bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeny, classification, and species-level taxonomy of ants (Hymenoptera: Formicidae)*
Authors: WARD PHILIP S.
Published: October 2019

The current state of ant systematics is reviewed.  In recent years substantial progress has been made in identifying the major clades of ants and the relationships among them.  Earlier inferences about ant phylogeny based on morphology have been refined and modified as a result of a recent influx of molecular (DNA sequence) data and new fossil discoveries.  It is now apparent that much of the biological and taxonomic diversity of ants is contained within the “formicoid clade” which comprises 14 of the 20 extant subfamilies and about 90% of all species.  Whether the remaining groups of extant ants (Leptanillinae and the poneroid subfamilies) represent a clade or a grade at the base of the ant tree remains unresolved.  The fossil record for crown group ants extends back to 90–100 mya.  Stem ants (sphecomyrmines, armaniines) were also present during this period.  Molecular divergence date estimates that take into account the fossil record of both ants and other Hymenoptera suggest that crown group ants arose ~115–135 mya.  Most of the extant ant subfamilies and genera are well defined morphologically and likely monophyletic, but there are some notable exceptions including the subfamily Cerapachyinae and several large and ambiguously delimited genera such as Pachycondyla.  Several tribes in the large subfamilies Formicinae and Myrmicinae also represent artificial assemblages.  Finally, while the species-level taxonomy of some ant genera is in a satisfactory state, taxonomic anarchy reigns in others, with numerous ill-defined species and many names of uncertain applicability.  Progress in this area of ant systematics will require sustained individual efforts, expansion of job opportunities, enlistment of new technologies, and a deeper understanding of the nature of ant species and the differences between them.
When half a glass of STAT3 is just not enough
Authors: Laurence Arian D. J. , Uhlig Holm H.
Published: October 2019

In this issue of Blood, Bocchini et al report a novel mechanism by which STAT3 mutations result in an unstable protein and give rise to a reduction in STAT3 signaling, suggesting that pathogenic mutations do not always confer dominant-negative effects via forming of nonfunctional STAT3 dimers but some may limit availability of total protein causing STAT3 haploinsufficiency.1
Molecular Pathways of Response to Recombinant Human Erythropoietin (rHuEpo) in Myelodysplastic patients Assayed with Macroarrays.
Authors: Agostino Cortelezzi , Gualtiero Colombo , Caterina Pellegrini , Ilaria Silvestris , Silvano Bosari , Giorgio Lambertenghi Deliliers , Nicola Fracchiolla S.
Published: October 2019

Recently, among myelodysplastic syndromes with IPSS low/intermediate 1 risk classes, promising results have been obtained in the treatment of anemia by high dosage recombinant human erythropoietin (rHuEPO) treatment, in the cases with low endogenous erythropoietin (EPO). We have reproduced these clinical results in the patients treated at our Institution. Based on these premises, in order to evaluate the molecular basis of the rHuEPO response in MDS patients, we investigated the differential gene expression in the bone marrow erythroid cells expressing glycophorin (Gly+) of EPO-responders (ER) and EPO-non responders (ENR) MDS patients, using commercially available macroarrays (Clontech), comparing gene level of expression with a baseline represented by a pool of erythoid cells isolated from normal bone marrow donors. We analysed 4 ER and 4 ENR MDS patients (RA and RARS). BM erythroid cells, after Ficoll density gradient, were separated by positive selection using an immunomagnetic procedure (MACS, glycophorin isolation kit; Miltenyi Biotech, Auburn, CA). The purity of Gly+ cells after magnetic immunosorting was tested by flow-cytometry and was superior to 97%, in all the cases. RNA extraction, cDNA synthesis and macroarrays hybridization and analysis were performed following manufacturer instructions (Clontech Laboratories, Palo Alto, CA). The data were validated on 4 differentially expressed genes by real time RT-PCR TaqMan technology, obtaining results that were superimposable to the macroarrays ones. Using a cut off of an at least two fold difference in gene expression, we obtained a differential pattern of expression in ER and ENR patients, BM Gly+ erythroid cells, with respect to normal donors. In particular, ER patients presented the up-regulation of interleukin-1 beta, apolipoprotein E precursor, CREB2, high mobility group protein (HMG-I), transducin beta-1 subunit and the down regulation of protein kinase MLK-3, cAMP-dependent phosphodiesterase (PDE43), IL-5R-alpha, caspase-9. On the other hand, in ENR group, we found a down regulation of proliferating cyclic nuclear antigen (PCNA), B-myb, prothymosin alpha, phenylethanolamine N-methyltransferase, thymosin beta-10 and an up regulation of homeobox protein HOX-A5, monocarboxylate transporter 1, interleukin-6, deoxyribonuclease II, sterol regulatory element-binding protein, metalloproteinase inhibitor 1 precursor, rap1 GTPase activating protein 1 (RAP1GAP). In conclusion, ER and ENR MDS patients erythroid cells present a different pattern of gene expression with respect to normal. These results may provide the basis for early testing of the genes differentially expressed in ER and ENR patients in order to elucidate their role in the prognostication of rHuEPO response in MDS patients with low endogenous EPO levels.
Telomere Length Reduction in Patients with Early Myelodysplastic Syndrome (MDS) Is Connected with Presence of Karyotype Abnormalities and with Shortened Survival and May Serve as an Additional Prognostic Factor.
Authors: Sieglova Zuzana , Cermak Jaroslav , Zilovcova Sona , Zizkova Hana , Michalova Kyra
Published: October 2019

