bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Acute Lymphoblastic Leukemia and Myeloproliferative Disorder in Transgenic Mice Expressing p210bcr/abl: A Novel Transgenic Model for Human Ph1-Positive Leukemias
Authors: Honda Hiroaki , Oda Hideaki , Suzuki Takahiro , Takahashi Tsuyoshi , Witte Owen N. , Ozawa Keiya , Ishikawa Takatoshi , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

The Philadelphia (Ph1) chromosome can be detected in chronic myelogenous leukemia (CML) and a significant number of acute lymphoblastic leukemia (ALL) cases. Generation of p210bcr/abl, a chimeric protein with enhanced kinase activity, is thought to be involved in the pathogenesis of these diseases. To elucidate the biological properties of p210bcr/abl and to create an animal model for human Ph1-positive leukemias, we generated transgenic mice expressing p210bcr/abl driven by the promoter of the tec gene, a cytoplasmic tyrosine-kinase preferentially expressed in the hematopoietic lineage. The founder mice showed excessive proliferation of lymphoblasts shortly after birth and were diagnosed as suffering from ALL based on surface marker and Southern blot analyses. Expression and enhanced kinase activity of the p210bcr/abl transgene product were detected in the leukemic tissues. In contrast, transgenic progeny exhibited marked granulocyte hyperplasia with thrombocytosis after a long latent period and developed myeloproliferative disorders (MPDs) closely resembling human CML. Expression of p210bcr/abl mRNA in the proliferating granulocytes was detected by RT-PCR. In particular, one MPD mouse showed remarkable proliferation of blast cells in the lung, which might represent an extramedullar blast crisis. The results demonstrate that the expression of p210bcr/abl in hematopoietic progenitor cells in transgenic mice can contribute to two clinically distinct hematopoietic malignancies, CML and ALL, indicating that this transgenic system provides a novel transgenic model for human Ph1-positive leukemias.
ALK-Positive Lymphoma: A Single Disease With a Broad Spectrum of Morphology
Authors: Benharroch Daniel , Meguerian-Bedoyan Zarouhie , Lamant Laurence , Amin Chauki , Brugières Laurence , Terrier-Lacombe Marie-Jose , Haralambieva Eugenia , Pulford Karen , Pileri Stefano , Morris Stephan W. , Mason David Y. , Delsol Georges
Published: October 2019

The t(2;5)(p23;q35) translocation, associated with anaplastic large-cell lymphoma (ALCL), results in the expression of a chimeric NPM-ALK protein that can be detected by the ALK1 monoclonal antibody. This report describes the morphologic and phenotypic spectrum of 123 cases of lymphoma that all express ALK protein. The results provide strong evidence that the morphologic patterns of ALCL described in previous reports as representing possible subtypes of ALCL, eg, common type, lymphohistiocytic, or small cell patterns, are morphologic variants of the same disease entity. All of these morphologic patterns could be found within this series, and in some patients different subtypes coexisted in a single biopsy or were found in successive biopsies from a single patient. The link between these morphologic subtypes is further reinforced by the presence in all cases of a highly characteristic large cell, with an eccentric nucleus and an eosinophilic paranuclear region. We suggest that this cell can be considered as a major distinguishing feature of ALK-positive lymphomas. Another characteristic of these tumors was the perivascular pattern of neoplastic cell infiltration seen in a significant number of cases. In addition to ALK protein, all tumors expressed epithelial membrane antigen and lacked CD15, features that may be of value in differentiating ALCL from Hodgkin's disease. In the majority of cases (84%), malignant cells showed both a cytoplasmic and nuclear staining for ALK1 and thus presumably carried the 2;5 translocation, but staining was restricted to the cytoplasm in a few cases, suggesting that translocations other than t(2;5) may induce expression of ALK protein. We conclude from this study that ALK-positive neoplasms represent a distinct entity. Because their morphology is often neither anaplastic nor large cell, we suggest that they should henceforward be referred to as ALK lymphomas.
Adenovirus-Mediated Human Immunodeficiency Virus-1 Nef Expression in Human Monocytes/Macrophages and Effect of Nef on Downmodulation of Fcγ Receptors and Expression of Monokines
Authors: De Swapan K. , Venkateshan Chettemgere N.S. , Seth Prem , Carleton Gajdusek D. , Gibbs Clarence J.
Published: October 2019

