bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors Affecting Outcome of Patients with Refractory/Relapsed Hodgkin’s Disease (HD) Treated with IGEV Chemotherapy (CT) and High Dose Consolidation Therapy (HDT) with Stem Cell Rescue.
Authors: Balzarotti Monica , Spina Michele , Magagnoli Massimo , Chisesi Teodoro , Pinto Antonello , Castagna Luca , Demarco Monica , Michieli Mariagrazia , Nozza Andrea , Tirelli Umberto , Santoro Armando
Published: October 2019

Purpose: to identify prognostic factors in pts with relapsing/refractory HD treated with IGEV CT and HDT

Methods: From 01/98 to 01/05 104 patients (pts) from our institutions with relapsed/refractory HD received 4 induction cycles of IGEV (ifosfamide 2000 mg/mq d 1–4; gemcitabine 800 mg/mq d 1& 4; vinorelbine 20 mg/mq d 1; prednisolone 100 mg/mq d 1–4, and G-CSF) and HDT consolidation. Prognostic factors for freedom for progression (FFP) were analyzed by means of the chi-square and Fisher’s exact tests. Multivariate regression logistic analysis was then applied for variable associated with outcome.

Results: Main pt characteristics at accrual: M/F 56/48, median age 31 (range 17–65), refractory/relapsed following last CT course 40/64, median previous CT regimens: 1 (range 1–2), simptoms A/B 32/71, Hasenclever (IPS) score <3/≥ 4: 72/24 (8 pts not evaluable), LDH ratio ≤ 1/> 1: 68/35, previous radiotherapy (RT) yes/no: 62/42. After induction, 45 pts (43%) obtained complete remission (CR), 41(40%) partial remission (PR) and18 (17)% di not response (NR). Overall 89 pts received HDT and at the end of the treatment program 76 out of 104 pts were in CR. On an intent-to treat analysis, two-year freedom from progression (FFP) and overall survival (OS) for the whole series were 47% and 74,6%, respectively.

In univariate analysis FFP was negatively influenced by B symptoms, IPS score ≥ 4, LDH ratio > 1, refractory disease, and by response to IGEV (see table).

Multivariate analysis revealed that factors retaining statistical significance were refractory disease, B symptoms, high IPS score and chemoresistance (see table).

Conclusions: based on this analysis, a prognostic model for IGEV-treated patients with refractory/relapsed HD can be constructed.

Factors influencing FFP Factor ≥ FFP p (univariate) p(multivariate) Symptoms B/A 16/57% 0.003 0.007 IPS > 4/< 3 32/51% 0.0035 0.051 LDH >1/<1− 19/61% 0.003 n.s. refrac/relaps 22.5/62% < 0.001 0.012 < CR/CR to IGEV 32/68% 0.002 n.s. NR/CR + PR 23.5/52% 0.001 0.005
Epstein-Barr Virus in Patients with Chronic Lymphocytic Leukemia.
Authors: Tsimberidou Apostolia-Maria , Keating Michael J. , Bueso-Ramos Carlos , Kurzrock Razelle
Published: October 2019

Purpose: The Epstein-Barr virus (EBV) is implicated in the development of Richter’s transformation in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). The objective of this study was to assess the incidence and the clinical significance of EBV in patients with CLL/SLL.

Patients and Methods: Patients with CLL/SLL who presented at The University of Texas M. D. Anderson Cancer Center over a two-year period, and had available marrow paraffin blocks were studied for evidence of EBV infection using a highly specific in-situ hybridization assay for detection of EBV encoded RNA (EBERs). Results were analyzed in relation to other presenting characteristics and outcome.

Results: Thirty-two patients were examined. EBERs were detected in 12 of 32 (38%) CLL/SLL marrows versus 0 of 20 normal marrows (p = 0.002). EBERs were observed in sporadic granulocytes alone or in addition to its presence in lymphocytes in nine of the twelve EBV-positive patients. EBERs were detected less frequently in patients with Rai stage 0-1 disease (20%) compared with Rai stage 2–4 (66%; p=0.008); EBER-positive patients tended to have higher lactate dehydrogenase (LDH) levels (p=0.053). The 10-year survival rate was 22% versus 58% for patients with and without discernible EBERs (log-rank, p=0.08).

Figure Figure

Conclusions: Evidence of EBV infection was found in 38% of CLL/SLL patients assessed. Despite the small number of patients tested, discernable EBERs were significantly more common in individuals with more advanced Rai stage, and there was a trend toward shorter survival in patients in whom EBV EBERs were discerned. Larger studies are needed to determine the prognostic value and role of EBV infection in patients with CLL/SLL.
The C1542G and G505A Polymorphisms of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) in Patients with Thrombotic Microangiopathies.
Authors: Sucker Christoph , Farokhzad Firuseh , Dahhan Fieras , Schmitz Michael , Hetzel Gerd R. , Grabensee Bernd , Zotz Rainer B. , Scharf Rudiger E.
Published: October 2019

Background Thrombotic microangiopathies are characterized by vascular microthromboses, microangiopathic hemolytic anemia, and thrombocytopenia. Although recent research has elucidated the pathogenesis of these rare thrombotic disorders to some extent, the determinants contributing to the onset and modulating the severity are largely unknown. It is likely that risk factors of venous and arterial thrombosis also play a role in this clinical setting.

Patients and Methods In the present study, we used a case-control and a case-only design, enrolling 23 patients (mean age [± SD] 35 ± 11 years) with a history of thrombotic microangiopathy and 689 control subjects to assess the role of gene polymorphisms of the thrombin-activatable fibrinolysis inhibitor (TAFI).