The degree of telomere shortening was studied in 74 patients with primary MDS or acute myeloid leukemia arising from MDS (sAML) classified according to either FAB ( 30xRA, 3xRARS, 19xRAEB, 3xRAEB-T, 6xCMML, 13x sAML) or WHO classification (11xRA, 3xRARS, 17xRCMD, 2x5q-syndrome, 13xRAEB I, 6xRAEB II, 16x sAML). The telomere lengths were measured in mononuclear bone marrow cell fraction using Terminal Repeat Fragment method (TRF) based on non-radioactive chemiluminiscent detection of hybridization products obtained by digestion of high molecular weight DNA with Hinf I and RsaI restriction enzymes. In accordance with our previous studies performed on age-matched healthy controls, TRF shorter than 7.5 kbp was considered a significant marker of telomere reduction. A significant difference in TRF was found between RA/RARS patients and RAEB/RAEB-T/sAML patients according to FAB criteria ( P=0.048) and between RA/RARS/RCMD and RAEB/sAML patients according to WHO criteria (P=0.035). These results reflected a gradual shortening of telomeres in increased number of patients observed during progression of the disease as well as an inverse correlation between telomere length and number of bone marrow blasts (r = −0.427, P=0.048). Significantly shorter telomeres in a subgroup of patients with karyotype abnormalities (detected by routine cytogenetics and by FISH) in comparison to patients with normal karyotype were present in RA/RARS subtype according to FAB (P=0.042) and in RCMD subgroup according to WHO (P=0.010). Median survival and estimated 3 years survival in RA/RARS patients (FAB) with reduced telomere length were 25,3 months and 76% compared to 48,5 months and 86% in those with normal telomeres (P=0.01). In patients classified according to WHO, median survival and estimated 3 years survival were: RA/RARS+ shortened TRF - 27,0 months,82%, RA/RARS+normal TRF - 92,5 months,100%, RCMD+ shortened TRF - 19,0 months, 50%, RCMD+ normal TRF - 48,5 months, 86%. In patients with advanced disease (RAEB, RAEB-T, sAML), no significant differences were observed either in TRF between patients with normal or abnormal karyotype or in survival between patients with normal or reduced TRF. Since the reduction of telomere length in MDS patients probably represents later but important step in development of pathological clone leading to genetic instability and disease progression, the occurrence of shortened TRF in early MDS may be a consequence of an underlying complex disorder of hematopoiesis with potencially adverse outcome. Thus, measurement of TRF in patients with early disease may serve as an additional prognostic factor.
Phagocytic Signaling Molecules in Lipid Rafts of COS-1 Cells Transfected with FcγRIIA.
Authors: Mansfield Pamela J. , Hinkovska-Galcheva Vania , Borofsky Michael , Shayman James A. , Boxer Laurence A.
Published: October 2019

FcγRIIA-mediated phagocytosis involves a number of signaling proteins and lipids, which increasingly are viewed as localizing subcellularly in plasma membrane microdomains providing a framework for their interaction. COS-1 cells stably transfected with FcγRIIA were used as a model to demonstrate co-localization of several enzymes shown to be important in polymorphonuclear leukocyte (PMN) phagocytic signaling. Previously we developed a model wherein FcγRIIA engagement in PMNs resulted in activation of phospholipase D (PLD), producing phosphatidic acid, which is hydrolyzed to diglyceride, an activator of PKC. PKCδ and Raf-1 then activate the MAP kinase pathway and subsequently myosin to allow pseudopod formation. In COS-1 cells as in PMNs, PLD in the membrane fraction was activated during phagocytosis. COS-1 PLD was found almost exclusively in lipid rafts identified by the presence of caveolin, while two of its cofactors, RhoA and ARF1, were enriched in rafts. PKCδ and Raf-1 translocated to the plasma membrane, and were enriched in lipid rafts, reaching highest levels 5 to 10 min after phagocytosis was initiated. Rottlerin, a specific inhibitor of PKCδ, completely inhibited phagocytosis, suggesting that PKCδ regulates phagocytosis in COS-1 cells; however, translocation of PKCδ to rafts was not inhibited by rottlerin. Chelation of intracellular calcium with BAPTA-AM inhibited phagocytosis by only 25%, suggesting that PKCα or PKCβ, which both require calcium, were not important regulators of phagocytosis. A specific inhibitor of MEK (which is activated by Raf and activates MAP kinase), PD098059, inhibited phagocytosis by about 35%, suggesting that the MAP kinase pathway is involved in, but not the key factor required for, COS-1 cell phagocytosis. Extracellular signal-regulated kinase-2 (ERK2), a MAP kinase, was present in the raft fraction. In PMNs, phagocytosis and activation of MAP kinase are inhibited by exogenous ceramide, and endogenous ceramide levels increase during phagocytosis, indicating that FcγRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcγRIIA that had been mutated in the ITAM region (Y282F and Y298F), rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAP kinase was activated normally, thus these processes did not require actual ingestion of particles. Previously we showed that ceramide 1-phosphate (C1P), the product of ceramide kinase (CERK), promotes membrane fusion in PMNs. Here we found that C1P increased in COS-1 cells during phagocytosis. CERK was found to be enriched in lipid rafts, translocating during phagocytosis. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.
Effects of the Genetic Background on the Myelofibrosis Induced by the GATA-1Low Mutation.
Authors: Migliaccio Anna Rita , Martelli Fabrizio , Panetta Barbara , Alfani Elena , Migliaccio Giovanni , Gatta Valentina , Di Baldassarre Angela , Pancrazi Alessandro , Bogani Costanza , Paoletti Francesco , Vannucchi Alessandro M.
Published: October 2019

