bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Biochemical Changes During In Vivo Erythrocyte Senescence in the Dog
Authors: Rettig Michael P. , Low Philip S. , Gimm J. Aura , Mohandas Narla , Wang Jiazhen , Christian John A.
Published: October 2019

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC’s life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, ≈115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age ≥110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs ≥112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.
Total Therapy With Tandem Transplants for Newly Diagnosed Multiple Myeloma
Authors: Barlogie B. , Jagannath S. , Desikan K.R. , Mattox S. , Vesole D. , Siegel D. , Tricot G. , Munshi N. , Fassas A. , Singhal S. , Mehta J. , Anaissie E. , Dhodapkar D. , Naucke S. , Cromer J. , Sawyer J. , Epstein J. , Spoon D. , Ayers D. , Cheson B. , Crowley J.
Published: October 2019

Between August 1990 and August 1995, 231 patients (median age 51, 53% Durie-Salmon stage III, median serum β-2-microglobulin 3.1 g/L, median C-reactive protein 4 g/L) with symptomatic multiple myeloma were enrolled in a program that used a series of induction regimens and two cycles of high-dose therapy (“Total Therapy”). Remission induction utilized non–cross-resistant regimens (vincristine-doxorubicin-dexamethasone [VAD], high-dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor with peripheral blood stem cell collection, and etoposide-dexamethasone-cytarabine-cisplatin). The first high-dose treatment comprised melphalan 200 mg/m2 and was repeated if complete (CR) or partial (PR) remission was maintained after the first transplant; in case of less than PR, total body irradiation or cyclophosphamide was added. Interferon--2b maintenance was used after the second autotransplant. Fourteen patients with HLA-compatible donors underwent an allograft as their second high-dose therapy cycle. Eighty-eight percent completed induction therapy whereas first and second transplants were performed in 84% and 71% (the majority within 8 and 15 months, respectively). Eight patients (3%) died of toxicity during induction, and 2 (1%) and 6 (4%) during the two transplants. True CR and at least a PR (PR plus CR) were obtained in 5% (34%) after VAD, 15% (65%) at the end of induction, and 26% (75%) after the first and 41% (83%) after the second transplants (intent-to-treat). Median overall (OS) and event-free (EFS) survival durations were 68 and 43 months, respectively. Actuarial 5-year OS and EFS rates were 58% and 42%, respectively. The median time to disease progression or relapse was 52 months. Among the 94 patients achieving CR, the median CR duration was 50 months. On multivariate analysis, superior EFS and OS were observed in the absence of unfavorable karyotypes (11q breakpoint abnormalities, -13 or 13-q) and with low β-2-microglobulin at diagnosis. CR duration was significantly longer with early onset of CR and favorable karyotypes. Time-dependent covariate analysis suggested that timely application of a second transplant extended both EFS and OS significantly, independent of cytogenetics and β-2-microglobulin. Total Therapy represents a comprehensive treatment approach for newly diagnosed myeloma patients, using multi-regimen induction and tandem transplantation followed by interferon maintenance. As a result, the proportion of patients attaining CR increased progressively with continuing therapy. This observation is particularly important because CR is a sine qua non for long-term disease control and, eventually, cure.
Etretinate Therapy for Refractory Sclerodermatous Chronic Graft-Versus-Host Disease
Authors: Marcellus D.C. , Altomonte V.L. , Farmer E.R. , Horn T.D. , Freemer C.S. , Grant J. , Vogelsang G.B.
Published: October 2019