Results The prevalence of the TAFI decreasing G/G genotype of the C1542G polymorphism was significantly higher in patients compared to controls (odds ratio 3.88; 95 % CI 1.07 – 11.47; p=0.02). In addition, in a case-only design the TAFI 1542 G allele was more prevalent in patients suffering from a severe course compared to those with a mild or moderate course (odds ratio 20; 95 % CI 1.85 – 216.17; p=0.009). By contrast, no such association was found for the TAFI G505A polymorphism.

Conclusions Our study shows an association of the TAFI decreasing 1542 G/G genotype with an increased risk for thrombotic microangiopathies and a more severe course. This finding might be explained by the role of TAFI as an inhibitor of local inflammatory processes.
Novel Evidence That Statin-Mediated Perturbation of Lipid Raft Formation Ameliorates Bleeding- Related Thrombocytosis.
Authors: Wysoczynski Marcin , Reca Ryan , Kucia Magda , Ratajczak Janina , Ratajczak Mariusz Z.
Published: October 2019

The a-chemokine stromal derived factor 1 (SDF-1) plays an important role in maturation/platelet formation of megakaryocytes (Megs), and we recently reported that the responsiveness of hematopoietic cells to SDF-1 is optimal when its corresponding CXCR4 receptor is included into membrane lipid rafts (Blood2005; 105:40). The formation of lipid rafts in vivo may be perturbed by cholesterol-lowering drugs, e.g., statins. Statins are effective in lowering LDL cholesterol and exert several pleiotropic effects on mature platelets, e.g., inhibit their activation. This latter effect is probably due to lowering of the cholesterol content in the membranes of mature platelets leading to the perturbation of membrane lipid raft formation which is required for proper platelet activation and signaling. However, under steady state conditions statins do not influence platelet production, no studies have been performed on their effects on platelet production in reactive thrombocytosis. In determining whether lipid raft formation plays a role in SDF-1- and stress-dependent thrombocytosis, we found that SDF-1 most efficiently stimulates in vitro platelet production when CXCR4 is included into membrane lipid rafts on Megs. At the molecular level, depletion of cholesterol from Megs membranes i) perturbed the responsiveness of megakaryocytic progenitors to an SDF-1 gradient, ii) inhibited SDF-1-mediated calcium flux and MAPKp42/44, AKT and STAT 1–6 phosphorylation in normal human Megs and (iii) inhibited F-actin polymerization, MMP-9 and VEGF secretion, and adhesion to endothelium and fibrinogen. More importantly we found that ex vivo-expanded human Megs, produced significantly fewer platelets during their transendothelial migration after preincubation with cholesterol-lowering MbCD. To evaluate whether cholesterol depletion from Megs affects platelet production we exposed C57Bl6 mice to statins (orally, 21 days, 750 mg/mouse) and observed that statins did not influence peripheral blood cell counts. However, when mice were acutely bled this treatment led to a significant amelioration of post-bleeding thrombocytosis as compared to untreated (control) mice. At the same time, recovery of erythrocyte and leukocyte counts was unaffected. Thus we demonstrated for the first time that statins affect CXCR4-mediated thrombopoiesis by perturbing lipid raft formation and should be considered as potential drugs to prevent post-trauma or post-operative thrombocytosis.
Inhibition of Immune Thrombocytopenic Purpura (ITP) by an Orally Bioavailable Inhibitor of Syk Kinase Activity.
Authors: Crow Andrew R. , Siragam Vinayakumar , Freedman John , Grossbard Elliott B. , Masuda Esteban S. , Pine Polly , Lazarus Alan H.
Published: October 2019

Immune Thrombocytopenic Purpura (ITP) is an autoimmune disease characterised by the destruction of platelets by pathogenic autoantibodies. Clearance of platelets by phagocytic cells such as macrophages occurs through activating receptors for the Fc portion of IgG (FcγR). A central tyrosine kinase in this activation pathway is Syk, which is required for FcγR-mediated phagocytosis; thus, we questioned whether inhibition of Syk would ameliorate ITP. R406 is an orally bioavailable Syk-kinase inhibitor which is well tolerated by human volunteers. Using a well-described murine model of ITP, we tested the ability of R788 (the prodrug of R406) to ameliorate thrombocytopenia. Mice injected with an antibody directed to integrin αIIβ were profoundly thrombocytopenic when platelets were enumerated 24 hr post injection. However, mice pre-treated with 25 or 40 mg/kg R788 were protected from thrombocytopenia. Mice pre-treated with the vehicle alone displayed no protection. It has been demonstrated that chimeric Syk deficient mice display a bleeding diathesis; it was therefore questioned whether treatment of mice would demonstrate prolonged tail vein bleeding time. In independent experiments, mice treated with the with the bioavailable inhibitor R406 had no significant difference in bleeding time compared to untreated mice or mice treated with vehicle alone. Syk is also involved in collagen-induced platelet secretion and aggregation. To further the potential use of this drug in humans, the ability of platelets derived from human volunteers treated with R788 to respond to in vitro collagen-induced aggregation was analysed. Platelets from volunteers treated with the drug displayed normal collagen (as well as ADP)-induced platelet aggregation compared to untreated volunteers or those treated with the vehicle alone. These data suggest that future investigation of Syk inhibition in autoimmune diseases such as ITP is warranted.
The Role of Fcγ Receptor Signaling, Tissue Factor Expression and Monocyte-Platelet Cross-Talk in the Initiation of Thrombosis by Immune Complexes.
Authors: Casasanto Peter , Reilly Michael P. , Liu Ming-Lin , Williams Kevin Jon , McKenzie Steven E.
Published: October 2019