We have reported that CD1 mice harboring the GATA-1low mutation develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, that manifests itself at 12 months of age with marrow fibrosis and with anemia, increased stem/progenitor cell trafficking and extramedullary hemopoiesis in the liver from 15 months onward (Vannucchi et al., Blood100: 1123–1132, 2002). The trait is expressed by homozygote and heterozygote females alike with 100% penetrance. The aim of these experiments was to assess the eventual effects of the genetic background on the severity of the myelofibrotic trait induced by the GATA-1low mutation. To harbour the GATA-1low in different backgrounds, it was exploited the fact that the GATA-1 gene is located on the X chromosome. Therefore, normal C57BL/6 and DBA2 females were crossed with GATA-1low males. Their heterozygote F1 female offspring (50% C57BL/6 or DBA/2, respectively) was backcrossed with wild type males (either C57BL/6 or DBA/2, as appropriate) and the F2 offspring (75% C57BL/6 or DBA72) analysed at 12 (by histological criteria) and at 15 months (by cytofluorimetric criteria) for the expression of the myelofibrotic trait. The results obtained are summarized in the Table below. In summary, all of the mice (independently from the background) developed marrow fibrosis while aging. However, a great variegation was observed in the progression of the disease in the different strains. It is proposed that such a variegation may represent a model to investigate the extreme variability of the clinical picture of the disease in humans.

Summary of the GATA-1low trait in the different backgrounds

C57BL/6 CD1 DBA2 Perinatal Mortality yes no no Thrombocytopenia yes yes yes Myelofibrosis Anemia no 15 months no Tear drop poikilocytes 12 months 15 months no Marrow fibrosis yes yes yes Osteosclerosis yes limited no Progenitor cell trafficking no yes no Extramedullary hemopoiesis no yes no
Mitochondria and Caspase-Independent Cell-Death Triggered by GCS-100, a Novel Carbohydrate-Based Therapeutic in Multiple Myeloma (MM) Cells.
Authors: Chauhan Dharminder , Li Guilan , Podar Klaus , Hideshima Teru , Ishitsuka Kenji , Catley Laurence , Sattler Martin , Tai Yu-Tzu , Burger Renate , Richardson Paul , Chang Yan , Schindler Joanne , Carver Bradley , Anderson Kenneth C.
Published: October 2019

GCS-100 is a carbohydrate-based therapeutic in clinical development for the treatment of cancer. Here we show that GCS-100 directly targets MM cells and their bone marrow (BM) microenvironment. Treatment of various MM cell lines, including those resistant to conventional anti-MM agents such as, dexamethasone, melphalan, or doxorubicin, with GCS-100 triggers growth arrest and apoptosis. Examination of purified patient MM cells demonstrated similar results, without significant toxicity against normal peripheral blood mononuclear cells. Importantly, GCS-100 decreases viability of bortezomib-resistant MM patient cells. GCS-100 blocks MM cell growth induced by both adhesion to bone marrow stromal cells (BMSCs) and related cytokine secretion, thereby inhibiting growth and survival in MM cells conferred by the BM microenvironment. GCS-100 overcomes drug-resistance conferred by anti-apoptotic protein Bcl-2 and heat shock protein-27. Although GCS-100 induces MM cell death evidenced by morphological and DNA fragmentation analysis, no activation of caspases-8, -9 and -3 was noted at the IC50 dose. Conversely, the pan caspase inhibitor Z-VAD-fmk failed to abrogate GCS-100-induced apoptosis. Furthermore, GCS-100 does not alter mitochondrial membrane potential, suggesting a mitochondria- and caspase-independent cell death-signaling pathway. These data provided the rationale for combining GCS-100 with an agent that specifically triggers mitochondrial apoptotic signaling in MM cells. For example, combining a conventional agent dexamethasone, a known inducer of Smac release from mitochondria and activator of caspase-9/3 cascade, with GCS-100 shows additive anti-MM activity; and combining GCS-100 with an antagonist to mitochondrial peripheral benzodiazepine receptor PK-11195 shows synergistic anti-MM activity, associated with profound mitochondrial cell-death signaling and activation of caspase-9 and -3. Collectively, these findings lay the framework for clinical evaluation of GCS-100, either alone or in combination with dexamethasone or PK-11195, to overcome drug resistance and improve patient outcome in MM.
Ascorbic Acid Overcomes Drug Resistance in Myeloma and Significantly Increases the Anti-Myeloma Effects of both Arsenic Trioxide and Melphalan in Vitro and in Vivo.
Authors: Campbell Richard A. , Chen Haiming , Zhu Daocheng , Santos Janice C. , Bonavida Benjamin , Pang Shen , Said Jonathan , Berenson James R.
Published: October 2019