Chronic graft-versus-host disease (GVHD) is the most common late complication of allogeneic bone marrow transplantation (BMT). The sclerodermatous form of the disease is often refractory to standard treatment modalities. Based on reports of response to etretinate, a synthetic retinoid, among patients with scleroderma, we have added etretinate to the treatment regimen of 32 patients with refractory sclerodermatous chronic GVHD. This case series is comprised mainly of patients who had chronic GVHD of long duration (median of 30 months before the initiation of etretinate). Most had failed to respond to three or more agents before etretinate treatment was started. Clinical response was assessed after 3 months of therapy. Five patients did not complete a 3-month trial. Among the 27 patients evaluable for response, 20 showed improvement including softening of the skin, flattening of cutaneous lesions, increased range of motion, and improved performance status. Four showed no response after 3 months of therapy and 3 had progression of their sclerosis. Overall, etretinate has been fairly well tolerated in our patients, with skin breakdown and/or ulceration leading to its discontinuation in 6 patients. We believe the results in our patients are encouraging and suggest that further evaluation of etretinate in the treatment of sclerodermatous chronic GVHD is warranted.
Monocyte Arrest and Transmigration on Inflamed Endothelium in Shear Flow Is Inhibited by Adenovirus-Mediated Gene Transfer of IκB-
Authors: Weber Kim S.C. , Draude Georg , Erl Wolfgang , de Martin Rainer , Weber Christian
Published: October 2019

Mobilization of nuclear factor-κB (NF-κB) activates transcription of genes encoding endothelial adhesion molecules and chemokines that contribute to monocyte infiltration critical in atherogenesis. Inhibition of NF-κB has been achieved by pharmacological and genetic approaches; however, monocyte interactions with activated endothelium in shear flow following gene transfer of the NF-κB inhibitor IκB- have not been studied. We found that overexpression of IκB- in endothelial cells using a recombinant adenovirus prevented tumor necrosis factor- (TNF-)–induced degradation of IκB- and suppressed the upregulation of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin mRNA and surface protein expression and the upregulation of transcripts for the chemokines monocyte chemoattractant protein 1 (MCP-1) and growth-related activity- (GRO-) by TNF-. This was associated with a reduction in endothelial MCP-1 secretion and GRO- immobilization. Adhesion assays under physiological shear flow conditions showed that firm arrest, spreading, and transmigration of monocytes on TNF-–activated endothelium was markedly inhibited by IκB- overexpression. Inhibition with monoclonal antibodies and peptide antagonists inferred that this was due to reduced expression of Ig integrin ligand as well as of chemokines specifically involved in these events. In contrast, rolling of monocytes was increased by IκB- transfer and was partly mediated by P-selectin; however, it appeared to be unaffected by the inhibition of E-selectin induction. Thus, our data provide novel evidence that selective modulation of NF-κB by adenoviral transfer of IκB- impairs the expression of multiple endothelial gene products required for subsequent monocyte arrest and emigration in shear flow and thus for monocyte infiltration in atherosclerotic plaques.
CrkL Activates Integrin-Mediated Hematopoietic Cell Adhesion Through the Guanine Nucleotide Exchange Factor C3G
Authors: Arai Ayako , Nosaka Yurika , Kohsaka Hitoshi , Miyasaka Nobuyuki , Miura Osamu
Published: October 2019

CrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (4β1) and VLA-5 (5β1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.
Protein Kinase B (c-Akt), Phosphatidylinositol 3-Kinase, and STAT5 Are Activated by Erythropoietin (EPO) in HCD57 Erythroid Cells But Are Constitutively Active in an EPO-Independent, Apoptosis-Resistant Subclone (HCD57-SREI Cells)
Authors: Bao Haifeng , Jacobs-Helber Sarah M. , Lawson Amy E. , Penta Kalyani , Wickrema Amittha , Sawyer Stephen T.
Published: October 2019

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.
Vascular Endothelial Cell Growth Factor–Induced Tissue Factor Expression in Endothelial Cells Is Mediated by EGR-1
Authors: Mechtcheriakova Diana , Wlachos Alexander , Holzmüller Harry , Binder Bernd R. , Hofer Erhard
Published: October 2019

