bluuebunny/crossref_metadata_2025 huggingface dataset:
Randomized comparison of interferon-alpha with busulfan and hydroxyurea in chronic myelogenous leukemia. The German CML Study Group [see comments]
Authors: Hehlmann R , Heimpel H , Hasford J , Kolb HJ , Pralle H , Hossfeld DK , Queisser W , Loffler H , Hochhaus A , Heinze B
Published: October 2019

As curative bone marrow transplantation is available only to a minority of patients with chronic myelogenous leukemia (CML), drug therapy remains of central interest. Several nonrandomized studies have suggested that interferon-alpha (IFN) may prolong survival in CML. In a randomized multicenter study the influence of IFN versus busulfan or hydroxyurea (HU) on survival of Philadelphia-positive (Ph+) CML was examined. A total of 513 Ph+ patients were randomized for treatment as follows: 133 for IFN, 186 for busulfan, and 194 for HU. IFN-treated CML patients have a significant survival advantage over busulfan-treated (P = .008), but not over HU-treated patients (P = .44). The longer survival is due to slower progression to blast crisis. Median survival of IFN-treated patients is 5.5 years [5-year survival, 59%; 95% confidence interval (CI), 48%-70%], of busulfan-treated patients, 3.8 years (5-year survival, 32%; CI, 24%-40%), and of HU-treated patients, 4.7 years (5-year survival, 44%; CI, 36%-53%). Patients who continue on IFN survive longer than those in whom IFN is discontinued before blast crisis (P = .007). Complete hematologic IFN-responders have a survival advantage over partial responders or nonresponders (P = .02). Cytogenetic IFN-responders have no significant survival advantage over nonresponders (P = .2). Patients who attain white blood cell (WBC) counts of 10 x 10(9)/L or less have a survival advantage in the IFN (P = .007) and HU (P = .05) groups. Whereas toxicity in the IFN group was considerably higher than in the busulfan or HU groups, long-lasting cytopenias necessitating discontinuation of therapy as observed with busulfan have not been seen with IFN or HU. The problems of conventional prognostic scores (Sokal's score, Score 1) that we observed in IFN-treated patients support the idea that IFN changes the natural course of CML. We conclude that, with regard to survival of CML in the chronic phase, IFN is superior to busulfan and as effective as HU. Whether and to what extent IFN is superior to HU appears to depend, at least in part, on the degree of WBC suppression by HU-therapy and on the risk profile of the patients.
Molecular cloning, characterization, and chromosomal localization of a novel protein-tyrosine phosphatase, HPTP eta
Authors: Honda H , Inazawa J , Nishida J , Yazaki Y , Hirai H
Published: October 2019

Protein-tyrosine phosphatases (PTPases) are considered to play an important role in signal transduction. We previously identified partial sequences of three novel PTPases in a human leukemic cell line. F-36P. We describe here cloning, characterization, and chromosomal localization of one of the newly identified PTPases, termed as HPTP eta (human protein-tyrosine phosphatase eta). The deduced amino acid sequence was composed of an extracellular region homologous to fibronectin type III repeats, a transmembrane region, and a cytoplasmic region containing a single PTPase-like domain. Based on its primary structure, this clone belongs to type-III receptor-type PTPases. The PTPase-like domain showed PTPase activity when expressed in Escherichia coli. Antibody against the extracellular region detected a protein of 220 to 250 kD in human hematopoietic cell lines expressing HPTP eta mRNA. The antibody also recognized a protein of approximately the same molecular weight in COS cells transfected with HPTP eta cDNA, indicating that the antibody specifically recognized HPTP eta gene product and that the cloned cDNA contained full-length coding region. The chromosomal localization determined by fluorescence in situ hybridization showed that the HPTP eta gene was located at chromosome 11p11.2 on the short arm of chromosome 11, which is frequently lost or deleted in human carcinomas.
Identification of a novel thrombin receptor sequence required for activation-dependent responses
Authors: Bahou WF , Kutok JL , Wong A , Potter CL , Coller BS
Published: October 2019

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1–160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34–44, 48–67, 65–79, and 87–94. Soluble peptides derived from regions 83–94, but not those from other regions of the receptor, neutralized the ability of anti-TR1–160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67–82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42–47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N- terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.
Antibodies cross-reactive with E- and P-selectin block both E- and P- selectin functions
Authors: Berg EL , Fromm C , Melrose J , Tsurushita N
Published: October 2019