Glutathione levels have previously been shown to be associated with the development of resistance to a variety of anti-myeloma therapies. Ascorbic acid (AA) depletes intracellular glutathione levels which, in turn, should increase the sensitivity of tumor cells to anti-myeloma agents such as arsenic trioxide (ATO) and melphalan. To determine the synergistic effects of combining AA, with ATO and/or melphalan, we evaluated the effects of these combinations with MTT assays on myeloma cell lines in vitro and using our severe combined immunodeficient (SCID)-hu murine myeloma models. We determined the synergistic effects of combining AA with ATO and/or melphalan on the myeloma cell lines RPMI8226, 8226/dox, U266, and U266/dox in vitro. MTT assays demonstrated marked synergistic anti-proliferative effects of AA at 10 mM when added to these cell lines in the presence of ATO concentrations ranging from 5x10−5 M – 5x10−9 M, and melphalan concentrations ranging from 3x10−5 M – 3x10−9 M. In order to provide further evidence for the clinical relevance of these synergistic effects of AA, we investigated the potential of AA to increase the efficacy of current anti-myeloma therapies in our SCID-hu murine model of human myeloma LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Twenty-eight days following implantation, mice then received treatment intraperitoneally (IP) with either AA (300 mg/kg) daily x5/week, ATO (1.25 mg/kg) daily x5/week, or melphalan (3.0 mg/kg) x1/week, or the combination of these agents. AA, ATO, and melphalan alone have no anti-myeloma effects at these doses, whereas AA+melphalan results in significantly decreased tumor burden and paraprotein levels. The most profound anti-myeloma effects were observed in animals treated with all three drugs together. These data show not only the additional synergistic anti-myeloma effects of AA on both ATO and melphalan in vitro but for the first time suggest that these effects are also present in vivo. This provides the rationale for combining AA with these agents in myeloma patients with resistant disease. In support of this, early results of clinical trials using the combination of AA, ATO and low doses of oral melphalan are promising.
Rituximab, Gemcitabine and Oxaliplatin (R-GEMOX): A Promising Regimen for Refractory/Relapsed B-Cell Lymphoma.
Authors: El Gnaoui Taoufik , Dupuis Jehan , Joly Bertrand , Belhadj Karim , Rahmouni Alain , Copie-Bergman Christiane , Allain Antoine , Tabah-Fish Isabelle , Reyes Felix , Haioun Corinne
Published: October 2019

Background : High-dose therapy with autologous stem cell support (HDT) is an established treatment for chemosensitive relapse in aggressive lymphoma. However, not all patients are candidates for HDT because of age, comorbidities or previous HDT. Effective and well tolerated salvage therapies with minimal toxicities are thus needed.

Methods : We designed the R-GEMOX regimen, with rituximab 375mg/m2 d1, gemcitabine 1000 mg/m2 d2 and oxaliplatin 100 mg/m2 d2 (recycling on d15). Between January 2002 and April 2004, 31 patients with refractory/relapsing B-cell lymphoma not eligible for HDT were enrolled in an open unicenter pilot study whose primary objective was to determine the overall response rate (ORR) after 4 cycles (induction phase) of R-GEMOX. Patients were planned to receive 8 cycles if a good response (at least PR) was observed after 4 cycles. Median age was 63 years (range: 43–77) and histological subtypes were : diffuse large B-cell lymphoma (n=22), follicular (n=6) and mantle cell (n=3). Prior treatment included anthracyclin in 30 patients (98%), rituximab in 16 (52%) and HDT in 8 (26%). International prognostic index at enrollment was ≥ 2 in 13 patients (42%). The median number of prior treatments was 2 (range : 1 to 5) and 9 patients had received at least 3 prior regimens.

Results : 226 cycles were given. The dose administered was 100% of the intended dose for the three drugs in all patients but 6, for whom the dose of oxaliplatin was reduced due to neurotoxicity (n=5) or preexisting renal insufficiency (n=1). The median number of cycles per patient was 8 (range: 2–8).Three patients progressed during the induction phase. After 4 cycles, observed responses were : 6 CR, 8 CRu, 12 PR and 2 failures resulting in an ORR of 84 %. At the end of treatment, among the 26 responder-patients at 4 cycles, 23 patients achieved CR/CRu and one patient progressed. As of August 2004, 22 patients are alive, 17 in continuous complete remission and 5 with evolutive disease. NCIC grade 3–4 neutropenia and thrombocytopenia were reported in 49% and 23% of the cycles. Six patients developed a grade 4 infection during one cycle. There was no renal toxicity.

Conclusion : The R-GEMOX regimen shows promising activity with an acceptable toxicity. It is currently evaluated in an ongoing multicentric phase II study on diffuse large B-cell lymphoma patients at first relapse. (=69.03% taille max abstract)
Treatment Response to Single Agent Sphingosomal Vincristine in Patients with Relapsed and/or Refractory Diffuse Aggressive Non-Hodgkin’s Lymphoma (NHL): Subgroup Analyses.
Authors: Rodriguez Maria Alma , Winter Jane N.
Published: October 2019

There is a substantial need for new effective therapies for patients with recurrent or refractory diffuse aggressive NHL. Efficacy and safety of sphingosomal vincristine (SV), a novel formulation of vincristine encapsulated in sphingomyelin liposomes or ‘sphingosomes’, were evaluated in a multinational study in aggressive NHL patients with multiple unfavorable prognostic factors. Methods: 119 pts with relapsed or refractory aggressive NHL were enrolled. SV was administered at 2 mg/m2 without capping the dose as a 1 hr IV infusion every 2 weeks. The objective response rate (ORR) and 95% CI for the proportions and difference in proportions were calculated for the following subgroups: by age (≤ 60 vs. > 60); sensitive disease vs. resistant (refractory or relapsed within 3 months of prior treatment); IPI score 0–2 vs. 3–5; prior therapies 2 vs. >2; bone marrow involvement at baseline; and prior ABMT. Results: ORR in the entire group was 25% (7% CR/Cru and 18% PR) (ASH Abstr. 2353, 2003).

