bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of a Toll Receptor-7 Agonist (S28690) on the Immunogenicity of Chronic Lymphocytic Leukemia Cells.
Authors: Hicks Lisa , Shi Y. , Mena J. , Hammond C. , Tomic Y. , Tomai M. A. , Miller R. L. , Spaner D. E. 
Published: October 2019

Abstract
Background: Chronic lymphocytic leukemia (CLL) cells are weakly immunogenic, a property that may contribute to disease progression and inhibit the effectiveness of immunotherapies such as vaccines. Low surface expression of co-stimulatory molecules contributes to this poor immunogenicity. CLL cells express Toll-Like-Receptor-7 (TLR7), a powerful modulator of innate immunity. TLR7 agonists may be capable of enhancing the immunogenicity of CLL cells and thereby increasing T cell mediated killing of CLL.
Methods: Circulating CLL cells were isolated directly from consenting patients by negative selection. TLR7 mRNA expression by CLL cells was demonstrated by RT-PCR. CLL cells were then incubated with S28690 (a TLR7 agonist), or with a negative control for 24–72h. Expression of the costimulatory molecules CD80, 83, 86, and 54 was determined by flow cytometry pre and post-incubation. Experiments were repeated in the presence of a NFkB inhibitor (dexamethasone), a p38 MAPK inhibitor, and a protein kinase C agonist (PDB). The effects of S28690 on phosphorylated-IkB and phosphorylated-STAT3 levels were measured by immunoblotting. The capacity of S28690-incubated CLL cells to stimulate T cell proliferation and killing was determined in mixed lymphocyte responses.
Results: All tested CLL samples (n=20) expressed TLR7 mRNA, while Jurkat cells (T cell origin) did not. After incubation with S28690, CD80, 83, 86, and 54 surface expression increased on all CLL samples tested. The relative increase varied from 4 to 9-fold and was positively correlated with CD38 expression. NF-kB and p38 inhibitors decreased the effects of S28690 on co-stimulatory molecule expression while PDB amplified the effect. After incubation with S28690, IkB and STAT3 phosphorylation increased in CLL cells. S28690-incubated-CLL cells were able to stimulate moderate T cell proliferation, but did not increase T cell mediated killing of CLL cells. However, CLL cells incubated with both S28690 and PDB (a PKC agonist), exhibited much lower amounts of phosphorylated STAT3, triggered marked T cell proliferation, and stimulated T cell mediated killing of CLL cells.
Conclusions: S28690 (a TLR7 agonist) causes increased expression of co-stimulatory molecules by CLL cells in vitro and transforms CLL cells into moderate stimulators of T cell proliferation. The effects of S28690 are synergistic with PKC agonists, potentially as a result of S28690-mediated NFkB activation and concurrent PKC-mediated inhibition of STAT3. These findings may find clinical application in immunotherapeutic approaches to CLL.
The Sesquiterpene Lactone Parthenolide Rapidly and Selectively Induces Apoptosis of B-Chronic Lymphocytic Leukemia Cells.
Authors: Steele Andrew J. , Casanova Anouska A. , Hoffbrand A. Victor , Mehta Atul B. , Ganeshaguru Kanagasabai , Jones Dylan T. , Wickremasinghe R. Gitendra 
Published: October 2019