To characterize the effect of human immunodeficiency virus-1 (HIV-1)nef expression in human monocytes/macrophage (HMØ) and U937 on the levels of FcγRs, HLA antigens, and monokines, elutriated HMØs and U937 cells were transfected with an adenovirus-mediated Nef expression system. Nef-expressing cells downmodulated FcγRI, FcγRII, and upregulated HLA class I molecules. Nef-expressing HMØs, treated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), overexpressed tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10. However, IL-6 was induced by LPS and inhibited by PMA. Additionally, a subpopulation of Nef-expressing HMØs underwent apoptosis. Our data suggest that HIV-1 nefdownmodulated FcγRs in myeloid cells in a manner similar to that previously reported for its effect on CD4+ in T cells.
Anti–VLA4/VCAM-1—Induced Mobilization Requires Cooperative Signaling Through the kit/mkit Ligand Pathway
Authors: Papayannopoulou Thalia , Priestley Gregory V. , Nakamoto Betty
Published: October 2019

Although a large body of data on mobilization have yielded valuable clues, the mechanism(s) dictating egress of stem/progenitor cells during baseline hematopoiesis and after their mobilization are poorly understood. We have previously provided functional in vivo evidence that cytoadhesion molecules, specifically the β1integrins, are involved in mobilization; however, the mechanism by which this was achieved was unclear. To provide further insights into the anti–very late antigen 4 (VLA4)/anti–vascular cell adhesion molecule 1 (VCAM-1)—induced mobilization, we used these antibodies to treat mutant mice with compromised growth factor receptor function. We found that mobilization by anti-VLA4 does not depend on a functional granulocyte colony-stimulating factor, interleukin-7 (IL-7), or IL-3α receptor. By contrast, the functional kit receptor is required, because W/Wv mice responded minimally, whereas Steel-Dickie (Sl/Sld) responded normally. Both Wv and Sl/Sld mice did not respond to anti–VCAM-1 treatment, in contrast to their +/+ littermates and despite normal levels of VCAM-1 expression in bone marrow cells. The defective response to anti–VCAM-1 in W/Wv mice was corrected after their transplantation with +/+ cells. mev/mev mice showed increased numbers of circulating progenitors before treatment and a heightened response after anti-VLA4 or anti–VCAM-1 treatment. Downmodulation of kit expression was detected in normal bone marrow cells after anti-VLA4 treatment. On the strength of the above findings we conclude that (1) anti–VLA4/VCAM-1—induced mobilization likely requires signaling for stimulation of cell migration; (2) this cooperative signaling involves the kit/kit ligand pathway, and provides a novel example of integrin/cytokine crosstalk; and (3) migration mediated through the kit/kit ligand pathway may be a common contributor to different mobilization stimuli. Dissection of the exact molecular pathways that lead to mobilization remains a future challenge.
A Fifty Percent Reduction of Platelet Surface Glycoprotein Ib Does Not Affect Platelet Adhesion Under Flow Conditions
Authors: van Zanten G. Henrita , Heijnen Harry F.G. , Wu Yaping , Schut-Hese Karin M. , Slootweg Pieter J. , de Groot Philip G. , Sixma Jan J. , Nieuwland Rienk
Published: October 2019

Glycoprotein (GP) Ib is an adhesion receptor on the platelet surface that binds to von Willebrand Factor (vWF). vWF becomes attached to collagens and other adhesive proteins that become exposed when the vessel wall is damaged. Several investigators have shown that during cardiopulmonary bypass (CPB) surgery and also during platelet activation in vitro by thrombin or thrombin receptor activating peptide (TRAP) GPIb disappears from the platelet surface. Such a disappearance is presumed to lead to a decreased adhesive capacity. In the present study, we show that a 65% decrease in platelet surface expression of GPIb, due to stimulation of platelets in Orgaran anticoagulated whole blood with 15 μmol/L TRAP, had no effect on platelet adhesion to both collagen type III and the extracellular matrix (ECM) of human umbilical vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet agglutination was highly dependent on the presence of GPIb. Immunoelectron microscopic studies showed that GPIb almost immediately returned to the platelet surface once platelets had attached to collagen. In a subsequent series of experiments, we showed that when less than 50% of GPIb was blocked by an inhibitory monoclonal antibody against GPIb (6D1), platelet adhesion under flow conditions remained unaffected.
Isolation and Characterization of the cDNA for Mouse Neutrophil Collagenase: Demonstration of Shared Negative Regulatory Pathways for Neutrophil Secondary Granule Protein Gene Expression
Authors: Lawson Nathan D. , Khanna-Gupta Arati , Berliner Nancy
Published: October 2019

A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length (∼2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF–induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.
Decay-Accelerating Factor (CD55) and Membrane Inhibitor of Reactive Lysis (CD59) Are Released Within Exosomes During In Vitro Maturation of Reticulocytes
Authors: Rabesandratana Herisoa , Toutant Jean-Pierre , Reggio Hubert , Vidal Michel
Published: October 2019

Exosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.
Effect of Disease Stage on Clinical Outcome After Syngeneic Bone Marrow Transplantation for Relapsing Experimental Autoimmune Encephalomyelitis
Authors: Burt Richard K. , Padilla Josette , Begolka Wendy Smith , Canto Mauro C. Dal , Miller Stephen D.
Published: October 2019

Relapsing experimental autoimmune encephalomyelitis (R-EAE) is an immune-mediated demyelinating central nervous system (CNS) disease. Myeloablation and syngeneic bone marrow transplantation (SBMT), when performed at the peak of acute disease (day 14), prevented glial scarring and ameliorated the disease severity. In contrast, when syngeneic BMT was performed late in chronic phase (day 78), significant glial scarring remained and the clinical severity did not differ significantly from that of the controls. After SBMT in either the acute or chronic phase of disease, the posttransplant immune system remained responsive to myelin epitopes as determined by in vitro proliferation and interferon-γ (IFN-γ) production. However, in mice undergoing SBMT, in vivo delayed-type hypersensitivity (DTH) responses were significantly decreased while IFN-γ RNA levels and inflammatory infiltrates within the CNS were slightly improved. We conclude that failure of SBMT to improve the clinical disease when performed in chronic phase may be due to preexisting glial scarring. We also conclude that in the absence of glial scarring and irreversible neuronal injury, in vivo DTH responses and histology are better predictors of clinical improvement than in vitro proliferation or IFN-γ cytokine production.
Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic Adenosine Monophosphate–Dependent Protein Kinase
Authors: Casteel Darren , Suhasini Modem , Gudi Tanima , Naima Reza , Pilz Renate B.
Published: October 2019

Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase–deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser169; Ala substitution for Ser169 resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore β-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser169 → Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.
Systematic Method to Obtain Novel Genes That Are Regulated bymi Transcription Factor: Impaired Expression of Granzyme B and Tryptophan Hydroxylase in mi/mi Cultured Mast Cells
Authors: Ito Akihiko , Morii Eiichi , Maeyama Kazutaka , Jippo Tomoko , Kim Dae-Ki , Lee Young-Mi , Ogihara Hideki , Hashimoto Koji , Kitamura Yukihiko , Nojima Hiroshi
Published: October 2019

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). We have reported that the expression of several genes was impaired in cultured mast cells (CMCs) ofmi/mi genotype, and demonstrated the involvement of MITF in the transcription of these genes. To obtain new genes whose transcription may be regulated by MITF, we prepared a subtracted cDNA library using +/+ and mi/mi CMCs. We found two clones carrying the granzyme (Gr) B and tryptophan hydroxylase (TPH) cDNAs in the subtracted library. The expression of the Gr B and TPH genes decreased in mi/mi CMCs, and recovered to nearly normal level by the overexpression of normal (+) MITF but not of mutant (mi) MITF. The +-MITF bound three and one CANNTG motifs in the Gr B and TPH promoters, respectively, and transactivated these two genes, indicating the involvement of +-MITF in their expression. Because TPH is the rate-limiting enzyme for serotonin synthesis, we examined the serotonin content of +/+ and mi/mi CMCs. The serotonin content was significantly smaller in mi/mi CMCs than in +/+ CMCs. The introduction of +-MITF but not of mi-MITF normalized the serotonin content in mi/mi CMCs.
Identification of Human and Mouse Hematopoietic Stem Cell Populations Expressing High Levels of mRNA Encoding Retrovirus Receptors
Authors: Orlic Donald , Girard Laurie J. , Anderson Stacie M. , Pyle Louise C. , Yoder Mervin C. , Broxmeyer Hal E. , Bodine David M.
Published: October 2019

One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin−CD34+ CD38− cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin− CD34+ CD38− cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.
Detection of Lymphoma in Bone Marrow by Whole-Body Positron Emission Tomography
Authors: Carr Robert , Barrington Sally F. , Madan Bella , O'Doherty Michael J. , Saunders Catherine A.B. , van der Walt Jon , Timothy Adrian R.
Published: October 2019