No of Prior Regimens/Disease Sensitivity ORR % aone patient had 1 prior regimen Sensitive (n=39)  Resistant (n=80) 41%  18% 2 Regimens (n=24)a  -Sensitive (n=11)  -Resistant (n=13) 46%  64%  31% >2 Regimens (n=95)  -Sensitive (n=28)  -Resistant (n=67) 20%  32%  15%

Other Subgroup Analyses ORR % bnot statistically significant as 95% CI on the difference in ORR includes zero Bone Marrow Involved (n=20)  Not involved (n=93) 10%  27% Ageb ≥60 (n=60)  >60 (n=59) 25%  25% IPIb 0–2 (n=59)  3–5 (n=58) 19%  31% Prior ABMTb Yes (n=39)  No (n=80) 26%  25%

Conclusion: Single-agent SV was active in heavily pretreated patients with relapsed and/or refractory NHL. A multivariate analysis showed that the two strongest predictors for response were number of prior regimens and sensitivity to the last therapy. The highest level of activity was observed in patients with sensitive disease and 2 prior regimens (ORR 64%). Response to SV in older patients was the same as in younger patients. Prior ABMT did not impact response with ORR being similar in both groups. SV was well tolerated and the safety profile was similar to standard vincristine despite a doubling of dose intensity.
The Induction of Apoptosis in Lymphoma Cells by Methylseleninic Acid and Selenodiglutathione.
Authors: Last Kim , Perry Jackie , Maharaj Lenushska , Strauss Sandra , Davies Andrew , Skoulakis Spyros , Fitzgibbon Jude , Lister Andrew , Joel Simon
Published: October 2019

Introduction. We have previously demonstrated that presentation serum selenium predicted not only for response to treatment and duration of first remission, but also importantly for overall survival in aggressive non-Hodgkin’s lymphoma (Journal of Clinical Oncology2003, 21:2335–2341). To investigate a possible causal association as the explanation for these findings we have studied the activity of 2 selenium species, methylseleninic acid (MSA) and selenodiglutathione (SDG). We provide the first report of the activity of these two compounds in lymphoma, using a panel of human cell-lines (CRL, DHL4, SUD4 and DoHH2) and a primary human lymphoma culture system.

Methods. The effects of 3-day incubations with MSA or SDG on cell proliferation and cell viability (n = 3–6) were determined by Trypan blue exclusion assay. Cell cycle distribution (including a sub-G1 apoptotic fraction), generation of reactive oxygen species (ROS) and PI/annexin staining were analysed by flow cytometry. Apoptosis was further investigated by western blotting for poly-ADP ribose polymerase (PARP) and specific caspase (8 and 9) cleavage. The activity of MSA and SDG was also investigated in a primary lymphoma culture system utilising a feeder layer of CD40 ligand expressing CHO cells irradiated prior to plating primary lymphoma cells.

Results. Both MSA and SDG demonstrated cytostatic and cytotoxic effects in all 4 cell-lines studied. The EC50 values ranged from 1.0 – 10.2 μM, with the exception of the % viability EC50 for MSA in DHL4 cells (166 μM). Flow cytometry revealed cell death was associated with an increase in the sub-G1 (apoptotic) fraction, without an obvious preceding G1, S or G2/M cell cycle arrest. Apoptosis was confirmed by flow cytometric analysis of PI/annexin dual labelled cells, and was associated with PARP cleavage and involvement of both caspase 8 and 9. SDG exposure resulted in marked increases in ROS, particularly in CRL and SUD4 cells (3-5-fold increase after 30 minutes exposure to the day-3 viability EC50 concentration). The addition of n-acetyl-cysteine reduced the cytotoxic activity of SDG, but not MSA, confirming that ROS contribute to the effects seen with SDG. In 5 different 2-day primary lymphoma cultures (n = 4–6) MSA and SDG showed a concentration dependent reduction in % viability, with day-2 EC50 values of approximately 30 μM for MSA and 10 μM for SDG.

Conclusions. We have demonstrated for the first time the cytostatic and cytotoxic activity of two selenium species, MSA and SDG, in human lymphoma cell-lines and primary lymphoma cells and their induction of caspase mediated apoptosis. These data suggest that selenium species may play a causal role in the success of conventional chemotherapy in aggressive lymphoma. Selenium compounds merit further investigation in the clinical management of this disease.
Vaccination with Leukemia Cells Expressing Cell-Surface-Associated GM-CSF Blocks Leukemia Induction in Immunocompetent Mice.
Authors: Ling Xiaoyang , Wang Yan , Arlinghaus Ralph B.
Published: October 2019

The fundamental basis for immunotherapy of leukemia is that leukemia cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant of leukemia cells. To overcome this immune tolerance, we transduced WEHI-3B mouse monocyte leukemia cells (1) with a transmembrane form of GM-CSF (tmGM-CSF). The tmGM-CSF was constructed using the pDisplay vector for cell-surface targeting (Invitrogen) into the pLOX lentivirus gene transfer vector (2). After infection of WEHI-3B cells with a recombinant lentivirus encoding tmGM-CSF, nearly all the transduced cells expressed tmGM-CSF on the cell-surface, as determined by flow cytometry analysis using anti-GM-CSF. To determine whether vaccination with tmGM-CSF expressing WEHI-3B cells would prevent leukemia formation, immunocompetent BALB/c mice were immunized with lethally-irradiated WEHI-3B cells (106, 3 times 7 day intervals), which express tmGM-CSF, prior to challenging vaccinated mice with WEHI-3B cells (5x104) that express GFP as a marker. 100% of vaccinated mice were protected from leukemia. Non-vaccinated mice succumbed to leukemia within 50–55 days. Vaccination of mice with lethally-irradiated WEHI-3B cells expressing CD40L protected 80% of the mice from leukemia. In contrast, mice immunized with lethally-irradiated WEHI-3B/GFP cells lacking tmGM-CSF were not protected. Mice vaccinated three times at 5,12, 19 days after challenge with WEHI-3B/GFP cells had a significant increase in survival in that 60% of mice were alive and healthy at 16 days (to this date) after all control non-vaccinated mice had died. Similar vaccine studies were performed with BCR-ABL (b3a2)+ 32D cells (106) in immunocompetent C3H/HeJ mice (3). These mice die of leukemia within 35 days. After infection of BCR-ABL+ 32D cells with the lentivirus encoding tmGM-CSF/GFP, tmGM-CSF was expressed on the cell-surface. The C3H/HeJ mice challenged with BCR-ABL+32D/GFP cells (106) showed a significant level of protection by vaccination with lethally-irradiated tmGM-CSF+ 32D BCR-ABL cells (106, 2 times at 7 day intervals); 40% of the vaccinated mice remained healthy; all non-vaccinated mice died of leukemia. There was a significant difference in survival (P=0.03) between the vaccinated and non-vaccinated groups. Interestingly, the spleens of vaccinated C3H/HeJ mice that died of leukemia at the same time as non-vaccinated mice approached normal size whereas non-vaccinated mice had enlarged spleens. Our findings suggest that over-expression of cell-surface tmGM-CSF in leukemia cells can overcome immune tolerance, allowing the immune system to efficiently recognize and destroy the leukemia cells, providing extended survival of vaccinated mice. Because significant protection from death was achieved by vaccination after challenge with leukemia cells, tmGM-CSF expression in leukemia cells has potential as a therapeutic strategy for treatment of leukemia.
Anti-Von Willebrand Factor Antibody Is Responsible for Lower Levels of Plasma vWF in Blood Group O Individuals.
Authors: Dai Letian , Cotton Shawn , Macartney Alistair , Savidge Geoffrey , Alhaq Anwar
Published: October 2019