Thrombus formation in response to physical disruption of the vascular endothelium is an essential response to vessel injury. In contrast, thrombus formation is pathologic when the endothelium is physically intact but blood and endothelial cells are activated by inflammation. Thrombosis secondary to immune complexes is a major cause of morbidity and mortality in hospitalized patients. We recently generated and characterized the first transgenic mouse model of heparin-induced thrombocytopenia/thrombosis (HIT/T) to recapitulate the salient features of the disease and confirmed that complexes of heparin and platelet factor 4, antibodies to the complex, and FcγRIIa-dependent platelet activation are both necessary and sufficient to model the disease in vivo. It is also likely that immune complex activation of monocytes and endothelial cells occurs in HIT/T. However, the interaction between activated blood and endothelial cells and tissue factor positive microparticles (TF+-MP) that may result in thrombin generation is not clear. Recent studies (del Conde et al., Blood 2005) showed that phosphatidylserine and PSGL-1 on the surface of monocyte-derived TF+-MP enables their fusion with activated platelets. Collagen-activated platelets incubated with TF+-MP were reported to cause increased TF-VIIa procoagulant activity (PCA) compared to non-activated platelets. We hypothesized that platelets activated by HIT/T immune complexes would also result in increased TF-PCA when incubated with monocyte-derived TF+-MP. To test this hypothesis we generated TF+-MP from THP-1 cells, a human monocytic cell line, stimulated with LPS (6 hr) and A23187 (subsequent 15 min). TF+-MPs were co-incubated with untreated or agonist-treated platelets. The HIT/T immune complex was prepared by incubating optimal ratios of heparin and recombinant human PF4 with KKO, a mouse monoclonal anti-heparin-PF4 antibody. Other agonists included anti-CD9 (producing a particulate immune complex) and collagen. Using a chromogenic assay of Xa generation we found that TF+-MPs were necessary to detect TF-PCA. PCA increased by 12–25% when TF+-MPs were incubated with platelets stimulated by collagen or anti-CD9 as compared to untreated platelets. When TF+-MPs were incubated with platelets stimulated by the HIT/T immune complex, there was a 2-fold increase in the PCA. The increase in TF-PCA was observed to be proportional to the concentration of microparticles added. The results suggest an important role in platelet-monocyte cross-talk in initiating and increasing TF procoagulant activity upon immune complex stimulation.
Gene Expression Profiling of Multiple Patients with Cyclic Thrombocytopenia Reveals Consistent Profile.
Authors: Sood Ruchira , Gourley Erin , Schrier Stanley L. , Go Ronald , Zehnder James L.
Published: October 2019

Cyclic thrombocytopenia (CTP) is a rare disorder characterized by periodic changes in platelet count. While some previous reports suggest an association with several cytokines, the etiology of this disorder remains poorly characterized. Using DNA microarrays, we examined the gene expression profile in peripheral whole blood at multiple time points encompassing a cycle of platelet counts from two CTP patients. We hypothesized that the variation in gene expression program in whole blood would reflect on the transcriptional changes associated with or perhaps even underlying this disease. Genome-wide cDNA microarray analysis was performed using amplified RNA obtained from 11 and 8 whole blood samples from each patient. The first patient is a 41-year old male with a 2-year history of CTP while the second patient is a 54-year old male with a 3-year history of CTP. The period of both patients’ cycles is roughly 3 weeks. No associated underlying disease has been found in both patients. With a focus on 1500 genes that change 3 fold within each group of samples we observed clusters of gene expression in whole blood that correlate with changing platelet numbers in both patients. Significant variation in expression of a cluster of interferon responsive genes during the platelet count cycle was particularly striking in both samples. Interferon (IFN) therapy is known to suppress platelet counts, and this observation suggests that aberrant IFN levels and signalling could be in part responsible for CTP. At high platelet counts, platelet transcripts were detected in whole blood RNA as inferred by high expression of previously described platelet genes including TBXAS1, TUBB1, OAZ1, SEPT5, several mitochondrial genes, NRGN and F13A1. In addition, gene clusters including known genes as well as previously uncharacterized genes were found to correlate with the peak, increasing or decreasing trends of platelet counts. Briefly, GATA2 and NFE2 expression coincided with the platelet count peak, while Tyk2 and SOCS5 expression was consistent with a rising trend of platelet counts and GATA3 and JAK2 coincided with decreasing trend of platelet counts. These results show gene expression changes associated with CTP in all cell types in whole blood and pave the way for new investigation into regulation of platelet number in a rare and fascinating disease.

Gene expression profile of whole blood of two CTP patients with platelet counts ranging from high to low and then increasing again from left to right of each panel Gene expression profile of whole blood of two CTP patients with platelet counts ranging from high to low and then increasing again from left to right of each panel
Analysis of Factors Influencing Natural Killer (NK) Cell Activity during the Early Phase after Allogeneic Stem Cell Transplantation (SCT).
Authors: Fischer Lars , Penack Olaf , Gentilini Chiara , Thiel Eckhard , Lutz Uharek
Published: October 2019

Background: After allogeneic SCT, NK cell mediated cytotoxicity is an important defense mechanism against residual tumor cells and viral infections. Using a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation, we investigated the effect of in vivo T cell depletion and the type of conditioning on NK cell function in the early phase after transplantation.