Positron emission tomography (PET) is a whole-body imaging technique using 18 fluorine-fluorodeoxyglucose (FDG), whose uptake is increased in tumor cells. Published studies have shown PET to be an effective method of staging lymphoma and to be more sensitive than CT at detecting extranodal disease. The purpose of this study was to determine whether the increased marrow uptake of FDG observed in some lymphoma patients during routine staging PET scans represented marrow involvement by disease. PET scans of 50 patients with Hodgkin's (12) and non-Hodgkin's (38) lymphoma were analyzed by three independent observers and the marrow graded as normal or abnormal using a visual grading system. Unilateral iliac crest marrow aspirates and biopsies were performed on all patients. The PET scan and marrow histology agreed in 39 patients (78%), being concordant positive in 13 and concordant negative in 26 patients. In 8 patients the PET scan showed increased FDG uptake but staging biopsy was negative; in 4 of these 8 patients the PET scan showed a normal marrow background with focal FDG “hot spots” distant from the site biopsied. In 3 patients the marrow biopsy specimen was positive but the PET scan normal; 2 of these 3 patients had non-Hodgkin's lymphoma whose malignant cells did not take up FDG at lymph node or marrow disease sites. Therefore, there were only 5 patients (10%) in whom there was a difference between the PET scan and biopsy result which could not be fully explained. Visual interpretation of marrow FDG uptake during whole-body staging PET scans can correctly assess marrow disease status in a high proportion of lymphoma patients. PET has the potential to reduce the need for staging marrow biopsy.
A Strong Expression of CD44-6v Correlates With Shorter Survival of Patients With Acute Myeloid Leukemia
Authors: Legras S. , Günthert U. , Stauder R. , Curt F. , Oliferenko S. , Kluin-Nelemans H.C. , Marie J.P. , Proctor S. , Jasmin C. , Smadja-Joffe F.
Published: October 2019

CD44 is a ubiquitous cell-surface glycoprotein that displays many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v. The expression of variant isoforms is highly restricted and correlated with specific processes, such as leukocyte activation and malignant transformation. We have herein studied CD44v expression in acute myeloid leukemia (AML) and, for comparison, in normal myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and -9v isoforms has been measured using specific monoclonal antibodies and flow cytometry. The composition of variant exon transcripts has been analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction followed by Southern hybridization with exon-specific probes. Our data show that (1) CD44-6v isoforms are expressed on 12.0% ± 2.5% of normal CD34+ cells; this expression is sharply upregulated through monopoiesis and, inversely, downregulated during granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10% and 14% of normal monocytes, respectively. (2) Sixty-nine from a total of 95 AML patients display a variable proportion (range, 5% to 80%) of CD44-6v+ leukemic cells. (3) A shorter overall survival characterizes the group of AML patients displaying more than 20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time, that the protein expression of CD44-6v containing isoforms may serve as a new prognostic factor in AML.
Low Transplant Mortality in Allogeneic Bone Marrow Transplantation for Acute Myeloid Leukemia: A Randomized Study of Low-Dose Cyclosporin Versus Low-Dose Cyclosporin and Low-Dose Methotrexate
Authors: Zikos P. , Van Lint M.T. , Frassoni F. , Lamparelli T. , Gualandi F. , Occhini D. , Mordini N. , Berisso G. , Bregante S. , De Stefano F. , Soracco M. , Vitale V. , Bacigalupo A.
Published: October 2019

Sixty patients undergoing allogeneic bone marrow transplant for acute myeloid leukemia (AML) in first remission (CR1; n = 49) or more advanced phase (n = 11) were entered in a prospective trial of graft-versus-host disease (GvHD) prophylaxis: low-dose cyclosporin A (IdCSA; 1 mg/kg/d from day −1 to +20 day; n = 28) or IdCSA plus low-dose methotrexate (IdMTX; 10 mg/m2 for day +1, 8 mg/m2 for days +3, +6, and +11; n = 32). Primary end points were acute GvHD (aGvHD) and transplant-related mortality (TRM); secondary end points were relapse and survival. The conditioning regimen consisted of cyclophosphamide (120 mg/kg) and fractionated total body irradiation (3.3 Gy/d for 3 consecutive days). The actuarial risk of developing aGvHD grade II-III was 61% for IdCSA alone and 34% for IdCSA + IdMTX (P = .02). The actuarial risk of TRM at 1 year was 11% versus 13%, respectively, and older patients (≧29 years) had higher TRM than younger patients (22% v 5%,P = .01). The age effect was significant in the IdCSA group (P = .04) but not in the IdCSA + IdMTX group (P = .1). The median follow-up is 4.4 years, with an overall actuarial survival of 78% for CR1 patients and 36% for patients with advanced disease. For patients in CR1 the outcome of the two regimens was as follows: survival 77% versus 80% (P = .6), relapse 20% versus 9% (P = .1), and TRM 13% versus 17% (P = .6). This study suggests that TRM can be reduced in AML patients undergoing allogeneic marrow transplants with a mild conditioning regimen and low-dose immunosuppression, and this translates in a 78% 5-year survival for CR1 patients. Beyond CR1 the major obstacle remains leukemia relapse, which is not prevented by low-dose in vivo immunosuppression.
A Multicenter Study of Platelet Recovery and Utilization in Patients After Myeloablative Therapy and Hematopoietic Stem Cell Transplantation
Authors: Bernstein Steven H. , Nademanee Auayporn P. , Vose Julie M. , Tricot Guido , Fay Joseph W. , Negrin Robert S. , DiPersio John , Rondon Gabriela , Champlin Richard , Barnett Michael J. , Cornetta Kenneth , Herzig Geoffrey P. , Vaughan William , Geils George , Keating Armand , Messner Hans , Wolff Steven N. , Miller Kenneth B. , Linker Charles , Cairo Mitchell , Hellmann Susan , Ashby Mark , Stryker Scott , Nash Richard A.
Published: October 2019