Plasma levels of vWF are known to be influenced by ABO blood groups, although the mechanism remains unresolved. Group O individuals have a significantly lower level of plasma vWF than those with group A, B or AB. This relative lower level of plasma vWF may result in a bleeding tendency and a shorter half-life of infused factor VIII in group O individuals. The formation of immune complexes between vWF and autoantibodies has been shown to accelerate vWF clearance from plasma in acquired von Willebrand syndrome. However, so far no evidence has been presented that the presence of autoantibodies against vWF is involved in lowering plasma level of vWF in group O individuals. In the present study, plasma samples were obtained from 199 healthy blood donors of blood group O (50), group A (50), group B (49), and group AB (50). A time-resolved fluorescence immunoassay (TRFIA) was developed to detect anti-vWF IgG in plasma samples. Briefly, 100 μl of diluted plasma was loaded on to duplicated vWF-coated and untreated control wells of a microplate. After incubation and washing, 100 μl of Europium-labeled anti-human IgG conjugate (1:500 dilution) was added to the plate to detect vWF IgG. The time-delayed fluorescence was then measured with a Victor microplate reader (PerkinElmer, Turku, Finland). The fluorescence counts of the control wells were subtracted from those of the vWF-coated wells. The results show that anti-vWF IgG was present in all four blood groups (Table 1). Of these blood groups, group O had the highest anti-vWF IgG level with 9.8 x 105 fluorescence counts, which was 2.7- to 3.5-fold higher than that of group A, B or AB. There was a significant difference in the anti-vWF IgG levels between group O and the rest of group A, B or AB. Quantitative analysis of plasma vWF by ELISA showed that the concentration of plasma vWF of group O was 29 to 35% lower than that of group A, B or AB (Table 1). These results suggest that TRFIA is a sensitive assay for detection of anti-vWF IgG in plasma samples, and the presence of the high level of anti-vWF Ig G in group O individuals may be responsible for lowing plasma vWF by acceleration of vWF clearance.

Anti-VWF IgG levels and vWF concentrations in diffeent ABO blood groups

Group O (n=50) Group A (n=50) Group B (n=49) Group AB (n=50) Data are presented as a mean ± SD. *P<0.01 compared with group A, B or AB. +P<0.01 compared with group A,B or AB. Anti-vWF IgG (x 105 Fluorescence counts) 9.8 ± 6.9* 2.8 ± 2.8 3.1 ± 3.0 3.6 ± 3.5 Concentration of vWF (% of normal controls) 116 ± 42+ 165 ± 46 165 ± 42 179 ± 49
Increase of CD4+CD25high Regulatory T Cells in the Peripheral Blood of Acute Myeloid Leukemia Patients.
Authors: Wang Xingbing , Yao Junxia , Zheng Jine , Liu Jun , He Yanli , Li Xiaoqing , Yu Jingming , Wei Jun , Yang Jin , Liu Zhongping , Huang Shiang
Published: October 2019

PURPOSE: Although immunosuppression has long been recognized in acute myeloid leukemia (AML), the underlying basis for the lack of an effective immune response against the tumor remains unclear. T cells constitutively expressing both CD4 and CD25 are essential for maintenance of self-tolerance and therefore have been referred to as regulatory T cells (Treg). Experimental tumor models in mice revealed that Tregs are potent inhibitors of an antitumor immune response. The purpose of the current study was to demonstrate the possible involvement of CD4+CD25high regulatory T cells in immune system impairment in patients with acute myeloid leukemia.

METHODS: The frequency and phenotypes of CD4+CD25high T cells in the peripheral blood of 36 patients suffering from acute myeloid leukemia and from 15 healthy controls were determined by flow cytometry. To assess the functional activity of CD4+CD25high cells, CD4+CD25high or CD4+CD25− cells were purified from PBMCs by sorting with FACSVantage. The immunoregulatory properties of CD4+CD25high and CD4+CD25− T cells were characterized by proliferation and suppression assays. Also, cytokine production after in vitro stimulation was examined in CD4+CD25high and/or CD4+CD25− T cells isolated from AML patients. In addition, the frequency of apoptotic or proliferating T cells in CD4+CD25high T cells was determined by 7AAD or ki67 binding cells.