Vascular endothelial cell growth factor (VEGF) is a major regulator of angiogenesis. We report here that treatment of endothelial cells with VEGF leads to upregulation of tissue factor mRNA and protein expression on the cell surface. Reporter gene studies show that transcriptional activation of the tissue factor gene by VEGF is mediated by a GC-rich promoter element containing overlapping binding sites for Sp1 and EGR-1. As shown by immunofluorescence and electrophoretic mobility shift assays, upon VEGF treatment EGR-1 rapidly accumulates in the nucleus and binds to its respective recognition site in the tissue factor promoter. Sp1 occupies this element in unstimulated cells and seems to be partially displaced by increasing amounts of EGR-1. Transfection of endothelial cells with an EGR-1 expression plasmid mimics the upregulation of tissue factor transcription observed after VEGF treatment. In contrast, NFκB, the major transcription factor involved in tissue factor upregulation by inflammatory stimuli, is not activated by VEGF. These data show that VEGF induces a response in endothelial cells largely distinct from inflammatory stimuli, and suggest that EGR-1 is a major mediator of the activation of the tissue factor and possibly other VEGF-responsive genes.
Endothelial Cells Undergoing Apoptosis Become Proadhesive for Nonactivated Platelets
Authors: Bombeli Thomas , Schwartz Barbara R. , Harlan John M.
Published: October 2019

Under normal conditions, platelets do not adhere to endothelium. However, when platelets or endothelial cells are stimulated by thrombin or cytokines, respectively, platelets bind avidly to endothelium. Because there is accumulating evidence that endothelial cells may become apoptotic under certain proinflammatory or prothrombotic conditions, we investigated whether endothelial cells undergoing apoptosis may become proadhesive for nonactivated platelets. Human umbilical vein endothelial cells (HUVEC) were induced to undergo apoptosis by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum-deprivation. After treatment of HUVEC or platelets with different receptor antagonists, nonactivated, washed human platelets were allowed to adhere to HUVEC for 20 minutes. To exclude matrix involvement, platelet binding was measured in suspension by using flow cytometry. Independent of the method of apoptosis induction, there was a marked increase in platelet binding to apoptotic HUVEC. Although HUVEC exhibited maximal adhesiveness for platelets after 2 to 4 hours, complete DNA fragmentation of HUVEC occurred only several hours later. Adhesion assays after blockade of different platelet receptors showed only involvement of β1-integrins. Platelet binding to apoptotic HUVEC was inhibited by more than 70% when platelets were treated with blocking anti-β1 antibodies. Treatment of apoptotic HUVEC with blocking antibodies to different potential platelet receptors, including known ligands for β1-integrins, did not affect platelet binding. As assessed by determination of β-thromboglobulin and platelet factor 4 in the supernatants, platelets bound to apoptotic HUVEC became slightly activated. However, significant expression of platelet P-selectin (CD62P) was not found. These data provide further evidence that endothelial cells undergoing apoptosis may contribute to thrombotic events.
Somatic Mutations Within the Untranslated Regions of Rearranged Ig Genes in a Case of Classical Hodgkin’s Disease as a Potential Cause for the Absence of Ig in the Lymphoma Cells
Authors: Jox Andrea , Zander Thomas , Küppers Ralf , Irsch Johannes , Kanzler Holger , Kornacker Martin , Bohlen Heribert , Diehl Volker , Wolf Jürgen
Published: October 2019

Hodgkin–Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin’s disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vκ3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vκ3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vκ3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3′ splice site of the leader intron and adjacent nucleotides in the rearranged Vκ light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.
Effects of the Tyrosine Kinase Inhibitor AG957 and an Anti-Fas Receptor Antibody on CD34+ Chronic Myelogenous Leukemia Progenitor Cells
Authors: Carlo-Stella Carmelo , Regazzi Ester , Sammarelli Gabriella , Colla Simona , Garau Daniela , Gazit Aviv , Savoldo Barbara , Cilloni Daniela , Tabilio Antonio , Levitzki Alexander , Rizzoli Vittorio
Published: October 2019

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210BCR/ABLtyrosine phosphorylation. We report here that preincubation of CML or normal CD34+ cells with graded concentration of AG957 (1 to 100 μmol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L;P = .004), colony-forming unit–granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P = .004), and LTC-IC (43 v 181 μmol/L; P = .004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 μmol/L significantly reduced the mean (±SD) percentage of BCR/ABL-positive progenitors (92% ± 10% v 33 ± 5%;P = .001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34+ cells with AG957 (1 μmol/L) and CH11 (1 μg/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34+ cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti–Fas-R antibody CH11. These data may have significant therapeutic applications.
Cladribine Activity in Adult Langerhans-Cell Histiocytosis
Authors: Saven Alan , Burian Carol
Published: October 2019