Methods: At day +30 and day +90 after allogeneic SCT with regular (n=14) and dose reduced conditioning (n=8), PBMCs were coincubated at 37°C for 3 h with the NK sensitive cell line HL60. 20μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody (moAb) was added to each tube containing 400μl effector/target cell suspension (2x106 cells, E:T ratio 1:1) prior to incubation. After 1 hour, 10μl of monensin (2mM) was added. After incubation for 3 hours, the cells were stained with conjugated moAb (CD56, CD16, CD3) for flow cytometry. The percentage of CD107a expressing NK cells was assessed and the absolute number of degranulating NK cells /μl was calculated. Results were compared to values from 15 healthy controls.

Results: Twenty two patients (pts.) were investigated. Fourteen pts. received a conventional conditioning regimen and eight a reduced intensity conditioning. T cell depletion was applied in 15/22 pts. (ATG n=12, alemtuzumab n=2, 1 OKT-3 n=1). The type of donor included MRD (n=7) and MUD (n=15). At day +30, the proportion of NK cells with cytotoxic activity (indicated by the mean percentage of degranulating CD107a+/CD56+ cells) was significantly reduced as compared to normal donors (2.6% vs. 5.6%, p<0.001). At day +90 the percentage of degranulating NK cells was still decreased compared to normal (3.5%, p=0.007). The predominant proportion of degranulating cells was in the CD56dim/CD16− subpopulation (mean 9.8%). After conventional conditioning, the mean percentage of CD107a+ cells was 1,9% at day +30, compared to 4,0% in patients with reduced intensity conditioning (p=0.21). The absolute number of degranulating NK cells was significantly reduced after conventional conditioning (4.1/μl vs. 19.8/μl, p=0.011). Interestingly, we found no influence of in vivo T cell depletion with ATG on the mean value for CD107a+ cells at day +30 (2.5% vs. 2.9%, p=0.77).

Conclusion: Although the proportion of NK cells is increased after allogeneic SCT, our data suggest that the cytotoxic activity of these cells is considerably reduced. The absolute number of NK cells with cytotoxic activity is significantly higher after reduced intensity conditioning which may contribute to the effectiveness of these regimens. Antibody induced in vivo T cell depletion with ATG showed no impact on NK cell activity during the first two months post SCT.
Long-Term Dosing of AMG 531 Is Effective and Well Tolerated in Thrombocytopenic Patients with Immune Thrombocytopenic Purpura.
Authors: Bussel James B. , Kuter David J. , George James N. , Aledort Louis M. , Lichtin Alan E. , Lyons Roger M. , Nieva Jorge , Wasser Jeffrey S. , Bourgeois Emanuelle , Kappers-Klunne Mies , LeFrere Francois , Schipperus Martin R. , Kelly Reggie , Christal Jenny , Chen Chien-Feng , Nichol Janet L.
Published: October 2019

AMG 531 is a novel platelet-stimulating peptibody that targets the TPO receptor, resulting in increased production of platelets. This ongoing, open-label study assessed the safety and efficacy of long-term AMG 531 dosing in ITP patients. Eligible patients have completed a previous AMG 531 study in ITP, are ≥18 years of age, have a baseline platelet count ≤50 x 109/L, with no recent significant change in medical history. The AMG 531 starting dose is 1μg/kg by SC injection with dose adjustment to an original maximum of 30μg/kg, since reduced to 15μg/kg. Patients are treated weekly unless the platelet count is >400x109/L. Concurrent corticosteroids can be tapered when the platelet count is ≥50x109/L. Preliminary data are available for 26 patients treated for up to 24 weeks: 17 women and 9 men; mean age, 48.4±11.6 (SD) years; mean baseline platelet count, 18.5±12.3 (SD) x109/L. Twenty-one patients (80.8%) had undergone a splenectomy before study entry; 6 (23.1%) were receiving concurrent corticosteroids for ITP. Twenty-one of 26 patients (80.8%) had a protocol-defined platelet response to AMG 531 (doubling of the baseline platelet count and ≥50x109/L). The mean AMG 531 dose at the first response was 3.7±2.7 (SD) μg/kg (at median 5 weeks); the mean dose at week 24 was 7.2±4.2 (SD) μg/kg. Twelve of 26 patients (46.2%) had a durable platelet response (doubling of the baseline count and ≥50x109/L at 6 or more of weeks 17–24). Twenty patients (76.9%) had a platelet count ≥100x109/L at least once; 7 (26.9%) had a platelet count ≥400x109/L. Of 6 patients on concurrent corticosteroids, 3 discontinued treatment and 2 had a ≥25% dose reduction. At least 1 serious adverse event was reported in 4 patients: anal fistula (unrelated to treatment), adverse drug reaction (unrelated), multiple sclerosis relapse (unrelated), bone pain (related), and diffuse reticulin formation in the bone marrow reported as myelofibrosis (related). Reticulin formation is hypothesized as due to excessive accumulation of megakaryocytes in the bone marrow. AMG 531 was discontinued, and a bone marrow 3 months later showed improvement. One patient experienced adverse events (musculoskeletal pain and headache) resulting in withdrawal from the study. No neutralizing antibodies have been detected to date. In summary, repeated exposure to AMG 531 has been generally well tolerated in this ongoing study. A total 80.8% of patients achieved a platelet response, defined as doubling of the baseline count and ≥50x109/L. Individualized weekly doses of AMG 531 may provide a therapeutic option in ITP, potentially enabling patients to taper off long-term corticosteroid therapy.
Implications of RA-Induced CD38 Antigne in IFN gamma and IL-1 beta Induced Apoptosis of Endothelial Cells and Development of Retinoic Acid Syndrome.
Authors: Gao Yin , Mehta Kapil
Published: October 2019

Differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid (RA) represents a major advance in the treatment of myeloid leukemia. Despite high remission rates, treatment with RA is associated with the development of a unique constellation of syndromes collectively referred to as ’retinoic acid syndrome’ (RAS). RAS is characterized by fever, dyspnea, weight gain, pulmonary edema, pulmonary infiltrates, pleural and pericardial effusions, episodic hypotension, and acute renal failure, which often lead to a fatal outcome if left uncontrolled. The molecular mechanisms underlying the development of RAS, particularly the events that lead to apoptosis of endothelial cells during RAS development, remain elusive. Here, we report that RA could induce gene expressions of interferon (IFN) gamma and IL-1 beta in the peripheral blast cells of APL samples. By using human umbilical cord endothelial cells (HUVECs) and human lung microvascular endothelial cells (HLMVECs) in vitro, we observed that IFN gamma in combination with IL-1 beta exerted synergistic effect in inducing apoptosis of endothelial cells. In addition, RA treatment up-regulated the expression of CD38 antigen, an ectoenzyme involved in the metabolism of two calcium messengers, the cADPR and NAADP. RA-induced CD38 expression promoted intense attachment of maturing APL cells to the endothelial cells. Experimental data demonstrate that endothelial cells when treated with IFN gamma (10ng/ml) and IL-1 beta (10ng/ml) together undergo significant apoptosis with caspase 8 activation and CD38 expression. To our knowledge, this is the first report that demonstrates the regulation of IFN gamma gene expression in APL cells in response to RA treatment and the combined effect of cytokines and CD38 in driving endothelial cell into apoptosis. These events suggest a link between RA treatment and the development of retinoic acid syndrome and thus may offer novel approaches for controlling the development of RAS in APL patients.
Differential Requirement for Cell Division in the Development of Terminally Differentiated Neutrophils and Macrophages from Their Common Progenitors.
Authors: Sugimoto Yuka , Katayama Naoyuki , Masuya Masahiro , Shiku Hiroshi
Published: October 2019

Neutrophils and monocytes are the main population of peripheral blood nucleated cells and progenies of common progenitor cells, CFU-GM, that are derived from hematopoietic stem cells. Monocytes are non-dividing immediate precursors for macrophages. Common clinical observation that monocytes more rapidly recover than do neutrophils in myelosuppressive patients indicates the difference in regulatory mechanism of terminal differentiation between neutrophils and macrophages. In this study, we investigated the cell division profiles in the differentiation pathway from common progenitors for neutrophils and macrophages to respective post-mitotic cells in culture. We prepared lineage-negative (Lin−) bone marrow cells from normal C57BL/6 mice by using immunomagnetic beads. The Lin− cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a cytoplasmic dye that is equally diluted between daughter cells, and stained with anti-Sca-1 antibody and lineage antibodies. CFSEhighSca-1+Lin− cells were sorted on a FACSVantage or FACSAria. When the CFSEhighSca-1+Lin− cells were cultured in the presence of steel factor (SF) and IL-11 for 5 days, the resulting cells could be separated into four populations based on staining with anti-fms and anti-Gr-1 antibodies: fms-Gr-1−, fms+Gr-1−, fms−Gr-1+, and fms+Gr-1+ cells. The fms+Gr-1+ cells showed morphological features typical of macrophages. The commitment of fms−Gr-1+ cells into neutrophilic lineage was evident, although they exhibited an immature morphology. CFSE labeling pattern in fms+Gr-1+ macrophages was equal to that of fms−Gr-1+ neutrophilic cells, indicating that fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells passed through similar successive rounds of cell division in culture of CFSEhighSca-1+Lin− cells. These fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells were subsequently cultured for additional 3 days in the presence of SF, IL-11, IL-3, GM-CSF, G-CSF, M-CSF, erythropoietin, and thrombopoietin. While a decline in the number of fms+Gr-1+ macrophages was observed, the number of fms−Gr-1+ neutrophilic cells increased approximately 5-fold during subsequent culture. The proliferation of fms−Gr-1+ neutrophilic cells and no cell division of fms+Gr-1+ macrophages were confirmed by Ki-67 antibody staining, BrdU incorporation, and DNA staining. The cultured fms−Gr-1+ neutrophilic cells also predominantly acquired a mature morphology. These results suggest distinct cell division histories between terminally differentiated neutrophils and macrophages, possibly explaining the early appearance of monocytes in the blood after myeloablative therapy, as compared to neutrophils.
Truncated and Microparticle Free Soluble Tissue Factor Bound to Peripheral Monocytes Preferentially Activate Factor VII.
Authors: Khan Mohammad , Hattori Takashi , Niewiarowski Stefan , Edmunds L. Henry , Colman Robert W.
Published: October 2019

Soluble plasma tissue factor (TF) circulates in picomolar concentrations in healthy individuals and increases in a wide spectrum of diseases. This study tests the hypothesis that both truncated or long length TF antigens in low concentrations combine with monocytes or platelets to convert factor VII (fVII) to fVIIa. Both recombinant truncated TF (rsTF, kDa 33) and long length (47 kDa) plasma TF (pTF), obtained from pericardial wounds of patients having cardiac surgery using cardiopulmonary bypass, were studied in association with blood cells and TF bearing microparticles. Tissue factor was measured by ELISA. RsTF binds to erythrocytes, platelets, mononuclear cells and neutrophils. The rate of fVII conversion with rsTF (1.0 – 103nmoles/L) is fastest with mononuclear cells, less with platelets, minimal with neutrophils and undetectable with erythrocytes. Both unstimulated and stimulated mononuclear cells or platelets in the presence of 3.5 pmoles/L rsTF or pTF convert fVII (10 nmoles/L) to fVIIa, but the amounts of fVIIa produced is greater in mononuclear cells. When leukocytes or platelets are absent, microparticles associated with 3.5 pmoles/L TF antigen, derived from pericardial wound plasma, do not activate fVII. Stimulated mononuclear cells convert nearly all available fVII (10 nmoles/L) to fVIIa with 3.5 pmoles/L pTF. Unstimulated mononuclear cells convert small amounts of fVII with as little as 1 pmole/L rsTF but no VIIa is produced by platelets, neutrophils or erythrocytes under these conditions. At all concentrations (0.001 to 1m moles/L) mononuclear cells more efficiently convert fVII to fVIIa than platelets. This study shows that stimulated mononuclear cells provide the most efficient phospholipid platform for activation of truncated or long length molecules of soluble TF at low concentrations of TF antigen. The results suggest a new mechanism by which soluble TF, which is increased in clinical inflammatory diseases, may lead to a prothrombotic state.
Prodefensin in Plasma of Patients with Chronic Myeloproliferative Disorders. A Parameter of Myelopoietic Activity.
Authors: Kamp Søren , Jensen Morten Krogh , Borregaard Niels
Published: October 2019