An observational study was conducted at 18 transplant centers in the United States and Canada to characterize the platelet recovery of patients receiving myeloablative therapy and stem cell transplantation and to determine the clinical variables influencing recovery, determine platelet utilization and cost, and incidence of hemorrhagic events. The study included 789 evaluable patients transplanted in 1995. Clinical, laboratory, and outcome data were obtained from the medical records. Variables associated with accelerated recovery in multivariate models included (1) higher CD34 count; (2) higher platelet count at the start of myeloablative therapy; (3) graft from an HLA-identical sibling donor; and (4) prior stem cell transplant. Variables associated with delayed recovery were (1) prior radiation therapy; (2) posttransplant fever; (3) hepatic veno-occlusive disease; and (4) use of posttransplant growth factors. Disease type also influenced recovery. Recipients of peripheral blood stem cells (PBSC) had faster recovery and fewer platelet transfusion days than recipients of bone marrow (BM). The estimated average 60-day platelet transfusion cost per patient was $4,000 for autologous PBSC and $11,000 for allogeneic BM transplants. It was found that 11% of all patients had a significant hemorrhagic event during the first 60 days posttransplant, contributing to death in 2% of patients. In conclusion, clinical variables influencing platelet recovery should be considered in the design and interpretation of clinical strategies to accelerate recovery. Enhancing platelet recovery is not likely to have a significant impact on 60-day mortality but could significantly decrease health care costs and potentially improve patient quality of life.
Oligoclonal Protein Bands and Ig Isotype Switching in Multiple Myeloma Treated With High-Dose Therapy and Hematopoietic Cell Transplantation
Authors: Zent C.S. , Wilson C.S. , Tricot G. , Jagannath S. , Siegel D. , Desikan K.R. , Munshi N. , Bracy D. , Barlogie B. , Butch A.W.
Published: October 2019

Multiple myeloma (MM) is usually characterized by production of a single serum monoclonal protein of constant isotype and light-chain restriction. Multiple Ig isotypes and isotype switches, which are rare in untreated patients, are reported to be more common in patients undergoing myeloablative therapy. These additional protein bands, detected by immunofixation electrophoresis (IFE), could be due to altered paraprotein production by the malignant plasma cell clone or oligoclonal Ig production during recovery of B-cell function after myeloablative therapy. We analyzed abnormal protein bands (APB), distinct from the presenting paraprotein, in 550 patients receiving high-dose therapy with autologous hematopoietic cell transplantation at a single institution. Fifty-five patients (10%) had APB, 48 had oligoclonal bands (OB), and 23 had an apparent isotype switch (IS) on IFE (16 had both OB and IS). Morphologic and flow cytometric examination of bone marrow in 17 patients with IS showed no evidence of a clonal plasma cell isotype switch. Patients with APB had significantly higher complete response to therapy (67% v 37%,P = .001). To assess the independent prognostic relevance of APB, a multivariate analysis was performed among 471 patients surviving at least 12 months from first transplant (all patients developing APB had done so by 12 months from first transplant). APB (in 50 patients) was a favorable feature for both event-free (rank 3, P = .004) and overall survival (rank 3, P = .0005). We propose that OB and IS are likely to be due to recovery of Ig production rather than alterations in the biology of the malignant plasma cell clone.
Thrombopoietin Promotes the Survival of Murine Hematopoietic Long-Term Reconstituting Cells: Comparison With the Effects of FLT3/FLK-2 Ligand and Interleukin-6
Authors: Matsunaga Takuya , Kato Takashi , Miyazaki Hiroshi , Ogawa Makio
Published: October 2019