Langerhans-cell histiocytosis (LCH) results from the accumulation of tissue histiocytes derived from the same progenitor cells as monocytes. Because cladribine is potently toxic to monocytes, we conducted a phase II trial of cladribine. Cladribine was administered to 13 LCH patients at 0.14 mg/kg per day by 2-hour intravenous infusion for 5 consecutive days, every 4 weeks for a maximum of six courses. Median age was 42 years (range, 19 to 72) and median pretreatment disease duration was 99 months (range, 6 to 252). One patient was untreated, one had received prior prednisone only, one prior radiation only, six prior radiation and chemotherapy, and four prior surgery, radiation, and chemotherapy. Seven patients had cutaneous involvement, six multifocal osseous, six pulmonary, two each with soft tissue and nodal involvement, and four had diabetes insipidus. Of 13 patients, 12 were evaluable for response and all for toxicity. After a median of three courses (range, 1 to 6), seven (58%) patients achieved complete responses (two pathologic and five clinical) and two (17%) patients achieved partial responses; overall response rate, 75%. Median response follow-up duration was 33 months (range, 1 to 65). Seven patients experienced grade 3 to 4 neutropenia. Only one patient had a documented infection, dermatomal herpes zoster. At a median follow-up of 42 months (range, 5 to 76), 12 patients remain alive and one patient has died. Thus, cladribine has major activity in adult LCH and warrants further investigation in both pediatric and adult LCH as a single agent and in combination with other drugs.
Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation
Authors: Ilaria Robert L. , Hawley Robert G. , Van Etten Richard A.
Published: October 2019

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.
The Expression of Human Blood Group Antigens During Erythropoiesis in a Cell Culture System
Authors: Southcott Mark J.G. , Tanner Michael J.A. , Anstee David J.
Published: October 2019

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.
Enhanced Endothelial Cell Apoptosis in Splenic Tissues of Patients With Thrombotic Thrombocytopenic Purpura
Authors: Dang Chau T. , Magid Margret S. , Weksler Babette , Chadburn Amy , Laurence Jeffrey
Published: October 2019

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy of obscure etiology. The fundamental pathologic lesion is a hyaline thrombus composed of platelets and some fibrin accompanied by endothelial cell proliferation and detachment, in the absence of an inflammatory response. We have previously demonstrated that plasmas from patients with both idiopathic TTP and a related disorder, sporadic hemolytic-uremic syndrome (HUS), induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) in vitro in those lineages of microvascular endothelial cells (MVECs) that are affected pathologically. We now demonstrate the presence of enhanced MVEC apoptosis in splenic tissues from patients with TTP, documented by terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL) and morphology. This is accompanied by elevated Fas expression. It contrasts with the absence of apoptosis in splenic tissues obtained after splenectomy for trauma or immune thrombocytopenic purpura. TUNEL-positive cells, identified by immunohistochemistry as MVECs or macrophages, presumably engulfing apoptotic ECs, are noted in numerous areas, including those apart from microthrombi. Thus, it is unlikely that EC apoptosis is simply a sequela of thrombus formation. Based on these data, we propose that MVEC apoptosis is of pathophysiologic significance in idiopathic TTP/sporadic HUS.
Muc-1 Core Protein Is Expressed on Multiple Myeloma Cells and Is Induced by Dexamethasone
Authors: Treon Steven P. , Mollick Joseph A. , Urashima Mitsuyoshi , Teoh Gerrard , Chauhan Dharminder , Ogata Atsushi , Raje Noopur , Hilgers Joseph H.M. , Nadler Lee , Belch Andrew R. , Pilarski Linda M. , Anderson Kenneth C.
Published: October 2019

