 Cells Stably Transfected with Oxidase Components Reduces NADPH Oxidase Activity in Response to PMA and fMLP.
Authors: Ambruso Daniel R. , Thurman Gail 
Published: April 2020

Abstract
Introduction: An intact respiratory burst is critical to the microbicidal activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase complex. We recently identified a 29 kDa neutrophil protein which binds p67phox, is identified as peroxiredoxin VI by sequence and activity, translocates to the plasma membrane of neutrophils during activation and enhances superoxide anion (O2−) production in a cell-free assay exhibiting saturable kinetics. The activity of p29 peroxiredoxin (Prx) requires cysteine residues of the protein. The present studies demonstrate the requirement of p29 Prx for optimal oxidase activity in intact cells.
Methods: K562 cells stably transfected with p47phox, p60phox, gp91phox and the low affinity fMLP receptor and expressing p22phox, p40phox and Rac were grown in tissue culture at 37°C in 5% CO2 with RPMI and 10% fetal calf serum. Three siRNA molecules (Q, A1, A2) as well as non-interfering RNA (Nsi) used as a control were obtained commercially. Transfection of K562 cells was completed with Nucleofector technology (Amaxa Biosystems, Gaithersburg, MD) using Solution L and protocol T020. Transfection was &gt;90% by expression of a GFP plasmid; viability after the procedure was &gt;90%. O2− was measured after stimulation with PMA (200 ng/ml) as SOD inhibitable cytochrome c reduction or in response to fMLP (1 μM) by the addition of Diogenes (National Diagnostics) and detection of chemiluminescence in a luminometer (FB12, Berthod Detection Systems, Pforzheim, Germany) as relative light units. Cell lysates were made with the addition of 10% Triton X-100 and stored at −70°C. Proteins (5–10μg of cell lysate) were separated on a 10% SDS-PAGE and blotted onto nitrocellulose. Detection of specific proteins was completed using polyclonal antibodies to p29 Prx and to other phox proteins and actin and secondary antibodies with a chemiluminescent technique.
Results: Transfection of the K561 cells with Q, A1, and A2 resulted in a knockdown of p29 Prx (25–40%) compared to Nsi. Optimal knockdown occurred in the presence of 2μM siRNA, at 48 hours after transfection. No changes in actin, p67phox, p47phox, p40phox, p22phox or gp91phox were documented with nucleofection, siRNAs or Nsi. In 5 separate experiments, knockdown of p29 Prx resulted in a decrease (p&lt;0.05) in O2− production (PMA) or O2− associated chemiluminescence (fMLP), see Figure (columns and bars represent mean ± SEM).
Conclusion: O2− production in response to PMA or fMLP was reduced in K562 cells with a knockdown of p29. These results extend previous studies and demonstrate the requirement of p29 Prx, peroxiredoxin VI, for optimal NADPH oxidase activity within intact cells.
Figure Figure
Further Evidence That MDS and AML Cells Exhibit a Differential Capacity To Activate the DNA-Damage Response (DDR) Pathways.
Authors: Boehrer Simone , Ades Lionel , Galluzzi Lorenzo , Olaussen Ken , Braun Thorsten , Grosjean Jennifer , Fabre Claire , de Botton Stephane , Fenaux Pierre , Kroemer Guido 
Published: April 2020

Abstract
Background: Findings, that activation of the DDR-pathway in preneoplastic lesions of solid neoplasms slows tumorigenesis, could also underly progression of MDS to AML. We previously showed (ASH 2006, abstr n° 2649), that - in contrast to the de novo AML-derived KG-1 cells - irradiated MDS/AML-derived cell lines P39 and MOLM-13exhibit earlier and greater cell cycle arrest in G2/M and a higher percentage of apoptotic cells;upregulate the crucial checkpoint kinase Chk1-P-Ser317;decrease G2/M-arrest upon inhibition of Chk1 (and to a lesser extent upon inhibition of ATM).