E- and P-selectin are inflammation-induced cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions. Monoclonal antibodies (MoAbs) specific for either E-selectin or P- selectin are protective in several animal models of inflammatory disease. To generate an MoAb with broader therapeutic potential, MoAbs that bind to both E- and P-selectin were generated by immunization of mice with mouse pre-B cell lines transfected with human E- and P- selectin. Interestingly, although the only selection criterion was the ability to bind both E- and P-selectin, all three antibodies obtained efficiently block both E- and P-selectin-mediated functions. The inhibited functions include neutrophil or HL-60 cell binding to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells, E- or P-selectin transfectant cell lines, and platelet-HL-60 rosetting. These antibodies, EP-5C7, EP-2C9, and EP-1D8, recognize the same or overlapping epitope within the lectin domains of E- and P-selectin. The data suggest that functionally important epitopes of homologous proteins can be targeted by selecting for antibodies with reactivity toward both proteins. Furthermore, a potent blocking antibody specific for both E- and P-selectin may provide a more effective and broadly useful reagent for treating acute and potentially certain chronic inflammatory conditions.
The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development
Authors: Young PE , Baumhueter S , Lasky LA
Published: October 2019

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.
Specific binding of leukemia oncogene fusion protein peptides to HLA class I molecules
Authors: Bocchia M , Wentworth PA , Southwood S , Sidney J , McGraw K , Scheinberg DA , Sette A
Published: October 2019

Many human leukemias are characterized by chromosomal translocations yielding hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion proteins represent true tumor-specific antigens that are potentially immunogenic. Although these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on leukemic cells. The ability of a series of synthetic peptides corresponding to the junctional sequences of chronic myelogenous leukemia (CML)-derived bcr-abl and acute promyelocytic leukemia (APL)-derived PML-RAR alpha fusion proteins to bind to purified class I molecules was studied. A series of 152 peptides 8, 9, 10, and 11 amino acids in length, spanning the b3a2 and b2a2 breakpoints for CML and PML-RAR alpha A and B breakpoints for APL were analyzed for HLA A1, A2.1, A3.2, A11, A24, B7, B8, and B27 binding motifs. Twenty-one CML peptides and 4 APL peptides were predicted to be potential HLA class I binders. The peptides were tested for binding to appropriate purified HLA molecules in a competition radioimmunoassay. Four peptides derived from b3a2 CML breakpoint bound with high (< 50 nmol/L) or intermediate (< or = 500 nmol/L) affinity to HLA A3, A11, and B8. None of the CML b2a2 or PML-RAR alpha A or B junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. This is the first evidence that tumor-specific breakpoint peptides can bind human MHC class I molecules and provides a rationale for developing a therapeutic vaccine strategy.
Assessment of aldehyde dehydrogenase in viable cells
Authors: Jones RJ , Barber JP , Vala MS , Collector MI , Kaufmann SH , Ludeman SM , Colvin OM , Hilton J
Published: October 2019

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.
SBH-1, a novel Reed-Sternberg-like cell line capable of inducing tumors in SCID mice: immunophenotypic, cytogenetic, and cytokine expression profiles
Authors: DeCoteau JF , Reis MD , Griesser H , Lorenzana A , al-Hashmi I , Hawley RG , Naumov A , White CA , Pinkerton PH
Published: October 2019

A new cell line, SBH-1, with the morphologic, immunophenotypic, and karyotypic features consistent with those of Reed-Sternberg (RS) and Hodgkin (H) cells, has been established from the pleural effusion of a patient. The cytologic appearance of SBH-1 cells is characteristic of multinucleate RS and mononuclear H cells, all containing inclusion-like nucleoli. The SBH-1 cells express CD30, CD15, CD25, CD71, CD45, CD20, CD22, and bcl-2 protein and are negative for epithelial membrane antigen. Cytogenetic analysis showed multiple clonal abnormalities with breakpoints at 14q32, 6q21, and 11q23. The Ig heavy chain genes and both Ig light chain genes were rearranged in SBH-1 cells, whereas the bcl-2 gene was in germline configuration. Messages for the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha, and transforming growth factor-beta and the cytokine receptors IL-2R, IL-4R, IL-6R, and IL-7R were detected by reverse transcription-polymerase chain reaction analysis. Xenotransplantation of SBH-1 cells into severe combined immunodeficient (SCID) mice led to local and disseminated tumor growth. The cytologic, histologic, and immunohistochemical features of SBH-1 cells in SCID mouse tumors were typical of RS and H cells. The SBH-1 cell line will be useful in the study of RS and H cell biology, inasmuch as it represents a cell line obtained from a previously untreated patient.
9-cis retinoic acid induces complete remission but does not reverse clinically acquired retinoid resistance in acute promyelocytic leukemia
Authors: Miller WH Jr , Jakubowski A , Tong WP , Miller VA , Rigas JR , Benedetti F , Gill GM , Truglia JA , Ulm E , Shirley M
Published: October 2019