Monoclonal antibodies (MoAbs) that selectively identify Muc-1 core protein (MoAbs DF3-P, VU-4H5) determinants were used to identify the Muc-1 glycoform present on 7 multiple myeloma (MM) cell lines, 5 MM patient plasma cells, 12 MM patient B cells, as well as 32 non-MM cell lines and normal hematopoietic cells. Flow cytometry studies demonstrated that all MM cell lines, MM patient plasma cells, and MM patient B cells expressed Muc-1 core protein epitopes. Circulating B cells from 4 normal donors also expressed Muc-1 core protein. In contrast, Muc-1 core protein was absent on 28 of 32 non-MM neoplastic cell lines, 17 of which expressed Muc-1. Splenic and tonsillar B cells, CD34+ stem cells, resting T cells, and bone marrow plasma cells obtained from normal donors both lacked Muc-1 glycoforms. We next studied the effects of estrogen, progesterone, and glucocorticoid receptor agonists and antagonists on Muc-1 expression, because consensus sequences for the response elements of these steroids are present on the Muc-1 gene promoter. These studies showed that dexamethasone (Dex) induced Muc-1 expression on MM cell lines, as determined by both flow cytometry and Western blot analyses. Dex also induced upregulation of Muc-1 on prostate and ovarian cancer cell lines. Time and dose-response studies demonstrated that Dex induced maximal cell surface Muc-1 expression by 24 hours at concentrations of 10−8 mol/L. Dex induced Muc-1 upregulation could be blocked with a 10-fold excess of the glucocorticoid receptor antagonist RU486, confirming that Dex was acting via the glucocorticoid receptor. No changes in Muc-1 expression were observed on MM cells treated with estrogen and progesterone receptor agonists and antagonists or with RU486. These studies provide the framework for targeting Muc-1 core protein in vaccination and serotherapy trials in MM. In addition, the finding that Muc-1 expression on MM cells can be augmented by Dex at pharmacologically achievable levels suggests their potential utility in enhancing treatments targeting Muc-1 in MM.
The t(6;8)(q27;p11) Translocation in a Stem Cell Myeloproliferative Disorder Fuses a Novel Gene, FOP, to Fibroblast Growth Factor Receptor 1
Authors: Popovici Cornel , Zhang Bin , Grégoire Marie-José , Jonveaux Philippe , Lafage-Pochitaloff Marina , Birnbaum Daniel , Pébusque Marie-Josèphe
Published: October 2019

In patients with an atypical stem-cell myeloproliferative disorder with lymphoma (B or T cell), myeloid hyperplasia, and eosinophilia, the chromosome 8p11-12 region is the site of a recurrent breakpoint that can be associated with three different partners, 6q27, 9q32-34, and 13q12. Rearrangements are supposed to affect a pluripotent stem cell capable of myeloid and lymphoid differentiation and to involve the same 8p11-12 gene. The t(8;13) translocation has recently been shown to result in a fusion between the FGFR1 gene that encodes a tyrosine kinase receptor for fibroblast growth factors and a novel gene, FIM (also called RAMP or ZNF198), belonging to a novel family of zinc finger genes. In the present study, we have cloned the t(6;8)(q27;p11) translocation in two patients and found a fusion between FGFR1 and a novel gene, FOP(FGFR1Oncogene Partner), located on chromosome band 6q27. This gene is alternatively spliced and ubiquitously expressed. It encodes a protein containing two regions of putative leucine-rich repeats putatively folding in -helices and separated by a hydrophobic spacer. The two reciprocal fusion transcripts were evidenced by reverse transcription-polymerase chain reaction in the tumoral cells of the patients. The predicted chimeric FOP-FGFR1 protein contains the FOP N-terminus leucine-rich region fused to the catalytic domain of FGFR1. It may promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1.
Prospective Randomized Multicenter Study Comparing Cyclosporin Alone Versus the Combination of Antithymocyte Globulin and Cyclosporin for Treatment of Patients With Nonsevere Aplastic Anemia: A Report From the European Blood and Marrow Transplant (EBMT) Severe Aplastic Anaemia Working Party
Authors: Marsh J. , Schrezenmeier H. , Marin P. , Ilhan O. , Ljungman P. , McCann S. , Socie G. , Tichelli A. , Passweg J. , Hows J. , Raghavachar A. , Locasciulli A. , Bacigalupo A.
Published: October 2019