Abstract
B-chronic lymphocytic leukaemia (CLL) is the most frequent leukaemia in the western world. CLL is incurable using conventional therapy. While the disease is controlled by treatment with chlorambucil (CHL) or fludarabine, extended exposure to these agents results in drug resistance. It is therefore important to identify novel agents which rapidly and selectively induce apoptosis of cells isolated from patients who have become resistant to conventional drugs. The sesquiterpene lactone parthenolide (PTL) is the active component of several medicinal plants which have been used for hundreds of years for the treatment of migraines, pain and inflammatory conditions. In some cell lines, PTL has been shown to block components of the NFκB pathway including the IκB kinase complex and/or the p65 subunit of NFκB. Here we investigated the sensitivity of isolates from 25 CLL patients (representing stages A, B and C) to PTL (1.25–80 μM) and CHL (3.125–200μM). IC50 values were evaluated by the 4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Apoptosis induction was quantified by FACScan analysis of annexin V binding and by western blot detection of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP). CHL resistance varied across the cohort from highly sensitive to highly resistant, with IC50 values in the range 5.3 to >200 μM. In contrast, all isolates tested were sensitive to PTL, with IC50 values in the range 3 to 20 μM. Strikingly, isolates from extensively-treated patients which were highly resistant to elevated levels of CHL in vitro were sensitive to PTL (Figure 1). PTL induced very rapid PARP cleavage which became clearly evident between 3–6 h of exposure to 5 μM PTL. In contrast, PARP cleavage was not detected until >12h exposure to 75 μM CHL in isolates which were sensitive to this drug. Furthermore, pulse/wash-out experiments showed that a very brief (1-2h) exposure to PTL was sufficient to induce commitment to the apoptotic programme. The annexin V-binding assay revealed that CLL cells were strikingly more sensitive to apoptosis induction by 2–5 μM PTL than were normal T or B lymphocytes or CD34+ bone marrow progenitor cells (Figure 2). Western blot analysis revealed that PTL-induced PARP cleavage was preceded by downregulation of anti-apoptotic mcl-1, a key mediator of the survival of B lineage lymphoid cells. However, we failed to detect a PTL-induced decrease in IκBαphosphorylation, suggesting that inhibition of IκB kinase did not contribute to the mechanism of CLL cell killing. In conclusion, the rapid, selective action of PTL and its ability to kill CLL isolates refractory to cytotoxic drugs suggest that this agent may be of value in the treatment of CLL patients who have become resistant to conventional therapeutic regimes.
Figure Figure
Sequential Phase II Studies of Flavopiridol by 72-Hour Continuous Infusion and 1-Hour Intravenous Bolus for the Treatment of Relapsed B-Cell Chronic Lymphocytic Leukemia: Results from CALGB Study 19805.
Authors: Byrd John C. , Peterson Bercedis L. , Gabrilove Janice L. , Odenike Olatoyosi M. , Grever Michael R. , Rai Kanti R. , Vardiman James W. , Larson Richard A. ,   Cancer and Leukemia Group B
Published: October 2019

Abstract
Flavopiridol has in vitro activity in CLL and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol administered using two different schedules has activity in CLL. Patients with previously treated CLL were enrolled on two sequentially performed phase II studies. Patients in the first trial received flavopiridol (50 mg/m2 daily) as a continuous infusion (CI) over 72-hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour intravenous bolus (IVB) daily for three days repeated every 3 weeks. Patients received up to 6 (CI cohort) or 8 (IVB cohort) cycles of therapy. Fifteen patients enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate (Rai stage I or II) and 9 (60%) high (Rai stage III and IV) risk stages. No responses were noted in this group with 27% having stable disease (SD) and 73% progressive disease (PD). Thirty-six patients enrolled in the IVB study, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) SD, and 13 (36%) PD. The progression-free survivals for responders in the IVB study were 2.9, 3.2, 8.7, and 19.3 months. The median progression-free survival was 2.1 months (95% confidence interval [CI] 1.8 – 3.8) for patients in the CI study and 3.2 months (95% CI [2.5 – 7.4]) for the IVB study. The median overall survival was 27 months (95% CI [20–42]) for the CI study and 24 months (95% CI [18–31]) for the IVB study. Toxicity was manageable and included mainly myelosuppression (granulocytopenia and thrombocytopenia), infections, diarrhea and fatigue. Grade 3 and 4 toxicities were 20% and 27%, respectively, on the CI study and 39% and 33% on the IVB study. One patient on the IVB study had tumor lysis syndrome that was managed medically and did not require dialysis. There was one on-study death following a myocardial infarction on the IVB study. We conclude that flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.
Heparin-Induced Thrombocytopenia Antibodies Do Not Predict Adverse Outcomes in End-Stage Renal Disease Patients on Dialysis.
Authors: Asmis Lars M. , Segal Jodi B. , Plantinga Laura C. , Fink Nancy E. , Kerman Jonathan S. , Kickler Thomas S. , Coresh Josef , Gardner Lawrence B. 
Published: October 2019