Methods: We further analyzed this differential checkpoint regulation in MDS versus AML cell lines by assessing irradiation-induced apoptosis in the presence of Chk1- and ATM-inhibitors and the functional role of H2AX, situated below the crucial checkpoint kinase Chk1. Myeloid cell lines P39, MOLM-13, MV4-11, and KG-1 were g-irradiated and their ability to undergo apoptosis (DIOC/PI staining) quantified in the absence and presence of the Chk1 inhibitor UCN-01 and the ATM inhibitor KU-55933. Protein expression of activated H2AX was quantified by immunoblot analysis with an anti-H2AX-P-Ser139 antibody. Histological staining of bone marrow biopsies from patients with low and high risk MDS, and AML was made with an anti-H2AX-P-Ser139 antibody. Staining was considered positive if at least 20% of the biopsy surface exhibited 50% or more stained cells.
Results: In the presence of the ATM inhibitor irradiated KG-1 cells retained their apoptosis-resistance, and irradiated P39, MOLM-13 and MV4-11 cells exhibited the same degree of apoptosis as seen in the absence of the inhibitor. In contrast, inhibition of Chk1 (newly) established apoptosis-sensitivity in KG-1 cells (which showed about 30% of DIOC-low cells following 24 hours of irradiation with 10 Gy), while having only minor effects on the other cell lines. Determining the concomitant activation status of H2AX, which is a downstream target of Chk1, showed that irradiation induced a comparable expression of H2AX-P-Ser139 in MDS and AML cell lines. Co-incubation with the Chk1 inhibitor was associated with lower expression of activated H2AX in the irradiated MDS cell lines P39 and MOLM-13 as compared to irradiated KG-1 cells. Histological staining of marrow biopsies with an anti-H2AX-P-Ser139 antibody demonstrated that activation of H2AX is less frequent in low risk MDS (1/10 samples) than in high risk MDS (9/18 samples) and AML (4/6 samples).
Conclusion: Further analyzing the different capacities of MDS and AML cells to employ DDR-pathways, we provide additional insights into the functional relevance of ATM and Chk1 inhibition in the apoptotic DDR, and give preliminary evidence for a differential activation of H2AX in irradiated cell lines and patient samples of MDS and AML.
MDS with Isolated Neutropenia or Thrombocytopenia. Incidence and Characteristics in the Groupe Francophone des Myélodysplasies (GFM) Registry.
Authors: Park Sophie , Andrieu V. , Sapena R. , Leroux G. , Stamatoullas A. , Callat M.P. , Beyne-Rauzy O. , Mairesse J. , Chaury M.P. , Dombret Herve , Siguret V. , Vey Norbert , Lepeu G. , Guerci A. , Cheze S. , Fenaux Pierre , Dreyfus F. 
Published: April 2020

Abstract
Background: MDS classification is regularly refined over time. WHO classification (2001) puts emphasis on cases with anemia and isolated dyserythropoiesis, as opposed to multilineage dysplasia, while the next modifications to WHO classification will probably consider cases with isolated or predominant granulocytic or thrombopoietic involvement.
Methods: In this context, we analyzed cases of IN and IT included in the GFM registry of MDS.
Results: From Jul 2003 to Jul 2007, 1009 newly diagnosed MDS patients (including 145 therapy MDS, and excluding CMML,) from 11 centers were included in the GFM registry. 22 of them (2%) presented with neutropenia (ANC &lt;1.5×109/l) (IN) and 24 (2.4%) with thrombocytopenia (plts&lt;100×109/l) (IT) as only cytopenia, respectively. IN included 13 F and 9 M and IT 17 F and 7 M. Median age was 70 years (range 54-89) in IN, and 72 years (range 50-84) in IT. Median ANC was 0.89×109 (range 0.14-1.48×109) in IN, and median platelet count 62×109 (range 7-99×109) in IT. According to WHO (2001), IN included 5 RA (diagnosis based in 4 pts and 1 pt on prominent unilineage dyserythropoiesis and unilineage dysmegacaryopoiesis respectively, and elimination of other causes of anemia, as karyotype was normal in all 5 pts), 1 RCMD, 8 RAEB-I, 2 RAEB-II, 1 patient with 5q- syndrome (with Hb=12.5g/dl), 1 unclassified MDS with marrow hypocellularity (of 39 U -MDS in the registry,) and 4 therapy-related MDS. IT included 13 RCMD, 7 RAEB-I, 4 RAEB-II. Cytogenetic findings were: for IN: good risk: 15 (normal: 13, del 5q, -Y), intermediate: 2 (+8), poor: 1, cytogenetic failure: 4 ; for IT: good risk: 13 (normal: 12, del 20q:1), intermediate: 5 (del 2, del3p,+8, del 9+t(9;14), del11+del13p), poor: 2, cytogenetic failure: 4. IPSS was: For IN: low (5), Int-1 (7), Int-2 (5), High (1) patients (4 ND); For IT: low (7), Int-1 (9), Int-2 (4) and High (0) (4 ND). Median follow-up of IN and IT was 23 months (range 2-48 mos). 5 IN pts progressed to AML and 4 of them died, 1 IT pt progressed to AML and 2 died.