We report the results of the first prospective randomized multicenter study of immunosuppressive treatment in patients with previously untreated nonsevere aplastic anemia (AA) as defined by a neutrophil count of at least 0.5 × 109/L and transfusion dependence. Patients were randomized to receive cyclosporin (CSA) alone or the combination of horse antithymocyte globulin ([ATG] Lymphoglobuline; Merieux, Lyon, France) and CSA. The endpoint of the study was the hematologic response at 6 months. One hundred fifteen patients were randomized and assessable with a median follow-up period of 36 months; 61 received CSA and 54 ATG and CSA. In the CSA group, the percentage of complete and partial responders was 23% and 23%, respectively, for an overall response rate of 46%. A significantly higher overall response rate of 74% was found in the ATG and CSA group, with 57% complete and 17% partial responders (P = .02). Compared with CSA alone, the combination of ATG and CSA resulted in a significantly higher median hemoglobin level and platelet count at 6 months. Fewer patients required a second course of treatment before 6 months due to a nonresponse. In the CSA group, 15 of 61 (25%) patients required a course of ATG before 6 months because of disease progression, compared with only 3 of 54 (6%) in the ATG and CSA group. The survival probabilities for the two groups were comparable, 93% (CSA group) and 91% (ATG and CSA group), but at 180 days, the prevalence of patients surviving free of transfusions, which excluded patients requiring second treatment because of nonresponse, death, disease progression, or relapse, was 67% in the CSA group and 90% in the ATG and CSA group (P = .001). We conclude that the combination of ATG and CSA is superior to CSA alone in terms of the hematologic response, the quality of response, and early mortality, and a second course of immunosuppression is less frequently required.
Dexamethasone-Induced Thymocyte Apoptosis: Apoptotic Signal Involves the Sequential Activation of Phosphoinositide-Specific Phospholipase C, Acidic Sphingomyelinase, and Caspases
Authors: Cifone Maria Grazia , Migliorati Graziella , Parroni Raffaella , Marchetti Cristina , Millimaggi Danilo , Santoni Angela , Riccardi Carlo
Published: October 2019

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.
Downregulation of JAK3 Protein Levels in T Lymphocytes by Prostaglandin E2 and Other Cyclic Adenosine Monophosphate-Elevating Agents: Impact on Interleukin-2 Receptor Signaling Pathway
Authors: Kolenko Vladimir , Rayman Patricia , Roy Biswajit , Cathcart Martha K. , O’Shea John , Tubbs Raymond , Rybicki Lisa , Bukowski Ronald , Finke James
Published: October 2019

The Janus kinase, JAK3 plays an important role in interleukin-2 (IL-2)–dependent signal transduction and proliferation of T lymphocytes. Our findings show that prostaglandin E2(PGE2) can inhibit upregulation of JAK3 protein in naive T cells and can downregulate its expression in primed cells. Reduction in JAK3 was selective because expression of other tyrosine kinases (JAK1, p56lck, and p59fyn) and signal transducer and activator of transcription (STAT)5, which are linked to IL-2 receptor (IL-2R) signaling pathway, were not affected. Inhibition of JAK3 may be controlled by intracellular cyclic adenosine monophosphate (cAMP) levels, as forskolin, a direct activator of adenylate cyclase and dibutyryl cAMP (dbcAMP), a membrane permeable analogue of cAMP suppressed JAK3 expression. Moreover, 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase, potentiated PGE2-induced suppression of JAK3. In naive T cells, but not primed T cells, PGE2 and other cAMP elevating agents also caused a modest reduction in surface expression of the common gamma chain (γc) that associates with JAK3. The absence of JAK3, but not IL-2R in T cells correlated with impaired IL-2–dependent signal transduction and proliferation. The alteration in IL-2 signaling included decreased tyrosine phosphorylation and DNA binding activity of STAT5 and poor induction of the c-Myc and c-Jun pathways. In contrast, IL-2–dependent induction of Bcl-2 was unaffected. These findings suggest that suppression of JAK3 levels may represent one mechanism by which PGE2 and other cAMP elevating agents can inhibit T-cell proliferation.
An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia
Authors: Alfarano A. , Indraccolo S. , Circosta P. , Minuzzo S. , Vallario A. , Zamarchi R. , Fregonese A. , Calderazzo F. , Faldella A. , Aragno M. , Camaschella C. , Amadori A. , Caligaris-Cappio F.
Published: October 2019