The effects of thrombopoietin (TPO; c-mpl ligand), FLT3/FLK-2 ligand (FL), and interleukin-6 (IL-6) on the survival of murine hematopoietic long-term reconstituting cells (LTRC) were studied by using lineage-negative, Sca-1–positive, c-kit–positive (Lin−Sca-1+c-kit+) marrow cells from 5-fluorouracil–treated mice. We tested the ability of these cytokines to maintain the viability of LTRC by transplanting the cultured cells to lethally irradiated Ly-5 congenic mice together with compromised marrow cells. As a single agent, only TPO could maintain the LTRC. Neither IL-6 nor FL was effective by itself, but they acted synergistically to maintain the LTRC. We examined whether the maintenance of LTRC by these cytokines was due to the survival of stem cells or was the result of active cell divisions and self-renewal. To monitor cell division, we used membrane dye PKH26. Enriched cells were stained with PKH26 on day 0 and incubated in suspension culture with TPO or with IL-6 and FL for 7 days. On day 7, PKH26low and PKH26high cells were prepared by sorting and their in vivo reconstituting abilities were tested by transplantation into lethally irradiated Ly-5 congenic mice together with compromised marrow cells. PKH26high populations cultured with both TPO alone and the combination of IL-6 and FL showed greater reconstitution activity than that of PKH26low populations. These data indicate that TPO alone and the combination of IL-6 and FL can support the survival of stem cells without stimulating their active cell proliferation.
Platelet-Endothelial Cell Interactions During Ischemia/Reperfusion: The Role of P-Selectin
Authors: Massberg Steffen , Enders Georg , Leiderer Rosmarie , Eisenmenger Simone , Vestweber Dietmar , Krombach Fritz , Messmer Konrad
Published: October 2019

Growing evidence supports a pathophysiological role for platelets during the manifestation of postischemic reperfusion injury; in the current study, we investigated the nature and the molecular determinants of platelet-endothelial cell interactions induced by ischemia/reperfusion (I/R). Platelet-endothelium and leukocyte-endothelium interactions after 1 hour of ischemia were monitored in vivo within mouse small intestine. By intravital fluorescence microscopy, we observed that platelets, like leukocytes, roll along or firmly adhere to postischemic microvascular endothelial cells. In contrast, few leukocyte-endothelial cell interactions were detected in sham-operated controls. Monoclonal antibodies against P-selectin significantly attenuated platelet rolling and adherence in response to I/R. To identify whether platelet or endothelial P-selectin plays the major role in mediating postischemic platelet-endothelial cell interactions, P-selectin-deficient or wild-type platelets were transfused into wild-type or P-selectin-deficient mice, respectively. Whereas platelets lacking P-selectin rolled along or adhered to postischemic wild-type endothelium, interactions between wild-type platelets with mutant endothelium were nearly absent, indicating that I/R-induced platelet-endothelium interactions are dependent on the expression of P-selectin by endothelial cells. Concomitantly, P-selectin expression in the intestinal microvasculature was enhanced in response to I/R, whereas no upregulation of P-selectin was observed on circulating platelets. In summary, we provide first in vivo evidence that platelets accumulate in the postischemic microvasculature early after reperfusion via P-selectin-ligand interactions. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of I/R injury.
High Prevalence of Serum Cryoglobulins in Multitransfused Hemophilic Patients With Chronic Hepatitis C
Authors: Santagostino E. , Colombo M. , Cultraro D. , Muça-Perja M. , Gringeri A. , Mannucci P.M.
Published: October 2019

The prevalence, clinical relevance, and risk factors of serum cryoglobulins in hemophilic patients with chronic hepatitis C virus (HCV) infection are unknown. We studied 135 consecutive hemophilic patients (median age, 31 years; range, 10 to 69 years) with chronic hepatitis C, exposed to the virus for 10 to 41 years. A total of 67 patients were coinfected with the human immunodeficiency virus (HIV), and 3 (2%) had signs of cirrhosis. Serum samples were tested for the presence of cryoglobulins, hepatitis B virus (HBV) markers, including HBV-DNA by hybridization assay, and antibody to HCV by enzyme immunoassay (EIA). Serum HCV-RNA was tested by polymerase chain reaction and typed with a hybridization technique. Samples were also tested for antitissue antibodies, immunoglobulins, rheumatoid factor, and C3 and C4 proteins of complement. Forty-two hemophiliacs (31%) circulated cryoglobulins (median levels, 166 mg/L; range, 66 to 480) predominantly type III (62%; and 29% type II). None of the patients had clinical signs or symptoms of systemic vasculitis. Cryoglobulinemic patients had more often serum HCV-RNA (95% v 80%, P < .05), rheumatoid factor (20% v 6%, P < .05), higher levels of IgG (2,354 ± 682 mg/dL v 1,928 ± 557 mg/dL,P < .0005) and IgM (323 ± 226 mg/dL v 244 ± 243 mg/dL, P < .05), and lower levels of serum C4 (19 ± 8 mg/dL v 24 ± 8 mg/dL, P < .05) than patients without cryoglobulins. The risk of producing cryoglobulins was greater for 114 patients circulating HCV-RNA than for 21 nonviremic patients (odds ratio [OR] = 4.9, 95% confidence interval [CI] = 1.1 to 22.0) and for the 31 patients with longer exposure to HCV (more than 26 years) than for the 24 patients with shorter (17 years or less) exposure (OR = 4.4 95% CI = 1.1 to 18.0). In conclusion a large number of multitransfused hemophiliacs with chronic HCV infection circulated serum cryoglobulins but none had clinical signs or symptoms of vasculitis. The risk of developing cryoglobulins parallels the duration of exposure to HCV.
Full Activity From Human β-Globin Locus Control Region Transgenes Requires 5′HS1, Distal β-Globin Promoter, and 3′ β-Globin Sequences
Authors: Pasceri Peter , Pannell Dylan , Wu Xiumei , Ellis James
Published: October 2019