9-cis retinoic acid (RA) is a high-affinity ligand for both retinoic acid receptors (RARs) and retinoid “X” receptors (RXRs). Although all-trans RA does not bind to RXRs, RAR/RXR heterodimers or RXR/RXR homodimers bind to specific DNA response elements and modulate proliferation and differentiation of normal and malignant cells. Because the development of clinical resistance to all-trans RA has been associated with a progressive decrease in plasma drug concentrations, we evaluated the ability of 9-cis RA to induce in vitro cytodifferentiation in subclones of a retinoid-sensitive and resistant APL cell line (NB4) and in short-term cultures of fresh leukemic cells aspirated from patients. We also evaluated the clinical activity and pharmacokinetics of 9-cis RA (LGD 1057) in patients with APL who were previously treated with all-trans RA. In vitro tests of both retinoid-sensitive NB4 cells, as well as samples of fresh cells from 11 patients with APL, showed relatively equivalent degrees of sensitivity to both 9-cis RA and all-trans RA at concentrations ranging from 10(-6) to 10(-8) mol/L; however, no substantial cytodifferentiation was observed using either drug alone or in combination (10(-6) mol/L of each) in retinoid-resistant NB4 cells. Seven patients with APL who had previously relapsed from a remission induced by all-trans RA were treated with 9-cis RA at daily oral doses ranging from 30 to 230 mg/m2. Pharmacokinetic studies showed that the mean terminal plasma half-life of 9-cis RA (1.3 hours) changed very little after several weeks of dosing, although the mean change per dose level in area under the plasma concentration x time curves and peak plasma concentrations showed a decrease by 49% and 45%, respectively. Peak plasma concentrations equaled or exceeded concentrations that were effective against retinoid-sensitive cells in vitro. Despite these favorable pharmacokinetic results, only one of the seven patients achieved complete remission, corroborating in vitro studies of blasts from three of the nonresponders that showed a relatively equivalent degree of resistance to both retinoids. Our results suggest that while 9-cis RA may not induce its own catabolism to the same degree as all-trans RA, this feature does not appear to overcome clinically acquired resistance to all-trans RA in APL. Nonetheless, the drug can induce complete remissions in patients with APL and may be useful for extended therapy in other diseases. Future studies should address the use of lower doses in patients who have not previously received retinoid therapy.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of treatment with low-dose warfarin-aspirin on activated factor VII
Authors: Raskob GE , Durica SS , Morrissey JH , Owen WL , Comp PC
Published: October 2019

Factor VII is an independent risk factor for ischemic heart disease. We performed a prospective study to evaluate the effect of combined low-dose warfarin-aspirin on activated factor VII (factor VIIa) and to determine if abruptly stopping this treatment is associated with a rebound in the level of factor VIIa. Thirty-three patients with clinically stable coronary artery disease were treated with combined 3 mg warfarin and 80 mg aspirin daily for 8 weeks. The factor VIIa level was measured before treatment, weekly during treatment, and 2 weeks after stopping treatment. The mean percent of pretreatment levels of factor VIIa for weeks 1 through 8 of treatment were 60%, 60%, 72%, 70%, 71%, 70%, 74%, and 87%, respectively (P < .05 compared with pretreatment for weeks 1 through 7 inclusive); 2 weeks after stopping treatment, the level was 122% (95% confidence interval [CI]; 111% to 133%; P < .001 compared with pretreatment). The mean percent level of factor VIIa on-treatment was 74% (P < .001). Factor VIIa is reduced by 26% on average during treatment. This finding provides further rationale for the antithrombotic effect of low-dose warfarin. The results suggest a rebound in the factor VIIa level may occur after treatment is stopped. The potential rebound and its clinical importance should be evaluated by further studies.
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation
Authors: Dunbar CE , Cottler-Fox M , O'Shaughnessy JA , Doren S , Carter C , Berenson R , Brown S , Moen RC , Greenblatt J , Stewart FM
Published: October 2019