Conclusion: IN and IT are rare in MDS, predominate in females, contrary to overall MDS, and are widely distributed in the current WHO classification. IT was always associated with multilineage dysplasia, and/or excess of marrow blasts contrary to some IN, which were therefore more difficult to diagnose, especially as karyotype was usually normal. Follow-up in the GFM registry is still limited to evaluate the prognosis of IN and IT. These findings are to be considered if new specific MDS categories are to be individualized.
Lenalidomide (Len) in Combination with Rituximab (R) Demonstrated Early Evidence of Efficacy in a Phase I/II Study in Relapsed/Refractory Mantle Cell Lymphoma (MCL).
Authors: Wang Michael , Fayad Luis , Hagemeister Frederick , Neelapu Sattva , Samuels Barry , Samanigo Felipe , Pro Barbara , Yi Qing , Bell Neda , Byrne Catriona , Weaver Pam , Hartig Kim , Knight Robert , Zeldis Jerry , Kwak Larry , Romaguera Jorge 
Published: April 2020

Abstract
Background: Relapsed/refractory MCL is difficult to treat. R targets CD20 antigen on the surface of MCL cells while Len may target the microenvironment of MCL cells and enhance the ADCC activity of R. To test this hypothesis, we initiated a single-center, phase I/II study. We now report the data from the completed phase I trial and the ongoing phase II trial.
Methods: Eighteen eligible patients (pts) with MCL had 1–4 lines of prior therapies. None had prior thalidomide and all received R. Treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks only during the first cycle with the first dose on Day 1 in Cycle 1. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. DLT was defined as grade (G) 3 or 4 non-hematologic or G4 hematologic toxicity during the first cycle. Phase I has been completed with 15 pts. Phase II is ongoing with 3 pts enrolled at MTD. Total 80 cycles of therapy were given to 18 pts. In addition to the regular radiology evaluation, a separate radiology evaluation was performed independently. EGD and colonoscopy with random biopsies were performed to confirm all CRs.
Results: In the phase I study, median age was 73 (62–84); median prior therapies were 3 (1–4); median cycles received to date (August 18, 2007) were 4 (range 1–14). Two DLT’s occurred at 25 mg. One G3 hypercalcemia and 1 G 4 non-neutropenic fever during the first cycle. After total 80 cycles of therapy in 18 patients, except for the 2 DLT’s, common non-hematologic toxic events included fatigue (23 G1-2), pruritis (18 G1-2), myalgias (5 G1-2, 1 G3) and rash (5 G1-2). There was no DVT/PE. Grade 3 hematologic toxic events included neutropenia (11), febrile neutropenia (2), and thrombocytopenia (2). The only grade 4 hematologic toxic events were neutropenia (5). There was no grade 3–4 anemia. There were no responses at 10 mg or 15 mg. Ten pts at MTD (20 mg) including 7 in phase I plus 3 in phase II were evaluable for response. Seven out of 10 pts achieved responses including 3 CRs (30%), 4 PR’s (40%), 1 SD and 2 PD’s. Therefore, the overall response rate was 70%. Media time to response was 2 cycles (range 2–4).
Conclusions: The MTD for Len with R in relapsed/refractory MCL was 20 mg, orally daily on days 1–21 of a 28-day cycle. Early evidence of response is promising with a very favorable toxicity profile at 20 mg and is being further evaluated in the ongoing phase II trial.
Lenalidomide Response in Relapsed/Refractory Diffuse Large B-Cell Non-Hodgkin’s Lymphoma.
Authors: Lossos Izidore S. , Wiernik Peter H. , Justice Glen , Tuscano Joseph M. , Cole Craig E. , Vose Julie M. , Pietronigro Dennis , Takeshita Kenichi , Ervin-Haynes Annette , Zeldis Jerome B. , Habermann Thomas M. 