Prodefensins (proHNPs) are the 74 aa antimicrobially inactive precursor peptides of defensins (HNPs). ProHNPs are synthesized in neutrophil precursors from the late promyelocytic stage and until the band cell stage. In promyelocytes, proHNP is proteolytically cleaved to yield 29–30 aa HNPs that are efficiently stored in primary (azurophil) granules. Beyond the promyelocytic stage, proHNPs are no longer proteolytically processed and significant amounts of proHNP escape the cell through constitutive exocytosis. The fate and biological implications of proHNP constitutively secreted from myeloid cells is unclear. We have raised antibodies against a synthetic propiece of proHNP (45 N-terminal amino acids) and developed an accurate, sensitive and specific ELISA for proHNP using these antibodies, with a detection limit of 4.0 ng/ml. We found levels of pro HNP in bone marrow plasma of 9 μg/ml and levels in blood plasma of 1.7 μg/ml. This indicates that the bone marrow is an important organ for generation of this pro-antibiotic molecule giving rise to plasma concentrations in the same order of magnitude as lysozyme and hCAP-18. Studies of plasma from patients with ph- chronic myeloproliferative disorders showed a mean concentration of 3.5 μg/ml indicating that proHNP is a parameter of myelopoietic activity. ProHNP in plasma was apparently not complexed to higher molecular weight structures, yet it was not present in urine. This indicates that proHNP from bone marrow is processed in circulation.
Hemoglobin and All-Cause Mortality: Results from the Baltimore Longitudinal Study of Aging (BLSA).
Authors: Longo Dan L. , Ble Alessandro , Metter Jeffrey E. , Guralnik Jack M. , Woodman Richard C. , Ferrucci Luigi
Published: October 2019

Whether mild-to-moderate hemoglobin (Hb) reduction is associated with increased mortality independent of underlying diseases is still debated. Two epidemiological studies addressing this issue in high risk populations (disabled older women, and persons 85 years or older) reported conflicting results. The BLSA offers the opportunity to investigate this association in an adult generally healthy population. The BLSA is a large cohort study on aging started in 1958. The participants are volunteers living in the Baltimore-Washington area (USA), well educated and self-describing as generally healthy persons. Participants are examined every 1-2 years. In the present analysis, all participants evaluated at least one occasion for Hb were included. Subjects were not excluded for any pre-existing conditions or specific outcomes. Hb levels were measured in 2979 participants recruited from 1958 to 2002 (1793 men and 1186 women, 78.7% Caucasians, age range: 18–96 years). Mean age at initial evaluation was 52.3 (±17.4) years for women and 52.1 (±17.9) for men, and the average time from baseline to death/censoring was 17.6 years for men and 12.4 years for women. Crude mortality rates were 21/1000 person years for men and 9.7/1000 person years for women. After adjusting for age, a U-shaped relationship (obtained fitting a Cox model with a Poisson regression approach according Therneau & Grambsch, 2000) was observed between Hb concentration and mortality with nadir at Hb=12.8g/dL in women and 15.0g in men. In the same model, significant excess of mortality was associated with Hb ≤ 11.2g/dL (95%CI 9.8g/dL–12.3 g/dL) and ≥ 14.0 g/dL (95%CI 13.5g/dL–14.9g/dL) in women, and ≤ 13.4 g/dL (95% CI 12.5g/dL–14.4g/dL) and ≥ 16.2g/dL (95% CI 15.4g/dL–17.2g/dL) in men. After adjusting for demographics, anthropometrics, smoking status, radiation exposure, and co-morbid diseases (heart disease, stroke, pulmonary disease, cancer, diabetes, hypertension), men with Hb ≤ 13.4 g/dL (mean: 12.7 g/dL, SD: 0.6) were at 30% higher risk (RR: 1.31, 95%CI: 1.08–1.58) compared to the reference group (Hb: 13.4–16.2). Accordingly, women with Hb ≤ 11.2 g/dL (mean: 10.2 g/dL, SD: 1.0) showed 100% greater risk compared to the reference group (Hb: 11.2–14.0), however this association was not statistically significant (RR: 2.01, 95% CI: 0.88–4.63). When men with Hb levels <10g/dL and women with <9 g/dL were excluded the results did not substantially change. Our findings showed that in the general population moderately-low Hb levels per se were associated with increased mortality risk. The impact of anemia correction on mortality in the elderly remains to be determined.
Homozygous JAK2 V617P Gain-of-Function Mutation Is Responsible for Constitutive JAK2/STAT5 Activation and Proliferation of HEL.92.1.7. Cell Line.
Authors: Estes Myka L. , Ozawa Yuki , Williams Ann H. , List Alan F. , Sokol Lubomir
Published: October 2019