RESULTS: Compared with healthy volunteers, patients with acute myeloid leukemia had a higher proportion of CD4+CD25high T cells (4.1±1.8% vs 2.0±0.5%) in peripheral blood with characteristics of Tregs, i.e., they are CD45-RA(−), CD69(−), CD45-RO(+), CD95(+), and intercellular CTLA-4(+), and secreted TGF-, TNF-α and IL-10 but did not secrete IL-2, IL-4, IL-5 or IFN-. When cocultured with CD4+25− cells, CD4+CD25high T cells potently suppressed their proliferation through a dose-dependent manner, thus behaving as Treg. In addition, In order to explain the increased prevalence of CD4+CD25high T cells in AML patients, we examined the apoptosis and proliferation variations of CD4+CD25high T cells in AML patients. Notably, in AML patients, both apoptosis and proliferation of CD4+CD25high T cells were significantly higher than that did normal donor.

CONCLUSIONS: we provide evidence of an increased pool of CD4+CD25high regulatory T cells in the peripheral blood of AML patients with potent immunosuppressive features, which maybe due to the proliferation of CD4+CD25high T cells. This finding suggests that the use of immunomodulatory therapy to treat patients with AML may be an effective strategy.
RUNX1-ETO Stimulates Wnt Signaling by Inhibiting the Function of ETO Family Member Proteins.
Authors: Moore Amy , Tahinci Emilios , Lee Ethan , Hiebert Scott
Published: October 2019

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myelogenous leukemia (AML). This translocation generates a fusion protein, RUNX1-ETO, consisting of the N-terminus of RUNX1 fused to a nearly full-length ETO protein. The RUNX1-ETO fusion protein stimulates the expression of genes that are regulated by Wnt signaling. The Wnt signaling pathway plays a key role in embryonic development and aberrations to this pathway are frequently involved in tumor formation. Therefore, we sought to define the molecular mechanism by which RUNX1-ETO may stimulate Wnt signaling. We have demonstrated that the ETO family member, Mtgr1, functions as a corepressor for TCF4 and that the levels of the TCF-regulated gene, c-Myc, are upregulated in Mtgr1-null mice. Here we show that the Xenopus homolog of Mtgr1, XETOR, can impair Wnt signaling and induce ventralization in a Xenopus axis duplication assay, a classical assay used to define the hierarchy of components in the Wnt pathway. Specifically, microinjection of in vitro transcribed XETOR mRNA was performed in the marginal zone of both dorsal blastomeres at the 2 to 4 cell stage with increasing amounts of XETOR. Embryos were monitored through stage 26. Compared to control embryos, the embryos injected with XETOR mRNA were ventralized and failed to develop head structures. Conversely, although each of the ETO family member proteins associated with TCF4, RUNX1-ETO failed to bind to TCF4 in co-immunoprecipitation experiments. Mtgr1 was originally identified as a RUNX1-ETO-associated protein. Therefore, we tested whether the fusion protein impairs the action of Mtgr1 as a co-repressor for TCF4. RUNX1-ETO associated with Mtgr1, and Mtgr1 failed to associate with TCF4 when RUNX1-ETO was co-expressed. Thus, RUNX1-ETO appears to stimulate TCF-dependent transcription by interfering with the action of the ETO family of transcriptional corepressors.
Egr-1 Abrogates the Block Imparted by c-Myc on Terminal M1 Myeloid Differentiation.
Authors: Hoffman Barbara , Shafarenko Marianna , Liebermann Dan A.
Published: October 2019

Both deregulated growth and blocks in differentiation cooperate in the multistage process of leukemogenesis. Thus, understanding functional interactions between genes that regulate normal blood cell development, including cell growth and differentiation, and how their altered expression contributes to leukemia, is important for rational drug design. Previously, we have shown that the zinc finger transcription factor Egr-1 plays a role in monocytic differentiation. Ectopic expression of Egr-1 in M1 myeloblastic leukemia cells was observed to activate the macrophage differentiation program in the absence of the differentiation inducer interleukin 6 (IL-6), and to promote terminal differentiation in its presence. In addition, we have shown that deregulated expression of the proto-oncogene c-myc blocks the myeloid terminal differentiation program. Here we show that restoring expression of Egr-1 in M1 cells that express deregulated c-Myc abrogates the c-Myc block in terminal differentiation, resulting in cells that undergo functional macrophage maturation. Interestingly, it was shown that the ability of Egr-1 to override the c-Myc block in terminal differentiation occurred in the absence of growth arrest and cell adhesion, resulting in cells with the characteristics of functionally mature macrophages. As with normal macrophages, the cells also underwent programmed cell death. It was also observed that Egr-1 diminished the aggressiveness of M1myc leukemias, and abrogated the leukemic potential of M1myc cells treated with IL-6. Taken together, these observations demonstrate that Egr-1 behaves like a tumor suppressor, suggesting that, on the one hand, genetic lesions which alter Egr-1 expression can cooperate with deregulated c-Myc in exacerbating the leukemic phenotype and, on the other hand, that enhancement of Egr-1 expression, or its downstream targets, can overcome blocks in differentiation due to deregulated c-Myc, as well as its associated leukemias These findings suggest that Egr-1 and/or Egr-1 targets may provide important tools for differentiation therapy in certain leukemic phenotypes
Association between Neuroleptics and Venous Thromboembolism: A Case-Control Study.
Authors: Lacut Karine , Le Gal Gregoire , Oger Emmanuel , Mottier Dominique
Published: October 2019

Background:

Preliminary reports suggest that use of antipsychotic drugs is associated with an increased risk of venous thromboembolism (VTE), but others did not confirm these results.

Objective:

To evaluate the relationship between antipsychotic drugs and VTE.

Design:

Case-control study (EDITH) designed to investigate genetic and environmental risk factors of VTE.

Setting:

Brest University Hospital.