Published: April 2020

Abstract
Background: Patients with diffuse large B-cell lymphoma (DLBCL) not cured by first line R-CHOP chemotherapy or second line high-dose chemotherapy with autologous stem cell rescue have a poor prognosis and represent a clear unmet medical need. Lenalidomide (Revlimid®), an immunomodulatory drug, has activity in hematological malignancies including relapsed/refractory multiple myeloma, chronic lymphocytic leukemia and cutaneous T-cell lymphoma.
Aim: To determine the activity and safety of lenalidomide monotherapy in relapsed/refractory DLBCL.
Methods: Patients with relapsed/refractory DLBCL with measurable disease after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology. Univariate analyses using Fisher’s exact test was conducted to investigate and characterize associations of prognostic variables with response.
Results: Twenty-six patients with DLBCL lymphoma were enrolled. The median age was 66 (45–86) and 13 were female. Median time from diagnosis to lenalidomide was 2.4 (0.5–12) years and median number of prior treatment regimens was 3 (1–6). Three patients (12%) exhibited an objective complete response (1 complete response (CR) and 2 complete responses unconfirmed (CRu)). Two patients had a partial response (PR) for an overall response rate of 19% and 7 had stable disease (SD). Response to lenalidomide was associated with low disease burden (33% for &lt; 50 cm2v 0% for ≥ 50 cm2, P=0.06) and longer time from rituximab to lenalidomide treatment (33% for ≥ 230 days v 0% for &lt; 230 days, P=0.05). Patients with favorable values for both these prognostic factors (N = 10) had a 50% response rate [RR] (30% CR/CRu RR) compared with a 0% RR for patients with unfavorable values (N = 16; P &lt; 0.004) and a progression free survival (ongoing) of 7.4 months v 1.9 months. Although there were no responses in the poor prognosis group, 4 patients exhibited stable disease with an ongoing median PFS of &gt; 4.2 months (&gt; 3.6 - &gt; 6.2). Eight patients (31%) required at least one dose reduction with a median time to first dose reduction of 1.8 months (0.4–2.9). One patient each (4%) had Grade 4 anemia, cauda equinine syndrome, febrile neutropenia, intermittent rash, lymphopenia, neutropenia, pneumonitis and thrombocytopenia. Most common Grade 3 adverse events were neutropenia (19%), thrombocytopenia (15%) and leukopenia (12%).
Conclusion: The prognostic factors of tumor burden and time from last dose of rituximab appear to identify relapsed/refractory DLBCL patients with a high likelihood of response to lenalidomide oral monotherapy.
Transplantation of Primary Human CD34+CD38 - Hematopoietic Stem Cells Recapitulates Idiopathic Myelofibrosis in the NOD/scid/IL2rgKO Mice.
Authors: Saito Noriyuki , Ishikawa Fumihiko , Shimoda Kazuya , Yoshida Shuro , Saito Yoriko , Fukata Mitsuhiro , Kawano Noriaki , Shulz Leonard D. , Akashi Koichi , Harada Mine 
Published: April 2020

Abstract
Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM leads to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38- cells from six IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Six out of thirteen recipients transplanted with patient HSCs exhibited human hematopoietic engraftment, and CD33+ myeloid cells accounted for 80.5+/−9.41% of all the engrafted CD45 + population (as compared with the recipients transplanted with normal HSCs). BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was &lt; 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. To investigate the origin of BM fibroblast, we performed FISH analysis using human and mouse centromeric probes and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of sixty fibroblasts examined, fifty-four cells were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38- HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.
A Prospective Clinical Trial of a Novel Epstein-Barr Virus (EBV) PCR Panel in Patients with EBV Associated Malignancies.