Abstract
Introduction: Heparin-induced thrombocytopenia (HIT) is a syndrome in which anti-platelet factor 4/heparin antibodies (HITab) and thrombocytopenia develop after heparin exposure. While HIT is an established risk factor for arterial and venous thromboses, HITab, in the absence of thrombocytopenia, may also be a prothrombotic risk factor. As patients with chronic kidney disease on hemodialysis are chronically exposed to heparin, we hypothesized that the presence of HITab may contribute to the high rates of thrombotic disease and mortality in these patients.
Patients and methods: We analyzed data from the CHOICE study, a prospective cohort study of incident dialysis patients. Clinical data were available from medical record review. Arterial cardiovascular events and events of vascular access occlusion were validated by chart review and adjudication. Venous thromboembolic events were defined by ICD-9 codes. Thrombocytopenia was defined as a platelet count <150,000/mm3 or a >50% decline from a previous platelet count. Median duration of follow-up is 2.7 years. We measured HITab using a commercial ELISA assay in stored serum obtained 6 months after cohort enrollment of 740 participants (596 on hemodialysis, 144 on peritoneal dialysis). ELISA results were corrected for inter-plate variability. A positive HITab test associated with thrombocytopenia in the subsequent three months was considered compatible with HIT. We used techniques of logistic regression and of survival analysis with adjustment for potential confounders in our analyses.
Results: 10.3% of all patients were positive for HITab at six months. HITab positivity was not associated with thrombocytopenia. HITab did not predict subsequent outcomes including cardiovascular (hazard ratio (HR) 0.96, 95% confidence interval (CI) 0.65–1.41), vascular access occlusive (HR 0.82, CI 0.40–1.71), or venous thromboembolic events (HR 1.39, CI 0.17–11.5) and did not predict mortality (HR 1.00, CI 0.66–1.53). 9 patients (1.2%) had results compatible with HIT (thrombocytopenia + HITab). 1 patient with HIT (0.13%) had venous thrombosis yielding a hazard ratio of 13.7 (CI 1.63–115.5) for HIT and thrombosis.
Conclusions: HITab seropositivity after six months of dialysis is frequent, but is not associated with thrombocytopenia. In end-stage renal disease patients treated by dialysis HITab seropositivity in the absence of thrombocytopenia is not an independent risk factor for cardiovascular morbidity or mortality. However, HIT may occur in this patient population and is associated with thrombosis.
Thrombophilic Genetic Mutations Have No Impact in the Pathogenesis of Thromboembolism in Gastro-Intestinal Cancer Patients.
Authors: Olimpieri Odoardo M. , Schiavon Gaia , Giannetti Barbara , Tirindelli Maria C. , Santini Daniele , Valeri Sergio , Vincenzi Bruno , Coppola Roberto , Tonini Giuseppe , Avvisati Giuseppe 
Published: October 2019

Abstract
Deep venous thrombosis and pulmonary embolism are well recognized and feared complications in cancer patients, with a considerable impact on the overall survival and the quality of life. During the last years many prothrombotic genetic mutations have been described, of these the most common are: Factor V Leiden, prothrombin G20210A gene mutation and the C677T mutation in the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene. Aim of this study was to asses the impact of these thrombophilic gene mutations on the development of VTE (Venous ThromboEmbolism) in patients with gastro-intestinal neoplasms. Moreover, we have tried to determine whether or not the C677T polymorphism of the MTHFR gene was associated with a higher toxicity during chemotherapy with antimetabolites such as 5-FU. From October 2002 to June 2004 48 patients (aged more than 18 years) not assuming anticoagulant therapy, with a new histopathologic diagnosis of gastro-intestinal cancer and no known hematologic or immunologic diseases were investigated by standard polymerase chain reaction techniques for the presence of the above thrombophilic gene mutations. Of the 48 tested patients, a homozygous or heterozygous Factor V Leiden was present respectively in 1 and 3, while the G20210A Factor II mutation was observed in 6 patients (all heterozygous). The C677T mutation in the MTHFR gene allelic frequency in the studied population was 46%. A VTE was observed in 7/48 patients (14.5%) and none of these patients had Factor V Leiden or G20210A Factor II mutations while 4 had a C677T MTHFR mutation. Therefore, none of the thrombophilic genetic mutations studied was associated to the development of VTE in the cohort of gastro-intestinal cancer patients. On the contrary chemotherapy (P=0.007) and advanced inoperable disease (P=0.009) were the only risk factors statistically significant for the development of VTE. As for toxicity during chemotherapy with antimetabolites, it was present in 25% of patients with the C677T polymorphism of the MTHFR while none of the patients without mutation had a toxicity (P=0.06). In conclusion, thrombophilic genetic mutations have no effect on the development of VTE in gastro-intestinal cancer patients. Moreover, we confirm that chemotherapy and an advanced disease play an important role in the pathogenesis of VTE in cancer patients. Furthermore, patients carrier of the C677T mutation in the MTHFR gene have a higher risk (OR: 1.4; C.I. 95%: 0.88–2.24) of toxicity during an antimetabolites-based chemotherapy.
A Dynamic Regulation of Platelet Secretion by Septin Proteins.
Authors: Martinez Constantino , Dent Judith A. , Russell Susan , Ware Jerry 
Published: October 2019