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.
Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V
Authors: Heeb MJ , Kojima Y , Greengard JS , Griffin JH
Published: October 2019

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid-dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506-FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.
Primitive human hematopoietic progenitors adhere to P-selectin (CD62P)
Authors: Zannettino AC , Berndt MC , Butcher C , Butcher EC , Vadas MA , Simmons PJ
Published: October 2019

P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P-selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX-1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells.
Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells
Authors: Ranheim EA , Cantwell MJ , Kipps TJ
Published: October 2019

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T-cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.
All-trans retinoic acid and cyclic adenosine monophosphate cooperate in the expression of leukocyte alkaline phosphatase in acute promyelocytic leukemia cells
Authors: Gianni M , Terao M , Norio P , Barbui T , Rambaldi A , Garattini E
Published: October 2019

Treatment of acute promyelocytic leukemia (APL) blasts with cyclic adenosine monophosphate (cAMP) analogs, in combination with all-trans retinoic acid (ATRA), results in the upregulation of the expression of leukocyte alkaline phosphatase (LAP), a marker for the differentiation of the granulocyte. The synergistic interaction between the cyclic nucleotide analogs and the retinoid is not unique to APL cells, as it is observed also in the peripheral granulocytes of chronic myelogenous leukemia (CML) patients. The molecular mechanisms underlying LAP induction were studied in NB4, an immortalized APL cell line. Induction of LAP enzymatic activity is dependent on the time of exposure and on the concentrations of dibutyryl-cAMP or 8-bromo-cAMP and ATRA, two factors that influence the kinetics of appearance of detectable levels of the enzyme. Augmentation of LAP levels by ATRA and cAMP is the result of both transcriptional and early posttranscriptional events and requires de novo protein synthesis. LAP induction correlates with augmentation in the levels of the type I catalytic subunit of cAMP-dependent protein kinase transcript and with granulocytic differentiation. The transcriptional component of the process leading to increased LAP gene expression was reproduced in its main features by transient transfection experiments performed in COS-7 cells using the normal retinoic acid receptor type alpha (RAR-alpha) or the APL-specific aberrant form (PML-RAR) and the upstream promoter of the liver/bone/kidney (L/B/K)-type alkaline phosphatase gene. The promoter is upregulated by treatment with ATRA, and this upregulation is further increased by cAMP analogs.
Ecotropic virus integration site-1 gene preferentially expressed in post-myelodysplasia acute myeloid leukemia: possible association with GATA-1, GATA-2, and stem cell leukemia gene expression
Authors: Ohyashiki JH , Ohyashiki K , Shimamoto T , Kawakubo K , Fujimura T , Nakazawa S , Toyama K
Published: October 2019

We investigated expression of the human ecotropic virus integration site-1 (EVI1) gene in patients with leukemia and myelodysplastic syndrome (MDS) using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The EVI1 transcripts were detected in 5 (10.0%) of 50 patients with de novo acute myeloid leukemia (AML), including two AML patients with trilineage myelodysplasia, and in 8 (34.8%) of 23 patients with post-myelodysplastic syndrome AML (post-MDS AML). EVI1 expression was also detected in 6 (35.3%) of 17 MDS patients and three of six patients with chronic myeloid leukemia (CML) in myelomegakaryoblast crisis. No EVI1 transcripts were detected in patients with acute lymphoid leukemia (n = 15) or CML in lymphoid blast crisis (n = 4). Chromosomal abnormalities at the 3q26 region, where the EVI1 gene is located, were found in one patient with MDS and two patients with CML myelomegakaryoblast crisis who had EVI1 expression. Our results showed that EVI1 expression was frequent in patients with post-MDS AML and AML with trilineage myelodysplasia, regardless of the presence or absence of 3q26 abnormalities. EVI1 expression was accompanied by expression of GATA-1 and GATA-2, and often by stem cell leukemia (SCL) gene expression. In patients with post-MDS AML, EVI1 expression was not always associated with a 3q26 abnormality, whereas EVI1 expression in CML myelomegakaryoblast crisis was often linked to a 3q26 abnormality. Our results suggest that the leukemogenic role of EVI1 expression may differ between post-MDS AML and leukemia, with EVI1 expression associated with a 3q26 abnormality.
Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support
Authors: Haas R , Witt B , Mohle R , Goldschmidt H , Hohaus S , Freuhauf S , Wannenmacher M , Hunstein W
Published: October 2019