Authors: Xavier Marin F. , Frank Dale , Nasta Sunita , Schuster Stephen , Andreadis Charalambos , Arnoldi Sharon , Sherman Eric , Berg Sigrid , Mato Anthony , Musher Benjamin , Downs Lisa , Porter David , Stadtmauer Edward , Goldstein Steven , Kim Ellen , Tsai Donald 
Published: April 2020

Abstract
EBV is known to be detectable within a number of different tumors, though the exact relationship between the malignant cells and the EBV virus is unknown in most cases. In post-transplant lymphoproliferative disorders, which are clearly EBV-driven, EBV PCR of peripheral blood has proven to be a clinically useful tumor marker. The goal of our study is to determine the utility of novel EBV PCR panel in EBV associated malignancies. Subjects with suspected active or untreated EBV-associated malignancies were evaluated with a 6 assay EBV PCR panel targeting LMP-1, EBER-1 and EBNA-1 within both whole blood cells and plasma. EBV positive subjects and EBV negative control tumor samples are tested for LMP and EBER using traditional histochemical staining. LDH, SPEP, and traditional EBV serology are also measured. Of the 40 patients who were evaluated to date, 18 subjects showed evidence of elevated blood EBV viral load: 3/5 nasopharyngeal carcinoma, 3/4 angioimmunoblastic lymphomas, 3/6 Hodgkin’s lymphomas, 4/10 peripheral T-cell lymphomas, 3/3 allogeneic bone marrow transplant lymphoproliferative disorders, 1/4 diffuse large B-cell lymphomas, 1/1 non-langerhans histiocytosis, and 0/7 low-grade B-cell NHL. Median DNA PCR copies/ml and ranges in EBV positive patients: EBNA plasma 500 (0–134,000), EBNA whole blood 700 (0–6,200), EBER plasma 200 (0–19,700), EBER whole blood 200 (0–1,000), LMP plasma 0 (0–600), LMP whole blood 0 (0–100). Patients were monitored with EBV PCR as they proceeded through treatment and 12 subjects are evaluable for response. Changes in viral load appear to be highly correlated with clinical tumor response (p=0.0013). 7/7 patients achieving CR had concurrent resolution of their plasma and whole blood EBV viral load. 5/5 patients with persistent disease had persistent elevated plasma and whole blood viral load. EBV histologic studies on the tumor samples are pending. Our data suggests that EBV PCR may be useful in the management of patients with EBV positive malignancies. Further patient enrollment and evaluation is ongoing.
Downregulation of a Candidate Myeloid Tumor Suppressor Gene Isolated from 7q21 Induces Abnormal Mitosis Due to Insufficient Centrosome Maturation.
Authors: Asou Hiroya , Matsui Hirotaka , Ozaki Yuko , Nagamachi Akiko , Aki Daisuke , Ohsugi Miho , Inaba Toshiya 
Published: April 2020

Abstract
To isolate myeloid tumor suppressor gene(s) in 7q, we searched microdeletions in a region spanning 21.7 Mb within 7q21.2–7q31.1 using a microarray-based CGH system. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we identified a common microdeletion cluster spanning approximately 120 Kb in the 7q21.3 subband. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Real-time quantitative PCR revealed that this region is also deleted in 9 (29%) out of 31 adult RAEB and AML patients. Database search revealed that this region contains three hypothetical genes. Among them, we chose one previously uncharacterized gene for further investigation and named Miki (mitotic kinetics regulator) for the function of its gene product, described below. Immunoblot analysis revealed high levels of Miki expression in most lymphoid leukemia cell lines, while half of myeloid leukemia cell lines expressed Miki at reduced levels. In six leukemia lines carrying monosomy 7, expression levels were generally low. Miki co-localized with the Golgi apparatus in the interphase and with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed insufficient maturation and disturbed positioning of centrosomes, resulting in unorganized spindles including loss of spindle tension, curled and fragile spindles, or even completely disturbed spindle formation. Time-lapse observation revealed prometaphase and/or metaphase delay with unaligned or even totally scattered chromosomes in prometaphase in virtually all cells in the mitotic phase. As a result, cells underwent pre-anaphase arrest and exited mitosis in the absence of chromosome segregation or terminated mitosis by cell death. In the interphase, there were many cells with chromatid bridges and/or bi- or tri-nuclear or even multinuclear cells with micronuclei that resembled pathological cells routinely observed in the bone marrow pictures of MDS patients. Interestingly, myeloid cell lines with low Miki expression, including those with monosomy 7, generally showed abnormal mitosis such as scattered chromosomes and abnormal nuclear morphologies (multi-nuclear cells with small nuclei) at higher frequency than cell lines expressing Miki at high levels. Moreover, induction of Miki restores normal mitosis in leukemia cells with monosomy 7. Miki was poly(ADP-ribosyl)ated (PARsylated) in late G2 to M phase by tankyrase-1, one of PAR polymerase (PARP), and tankyrase-1 activity was required for the binding of Miki to spindles and centrosomes. These data suggest that loss of Miki gene contributes to the development and progression of MDS by disturbing mitosis.