HEL (Human erythroleukemia) is a growth factor independent erythroleukemic cell line established from the bone marrow of a patient with relapsed Hodgkin disease after autologous bone marrow transplantation (Martin P & Papayannopoulou T: Science1982;216:1233–1235). HEL cells display a block in differentiation at the level of common erythroid-megakaryocytic progenitor and have been commonly used as a model to study erythroid and megakaryocytic differentiation. Liu RY et al. (Blood1999;93:2369–2379) reported that constitutive activation of JAK2 tyrosine kinase in Dami/HEL cell line correlated with factor independent growth. Recently, several groups of investigators described a point mutation V617P in the autoinhibitory regulatory (JH2) domain of JAK2 tyrosine kinase in patients with myeloproliferative disorders (MPD). This acquired mutation results in constitutive activation of the JAK2/STAT5 pathway in hematopoietic cells. To date, the V617P mutation represents the most common causative genetic defect detected in patients with Philadelphia chromosome negative MPD. Interestingly, rare cases of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) were found to have the same mutation. We tested several hematopoietic cell lines including HL-60, HEL, KG-1, K-562, TF-1, U937 and UT-7 for the presence of V617P mutation using RFLP and automatic DNA sequencing. We found the homozygous mutation V617P only in HEL cells. This cell line has a very complex karyotype suggesting that a defect of multiple oncogenes or antioncogenes could be responsible for sustained cell proliferation. We employed the JAK2 inhibitor, AG490 at concentrations ranging from 0 to 100 mM and measured proliferation of HEL and control K562 cell lines using MTT assay. We observed dose-dependent inhibition of HEL cell proliferation with low micromolar concentrations of AG490 in contrast to K562 control cell line, harboring bcr-abl fusion tyrosine kinase. IC50 was 13 mM for HEL cells and 65 mM for control K562 cells, respectively. Low concentrations of AG490 significantly decreased phosphorylation of JAK2 and STAT5 in HEL cells but not in control K562 cell line, where bcr-abl activates separately JAK2 and STAT5 pathway. Exposure of HEL cells to AG490 [0–50mM] induced apoptosis as measured by annexin V labeling in a concentration dependent fashion. No significant increase in apoptosis was detected in K562 cells using similar concentrations of the JAK2 inhibitor. Overall, these results suggest that the mechanism of proliferation of the HEL cell line is driven by constitutive activation of JAK2 tyrosine kinase secondary to V617P gain-of-function mutation. Our data provides further evidence that the JAK2/STAT5 intracellular signaling pathway is preserved in this cell line. Thus, HEL cells can serve as a model to test novel JAK2 specific inhibitors in preclinical studies.
Effect of Bax G125A Single Nucleotide Polymorphism (SNP) on Bax Gene Expression in Diffuse Large Cell Lymphoma.
Authors: Saxena Anurag , Bosch Mark , Qureshi Tania , Nokhrina Kateryna , Moshynska Oksana
Published: October 2019

Altered expression of Bax protein has been shown to be predictive of overall and disease free-survival of patients with diffuse large B-cell lymphoma (DLCL)(Am J Hematol, 2003; 73:101–7). Further, absence of Bax and low levels of Bcl-2 have been shown to be associated with a more aggressive phenotype (P<0.05) (Blood, 1997; 90:3173–8) in patients with DLCL. We have previously reported a single nucleotide polymorphim (SNP), G125A, in the 5′-untranslated region of the BAX promoter and its association with reduced protein expression and treatment resistance in B-cell chronic lymphocytic leukemia (Cancer Lett2002; 187:199–205). The aim of the present study was to evaluate BAX SNP incidence and its association with the protein expression in lymphomas. DNA was extracted from fifty lymphoma samples (31 DLCL, seven follicular lymphomas (FL), three marginal zone lymphomas (MZL), four T-cell lymphomas, three Hodgkin’s lymphomas, and two cases of Mantle cell lymphoma (MCL), and from 55 control samples. BAX SNP was detected by restriction enzyme analysis (J Clin Pathol, 2003; 56: 205–9). Fourteen out of 50 (28%) samples had G125A SNP, in all cases only one allele was replaced. It was present in eight (26%) DLCL, two (29%) FL, in two MZL, in one T-cell lymphoma, in one HL, and none of MCL had this SNP. Eight out of 55 (14.5%) controls had the SNP. The presence of BAX promoter SNP was associated with reduced Bax protein expression as detected by Western blot analysis (P=0.024). Two plasmids with G and A nucleotides at position 125 of the BAX promoter linked to luciferase as a reporter were constructed and used for transfection, as well as the pGL3-Basic and pGL3-Control plasmids; pRL-SV40 plasmid was used for co-transfection. Dual-Glo luciferase assay was performed and Firefly luciferase activity was normalized to Renilla luciferase. In transfection experiments, the G125A SNP reduced expression (P=0.0202) of BAX promoter. BAX SNP, G125A, present in approximately a quarter of DLCL has a direct negative effect on BAX gene expression.
Point Mutations Found in the Juxtamembrane Domain of FLT3 Define a New Class of Activating Mutations in AML.
Authors: Reindl Carola , Bagrintseva Ksenia , Schnittger Susanne , Ellwart Joachim W. , Wenig Katja , Hopfner Karl-Peter , Hiddemann Wolfgang , Spiekermann Karsten
Published: October 2019

In acute myeloid leukemia (AML) FMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one third of patients. Until now, two distinct activating mutations are known: FLT3-length mutations (FLT3-LM) in the juxtamembrane (JM) domain in 20–25% and FLT3-point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7–10% of patients. Here, we have characterized a new class of activating point mutations (PM) that cluster in a 16 amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PM).