Participants:

857 patients consecutively hospitalized for a documented venous thromboembolic event were included between May 2000 and May 2004. Controls were matched on age, sex and the main risk factors of venous thromboembolism (cancer, surgery, pregnancy…).

Results:

The mean age of patients was 67.7 year. No significant difference was found between cases and controls concerning the main characteristics, except for smocking and body mass index. Among cases, 89 (10.4%) were current users of neuroleptics compared to 35 (4.8%) among controls. Current use of neuroleptics was associated with a significant increased risk of venous thromboembolism (OR = 2.32, 95% CI: 1.55–3.48). Excluding neuroleptics used for non psychiatric disorders, and after adjustment on the main confounding factors, this association remained significant (OR = 3.48, 95% CI: 2.00–6.04). No difference was found between the different chemical categories of neuroleptics, but the number of patients in some groups had limited statistical power to demonstrate significant differences. Biological mechanisms of action have been proposed to explain this relation. Analyses are ongoing for anti-phospholipid antibodies and homocysteine.

Conclusion:

In this case-control study of hospitalized patients, neuroleptics use was associated with a significant increased risk of venous thromboembolism. These results are concordant with previous reports. Nevertheless, further investigations are needed to explain wich mechanisms may be involved in such association and before use of neuroleptics can be definitely considered as risk factor for venous thromboembolism.
Regulation of Integrin Expression and Activation by VEGF in Human Brain Microvascular Endothelial Cells: Implication of α6 Integrin in Angiogenesis.
Authors: Lee Tae-Hee , Avraham Hava Karsenty , Li Huchun , Kennel Stephen J. , Avraham Shalom
Published: October 2019

The precise role of vascular endothelial growth factor (VEGF) in the regulation of integrins is not well elucidated due to their high redundancy. Here, we examined the effects of VEGF on the expression and activation of integrins in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and Ribonuclease (RNase) protection assays which cover most of the known integrins, we observed that VEGF significantly upregulated the mRNA expression of α1, α2, and α6 integrins in HBMECs. While VEGF was reported to induce α1 and α2 integrins, the observation of α6 integrin induction by VEGF is novel. Using small interfering RNA (siRNA) oligonucleotides for α6 integrin, we observed downregulation of the cell surface expression of α6 integrin in HBMECs. This downregulation resulted in inhibition of both HBMEC capillary morphogenesis and of the VEGF-induced adhesion and migration of the cells. VEGF also induced the activation of α6 integrin in the HBMECs. Functional blocking of α6 integrin by its specific antibody led to inhibition of VEGF-induced adhesion and migration as well as of in vivo angiogenesis, and significantly suppressed tumor angiogenesis and breast carcinoma cell growth in vivo. These results indicate that VEGF can modulate the in vitro angiogenesis of HBMECs via increased expression and activation of the α6 integrin, and that this integrin participates in VEGF-driven angiogenesis in vivo.
Vascular Endothelial Cadherin-Expressing Cells Involved in Both Developmental and Adult Neovascularization.
Authors: Kogata Naoko , Arai Yuji , Hashimoto Kazuaki , Ogawa Minetaro , Mochizuki Naoki
Published: October 2019

Vessel formation, including vasculogenesis and angiogenesis, is thought to be directly mediated by adhesive molecules between vascular endothelial cells and adjacent cells. Vascular endothelial cadherin (VE-cadherin, CD144) is a adhesive molecule exclusively expressed in adherens junctions of cultured endothelial cells. VE-cadherin transcripts have been detected at first in mesodermal cells. However, no work has focused on which cell type expresses VE-cadherin during vessel formation and after vessel stabilization. To monitor VE-cadherin in vivo, we generated transgenic mice expressing a VE-cadherin promoter-driven Cre (VE-cad-Cre). By crossing VE-cad-Cre with loxP-based EGFP reporter mice (CAG-CAT-EGFP) and LacZ reporter mice (cAct-XstopX-LacZ), we obtained two double transgenic mice; one expressing EGFP (VE/EG) another expressing LacZ (VE/Z) dependently upon Cre driven by VE-cadherin promoter. Both reporter expression was increased in developing vasculature during embryogenesis, while it was decreased in postnatal vessel maturation. Intriguingly, LacZ reporter was found in the budding cells and lining cells of the dorsal aorta in the aorta-gonad-mesonephros region, suggesting that VE-cadherin-expressing cells belongs to hemangioblasts. Consistently, although VE-cadherin and its mRNA was detected in vasculature by immunohistochemistry and in situ hybridization, respectively, during embryogenesis, it was not detected in maturated vessels. These results indicated that VE-cadherin promoter activity reflects the endogeneous VE-cadherin expression and that VE-cadherin promoter is activated during developmental vasculogenesis and angiogenesis. To test whether the VE-cadherin promoter is re-activated in postnatal neovascularization, we used two models; a VEGF-induced cornea angiogenesis model and a myocardial ischemia model. VEGF implanted in the VE/EG eyes promoted the neovascularization visualized by EGFP expression. Myocardial ischemia in which the coronary artery was banded triggered the VE-cadherin promoter, thereby inducing reporter expression in vascular cells (endothelial cells and mural cells) of undamaged region and in infiltrated cells of damaged region. Furthermore, by flow cytometric analyses, we confirmed that myocardial ischemia re-activated the VE-cadherin promoter of both bone marrow cells and peripheral blood cells, as indicated by the remarkable increase of EGFP-positive cells. These data show that the VE-cadherin promoter is re-activated in pre-existing vascular cells and infiltrated cells during adult neovascularization. Collectively, VE-cadherin-expressing cells participates in both developmental and postnatal vasulogenesis and angiogenesis.