The locus control region (LCR) activates high-level human β-globin transgene expression. LCR cassettes composed of 5′HS2-4 linked to the 815 bp β-globin proximal promoter do not express fully. Here, we show that LCR (5′HS2-4) β-globin transgenes that also contain either 5′HS1 or the distal promoter fail to express fully in single- and low-copy transgenic mice. In contrast, full expression is obtained in the presence of both 5′HS1 and the distal promoter. Nine factor binding sites were identified in 5′HS1, using in vitro DNaseI footprint and gel retardation assays, and these include a strong Sp1/Sp3 site, four GATA-1 sites, and two sites that encompass an ACTAAC motif. LCR (5′HS1-4) β-globin transgene constructs with the distal promoter deleted or replaced by spacer DNA show that specific distal promoter sequences are required for full expression. An LCR (5′HS1-4) transgene construct with truncated downstream β-globin gene sequences indicates that 3′ sequences also play an important role. These results show that full expression of the β-globin gene directed by the LCR requires 5′HS1, the distal β-globin promoter, and 3′ sequences, and has implications for gene therapy construct design and models of LCR activation.
Detection of Microchimerism After Allogeneic Blood Transfusion Using Nested Polymerase Chain Reaction Amplification With Sequence-Specific Primers (PCR-SSP): A Cautionary Tale
Authors: Carter Anthony S. , Bunce Mike , Cerundolo Lucia , Welsh Ken I. , Morris Peter J. , Fuggle Susan V.
Published: October 2019

In bone marrow transplantation, the detection of chimerism is an important adjunct to the repertoire of tests available for determining acceptance of the graft. In solid organ transplantation, there is currently intense interest in the role that chimerism plays in both short- and long-term host reactivity to the graft. Allogeneic blood transfusion has been associated with a subtle immunosuppressive effect in renal transplantation and chimerism is implicated as a possible mechanism for this effect. To assess the survival of allogeneic cells after blood transfusion or transplantation, we have developed a technique based on molecular typing for HLA class II alleles, which enables the detection of donor-derived cells in patients receiving blood transfusions. While developing this technology, we investigated why we and others observe false amplification. Sequencing of false products has shown that they arise from amplification of both pseudogenes and non-pseudogenes present in the DNA under test. Elucidation of this phenomenon allows the amplification of these false products to be predicted in any given combination and hence avoided by the judicious selection of primers. Validation has been achieved by following donor alleles after transfusion of blood containing defined numbers of leukocytes expressing selected mismatched antigens.
Distinct Apoptotic Responses Imparted by c-myc and max
Authors: Nesbit Chadd E. , Fan Saijun , Zhang Hong , Prochownik Edward V.
Published: October 2019

The c-myc oncoprotein accelerates programmed cell death (apoptosis) after growth factor deprivation or pharmacological insult in many cell lines. We have shown that max, the obligate c-myc heterodimeric partner protein, also promotes apoptosis after serum withdrawal in NIH3T3 fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. We now show that c-myc– and max-overexpressing 32D cells differ in the nature of their apoptotic responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin (Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max overexpression increases sensitivity only to Camptothecin. Drug treatment of c-myc–overexpressing cells in the absence of IL-3 did not alter the spectrum of drug sensitivity other than to additively accelerate cell death. In contrast, enhanced sensitivity to Adriamycin, Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was revealed in max-overexpressing cells concurrently deprived of IL-3. Differential rates of apoptosis were not strictly correlated with the ability of the drugs to promote G1 or G2/M arrest. Ectopic expression of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc–overexpressing cells. These results provide mechanistic underpinnings for the idea that c-myc and max modulate distinct apoptotic pathways.