Stable expression of four distinct FLT3-JM-PM in IL-3 dependent murine Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3-ligand and resistance to apoptotic cell death compared to FLT3-WT-expressing cells. As a molecular mechanism, we could show activation of STAT5 and upregulation of Bcl-x(L) by all FLT3-JM-PM. A selective FLT3-inhibitor, PKC412, was able to abrogate the factor-independent growth of FLT3-JM-PM. Mapping of the FLT3-JM-PM on the crystal structure of FLT3 showed that these mutations probably reduce the stability of the JM domain in the autoinhibitory conformation, and provide a structural basis for their transforming capacity.

Our results show that point mutations in the autoinhibitory JM domain represent a new class of gain-of-function mutations able to activate the transforming potential of FLT3.
Aberrant Pre-mRNA Processing Due to Novel Splicing Mutations in the CBL Proto-Oncogene in Acute Myeloid Leukemia.
Authors: Arnoczky Kristy J. , Marburger Trent B. , Vukosavljevic Tamara , Caligiuri Michael A. , Whitman Susan P.
Published: October 2019

Ubiquitin (Ub)-dependent proteolysis is a highly controlled pathway that regulates almost all cellular processes by modulating the levels of key proteins. Several E3 Ub ligases are functionally abnormal in cancer. The E3 ubiquitin ligase, Casitas B-lineage lymphoma (CBL) protein, negatively regulates many receptor tyrosine kinases (RTKs) that are frequently over-expressed and/or constitutively activated (e.g., FLT3) in acute myeloid leukemia (AML). The CBL linker region, encoded in part by exon 8, lies between the tyrosine kinase binding and the RING finger domains and, mutations introduced into this linker region were previously shown to confer transforming ability. Herein, we describe novel splicing mutations in the c-CBL gene that lead to aberrant pre-mRNA processing in AML. DNA PCR revealed a splice site mutation whereby 14 nucleotides were deleted at the exon 8-intron 8 boundary in one CBL allele of the MOLM-13 cell line. Out of 18 patient samples, one primary AML blast sample had a 4 base pair deletion (16 bases upstream of the intron 7-exon 8 junction) which was replaced by a 12 base pair GC-rich insertion. We hypothesized that this mutation was an intronic splicing silencer element. RT-PCR/sequencing confirmed aberrant pre-mRNA processing in both the AML patient sample and MOLM-13 cells as they expressed a wild-type CBL transcript and an in-frame variant lacking exon 8. The splice variants are reminiscent of the oncogenic Cbl intragenic deletion mutation present in the murine pre-B cell line, p70Z. Such Cbl mutants are suggested to result in a structural alteration, allowing displacement of wild-type Cbl from the RTK complex thereby abrogating Cbl’s negative regulatory function. Thus it is possible that this mutant may enhance proliferation and survival of AML cells through attenuation of ubiquitin/proteosome-dependent degradation of receptor tyrosine kinases. Further investigation into the role of CBL and other players in the Ub-mediated proteolytic pathway is likely to provide additional insight into a subset of AML patients that in turn could lead to discovery of new therapeutic targets and strategies.
Increased Superoxide Levels, HO-1 and Fyn Protein Expression in Bcr/abl Overexpressing Cells: Implications for Adaptation and Survival in an Oxidatively Challenging Environment.
Authors: Chandra Joya , Ban Kechen
Published: October 2019

The bcr/abl oncogene is present in the majority of CML and in a subset of ALL patients. In addition to anti-apoptotic properties, proliferative advantages and growth factor independence, the presence of the Bcr/abl kinase raises intracellular levels of reactive oxygen species (ROS). Increased ROS in CML have been linked to DNA damage and DNA repair deficiencies with implications for promoting genomic instability and secondary mutations associated with disease progression. Here we show that superoxide is the specific ROS entity that is elevated when Bcr/abl is overexpressed in an IL-3 dependent pre-B cell line, BaF3. Strikingly, treatment with classical oxidants, such as hydrogen peroxide or tert-butyl peroxide, demonstrated an inherent resistance to oxidative stress induced apoptosis in Bcr/abl-containing cells, suggesting that antioxidant defenses activated by Bcr/abl as a response to the increased endogenous ROS production may promote survival in Bcr/abl positive populations.

In an effort to characterize the redox environment in Bcr/abl containing cells, we examined several antioxidant enzymes that are known downstream substrates of c-abl or bcr/abl, including peroxiredoxin, catalase and heme oxygenase-1 (HO-1). Of these, HO-1 protein expression was three fold higher in Bcr/abl transductants, consistent with a reported role for HO-1 as a Bcr/abl dependent survival factor. Interestingly, the Src family kinase, Fyn, which can be activated by oxidative stress, was increased four fold in Bcr/abl overexpressing cells. This is consistent with reported microarray data in which fyn mRNA levels are higher in Philadelphia positive leukemias than non-Philapdelphia positive leukemias. Other components of ROS responsive signaling pathways were also interrogated including the Mapk pathway and Akt pathway, with no difference noted between parental and Bcr/abl overexpressing cells.

Our data suggest a relationship between Bcr/abl-induced superoxide elevation, resistance to oxidative stress, elevation of HO-1 (which has antioxidant properties) and elevated Fyn (a putative downstream target for ROS production). These redox alterations and their consequences may equip Bcr/abl positive cells with a survival and/or proliferative advantage in an oxidatively challenging environment, thus promoting disease progression and blast crisis.