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.
Feasibility of Immunotherapy of Relapsed Leukemia With Ex Vivo–Generated Cytotoxic T Lymphocytes Specific for Hematopoietic System-Restricted Minor Histocompatibility Antigens
Authors: Mutis Tuna , Verdijk Rob , Schrama Ellen , Esendam Bennie , Brand Anneke , Goulmy Els
Published: October 2019

Allogeneic bone marrow transplantation (BMT) is a common treatment of hematologic malignancies. Recurrence of the underlying malignancy is a major cause of treatment failure. Donor-derived cytotoxic T lymphocytes (CTLs) specific for patients’ minor histocompatibility antigens (mHags) play an important role in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactivities. mHags HA-1 and HA-2 induce HLA-A*0201-restricted CTLs in vivo and are exclusively expressed on hematopoietic cells, including leukemic cells and leukemic precursors, but not on fibroblasts, keratinocytes, or liver cells. The chemical nature of the mHags HA-1 and HA-2 is known. We investigated the feasibility of ex vivo generation of mHag HA-1– and HA-2–specific CTLs from unprimed mHag HA-1– and/or HA-2–negative healthy blood donors. HA-1 and HA-2 synthetic peptide-pulsed dendritic cells (DCs) were used as antigen-presenting cells (APC) to stimulate autologous unprimed CD8+ T cells. The ex vivo–generated HA-1– and HA-2–specific CTLs efficiently lyse leukemic cells derived from acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patients. No lytic reactivity was detected against nonhematopoietic cells. Sufficient numbers of the CTLs can be obtained for the adoptive immunotherapy purposes. In conclusion, we present a feasible, novel therapy for the treatment for relapsed leukemia after BMT with a low risk of GVHD.
Influenza A Virus Accelerates Neutrophil Apoptosis and Markedly Potentiates Apoptotic Effects of Bacteria
Authors: Colamussi Maria Luisa , White Mitchell R. , Crouch Erika , Hartshorn Kevan L.
Published: October 2019

Neutrophils are recruited into the airway in the early phase of uncomplicated influenza A virus (IAV) infection and during the bacterial superinfections that are a significant cause of morbidity and mortality in IAV-infected subjects. In this report, we show that IAV accelerates neutrophil apoptosis. Unopsonized Escherichia colihad similar effects, although apoptotic effects of opsonized E coli were greater. When neutrophils were treated with both IAV and unopsonized E coli, a marked enhancement of the rate and extent of neutrophil apoptosis occured as compared with that caused by either pathogen alone. Treatment of neutrophils with IAV markedly increased phagocytosis of E coli. Simultaneous treatment of neutrophils with IAV and E coli also elicited greater hydrogen peroxide production than did either pathogen alone. IAV increased neutrophil expression of Fas antigen and Fas ligand, and it also increased release of Fas ligand into the cell supernatant. These findings may have relevance to the understanding of inflammatory responses to IAV in vivo and of bacterial superinfection of IAV-infected subjects.
Genetic and Pharmacological Analyses of Syk Function in IIbβ3 Signaling in Platelets
Authors: Law Debbie A. , Nannizzi-Alaimo Lisa , Ministri Kathleen , Hughes Paul E. , Forsyth Jane , Turner Martin , Shattil Sanford J. , Ginsberg Mark H. , Tybulewicz Victor L.J. , Phillips David R.
Published: October 2019

Agonists induce inside-out IIbβ3signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in IIbβ3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of IIbβ3 by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in IIbβ3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits IIbβ3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.