© 1998 by The American Society of Hematology.
FLT-3 Ligand Provides Hematopoietic Protection From Total Body Irradiation in Rabbits
Authors: Gratwohl A. , John L. , Baldomero H. , Roth J. , Tichelli A. , Nissen C. , Lyman S.D. , Wodnar-Filipowicz A.
Published: October 2019

Several hematopoietic cytokines have been investigated for their potential to provide protection from the lethal consequences of bone marrow aplasia after total body irradiation (TBI). Some can increase the dose of irradiation tolerated by the animals; none allow endogenous recovery after doses such as administered in clinical blood or marrow transplantation. We tested the radioprotective potential of FLT-3 ligand, an early acting hematopoietic cytokine, alone and in combination with a late acting cytokine, granulocyte-colony stimulating factor (G-CSF). Adult outbred New Zealand White rabbits were submitted to TBI of 1,200 or 1,400 cGy by a Co60 source. Recombinant human (rh) FLT-3 ligand at a dose of 500 μg/kg and/or rhG-CSF at a dose of 10 μg/kg were administered for 14 days subcutaneously daily, beginning either 2 days before or the day after TBI. All control animals given no growth factors died of aplasia at day 10 (range, 5 to 16). All 8 animals given G-CSF had severe aplasia and 7 died at day 8 (range, 5 to 10); 1 animal survived, with G-CSF being administered before TBI. In contrast, 11 of 12 animals given FLT-3 ligand, with or without G-CSF, survived. Radioprotection was best in the group given FLT-3 ligand together with G-CSF before TBI. In these animals median platelet counts were never <10 × 109/L and median white blood cell counts never <0.5 × 109/L. These data show that hematopoietic recovery can occur after 1,400 cGy TBI in rabbits, if protected by FLT-3 ligand, and suggest a radioprotective clinical potential of this cytokine.

© 1998 by The American Society of Hematology.
Presence of a p53 Gene Deletion in Patients With Multiple Myeloma Predicts for Short Survival After Conventional-Dose Chemotherapy
Authors: Drach Johannes , Ackermann Jutta , Fritz Elke , Krömer Elisabeth , Schuster Rudolf , Gisslinger Heinz , DeSantis Maria , Zojer Niklas , Fiegl Michael , Roka Sebastian , Schuster Judith , Heinz Renate , Ludwig Heinz , Huber Heinz
Published: October 2019

In multiple myeloma (MM), previous studies showed that mutations of the p53 gene are rare events in patients with newly diagnosed disease, but it is not known whether deletions of p53 are of any significance in MM. To address this question, we used interphase fluorescence in situ hybridization (FISH) with a DNA probe specific for the p53 locus at 17p13 and investigated bone marrow plasma cells from 72 patients with MM (59 patients = 81.9% before therapy). By FISH, deletions of p53, which were found to be predominantly monoallelic, were detected in 32.8% and 54.5% of patients with newly diagnosed and relapsed MM, respectively. Karyotypes from six of the patients with a p53 deletion by FISH showed a structural abnormality of 17p in only one of them. Additional FISH studies including a distal-17p probe (specific for theD17S34 locus) provided evidence for an interstitial deletion on 17p resulting in loss of p53 hybridization signals in myeloma cells. Among all 59 patients with newly diagnosed MM, presence of a p53 deletion was associated with stage III (P = .054), but not with other laboratory and clinical parameters. Patients with a p53 deletion had significantly shorter survival time compared with those without a deletion, both from the time of diagnosis (median 13.9v 38.7 months; P < .0001) and from the time of initiation of induction treatment consisting of conventional dose chemotherapy (median 15.9 months v median not reached at 38 months; P < .0002). On stepwise multivariate regression analysis, presence of a p53 deletion was the most significant independent parameter predicting for shortened survival (P = .002). We conclude that a p53 gene deletion, which can be identified by interphase FISH in almost a third of patients with newly diagnosed MM, is a novel prognostic factor predicting for short survival of MM patients treated with conventional-dose chemotherapy.

© 1998 by The American Society of Hematology.
Exclusion of the First EGF Domain of Factor VII by a Splice Site Mutation Causes Lethal Factor VII Deficiency
Authors: McVey John H. , Boswell Emma J. , Takamiya Osamu , Tamagnini Gabriel , Valente Victor , Fidalgo Teresa , Layton Mark , Tuddenham Edward G.D.
Published: October 2019

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.

© 1998 by The American Society of Hematology.