Abstract
Septins form a cytosolic protein family gaining recognition as important effectors in molecular mechanisms involving membrane rearrangements, such as cytokinesis and exocytosis. So far, twelve different members of the family (named SEPT1 to SEPT12) have been characterized in mammals, and their roles are not limited to those mentioned above, but embrace a large number of physiological and pathophysiological events. In particular, reports have associated septins with cancer, apoptosis and neurodegenerative diseases. In this study, we further characterize the platelet septin, SEPT5 (formerly CDCrel-1) as a regulator of platelet physiology. SEPT5 is highly expressed in megakaryocytes, heart and brain and is directly linked to secretion mechanisms in both platelets and neurons. In neurons, SEPT5 negatively modulates neurotransmitter release and has recently been implicated in mechanisms leading to development of schizophrenia in rat models. In platelets, we and others have shown that SEPT5 is preferentially located around α-granules and associates with syntaxin 4 and other members of the septin protein family (SEPT4 and SEPT8) to form a cytoplasmic macromolecular complex. Work from other laboratories has linked SEPT5 to different members of the SNARE family such as syntaxin 1A, the sec6/8 complex, and SNAP25. We have found the overexpression of mouse SEPT5 is associated with fewer α-granules but those present are larger in size suggesting levels of Sept5 maintain normal α-granule size. In a murine model of SEPT5 deficiency, platelets display an increased sensitivity to platelet agonists such as collagen and the thromboxane analogue U46619. To date, SEPT5 is the only mammalian septin whose relevance has been tested in a mouse knockout model. Our current studies use a lumi-aggregometer to document the active secretion of ATP in SEPT5 deficient platelets. Platelets lacking SEPT5 are more responsive to submaximal concentrations of acid insoluble fibrillar type I collagen (< 2.5 mg/mL). We observe a 2- to 3-fold increase in ATP secretion in deficient platelets as compared to their wild-type littermates. The increased dense granule release in the absence of SEPT5 also coincides with increased platelet aggregation. A maximum release of ATP was determined in the presence of the calcium ionophore (A23187, 40μM). Higher concentrations of collagen (5 μg/ml) produced a maximum level of ATP release in SEPT5 deficient mice unlike their wild-type littermates. Our current hypothesis places SEPT5 as a scaffolding protein that serves to anchor other proteins into higher-ordered molecular complexes. SEPT5 biology in platelets needs to be further studied to fully understand its role in granule secretion and formation. While important for platelet biology, these studies may also have implications for mechanisms associated with neurotransmitter release. As such, the platelet becomes an excellent model for analyzing SEPT5 function with implications for hemostasis and beyond.
Proteomic Pathway Discovery Reveals That C/EBPalpha Disperses PML Nuclear Bodies To Recruit Its Own Coactivators in a MAPK-Dependent Manner.
Authors: Meisel A. , Kachirin R. , Christopeit M. , Hiddemann W. , Behre G. 
Published: October 2019

Abstract
The downregulation of the tumor suppressor protein C/EBPalpha is a critical event during cancerogenesis. In contrast restoration of its expression leads to differentiation and ultimately apoptosis. To investigate the mechanisms mediating the antiproliferative and differentiation-inducing activity of C/EBPalpha, we performed a proteome-wide screen for C/EBPalpha target proteins using 2-D-gel electrophoresis and MALDI-TOF masspectrometry. Besides 80 other target proteins we identified the tumor suppressor PML as a protein being regulated by C/EBPalpha. In fluorescence microscopy studies we could show that the induction of C/EBPalpha leads to a dramatic decrease in the number of PML nuclear bodies. We found out that these changes occur due to a reduction in the slower migrating sumoylated isoforms of PML. In reporter assays overexpression of PML was able to increase the trans-activation capacity of C/EBPalpha. CBP was further augmenting this coactivating effect. Cadmium, which is like C/EBPalpha desumoylating the PML protein and dispersing the PML nuclear bodies was having the same activating effect. We could show that p38 was mediating the desumoylation of PML, while the p38 inhibitor SB203580 could partially abrogate the C/EBPalpha-induced effects on PML nuclear bodies. Taken together our investigations could identify a mechanism how C/EBPalpha is targeting PML and CBP to sites of active transcription.
The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.
Authors: Lee Aimee , Khanna-Gupta Arati , Zibello Terry , St.Clair Caryn , Berliner Nancy 
Published: October 2019

Abstract
CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.