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non- Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.
Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form
Authors: Miyazawa K , Williams DA , Gotoh A , Nishimaki J , Broxmeyer HE , Toyama K
Published: October 2019

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4- h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4- h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a “turn-off switch” for activated c-kit kinase.
Small noncleaved cell lymphoma and leukemia in adults. A retrospective study of 65 adults treated with the LMB pediatric protocols
Authors: Soussain C , Patte C , Ostronoff M , Delmer A , Rigal-Huguet F , Cambier N , Leprise PY , Francois S , Cony-Makhoul P , Harousseau JL
Published: October 2019

In France, more than 80% of children with Burkitt's lymphoma or Burkitt's leukemia (ALL3) are now cured with the LMB (B-cell non- Hodgkin's lymphoma and B-ALL) protocols of the Societe Francaise d'Oncologie Pediatrique, but so far, poor results have been obtained in the few adult studies available. We have analyzed the experience with LMB protocols in adult patients. This retrospective study involved 65 adult patients with small noncleaved cell lymphoma or ALL3 treated with the LMB protocols. They were 17 to 65 years old and not previously treated. Human immunodeficiency virus-infected patients were excluded. The diagnoses were made between September 1984 and August 1991. According to the Murphy classification, 12 patients (18%) had stage I or II disease, 25 (38%), stage III disease; 4 (6%), stage IV disease; and 24 (37%), ALL3 (> or = 25% blasts). According to the Ann Arbor classification, 9 patients had stage I disease; 8 patients, stage II; 5 patients, stage III; 21 patients, stage IV disease; and 22 patients, ALL (> or = 30% blasts). Twelve patients had central nervous system (CNS) involvement before treatment. Thirty-nine patients were treated according to the LMB 84 protocol scheme; 14 according to the LMB 86 protocol, and 12 patients received the LMB 84 induction courses followed by the LMB 86 consolidation courses. Three patients underwent bone marrow transplantation (BMT) while in second complete remission (CR) and 3 others had refractory disease. There were some protocol violations caused by empirical medical decisions: local irradiation was performed in 4 patients, 2 patients received prophylactic radiation to the brain that was not specified in the protocol, 13 patients underwent BMT in first CR, and methotrexate doses were modified in 10 patients. Fifty-eight patients (89%) achieved a CR. There were four (6%) primary induction treatment failures, and three (4%) early treatment-related deaths. Eight patients relapsed between 2 and 30 months after CR (median, 4.7 months). Forty-seven patients are alive in CR (45 first CR, 2 second CR) with a median follow-up of 57 months (24 to 93 months). There were five toxicity-related deaths among patients in CR including four BMT-related deaths and five deaths caused by refractory relapses. One patient died in CR at 62 months of rectal cancer. The 3- year overall survival rate is 74% (SE = 5). According to the stages in the Murphy classification, the 3-year survival rates are stages I and II, 100%; stage III, 80% (SE = 7); and stage IV and ALL, 57% (SE = 8). Seven of 12 patients with initial CNS disease are alive with a median survival of 56 months.(ABSTRACT TRUNCATED AT 400 WORDS)
Release of Fc gamma RIIa2 by activated platelets and inhibition of anti- CD9-mediated platelet aggregation by recombinant Fc gamma RIIa2
Authors: Gachet C , Astier A , de la Salle H , de la Salle C , Fridman WH , Cazenave JP , Hanau D , Teillaud JL
Published: October 2019

Thrombin-activated human platelets and megakaryocyte cell lines release soluble Fc gamma RII (Fc gamma RIIa2) containing the extracellular and intracellular regions of Fc gamma RIIa1, but lacking the transmembrane domain. Use of polyclonal antibodies directed either against the entire intracytoplasmic tail, or against a peptide located near the C-terminal part of the intracellular region of Fc gamma RIIa2, showed the presence of both a complete form of Fc gamma RIIa2 and a C-terminal truncated form in supernatants of platelets after release of their alpha granule contents and in culture supernatants of megakaryocyte cell lines. Furthermore, recombinant Fc gamma RIIa2 inhibited in a dose-dependent manner Fc-dependent anti-CD9 antibody-induced platelet aggregation. Thus, release of Fc gamma RIIa2 by activated platelets could play an important role in the regulation of platelet activation by immune complexes.
Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions
Authors: Harsfalvi J , Stassen JM , Hoylaerts MF , Van Houtte E , Sawyer RT , Vermylen J , Deckmyn H
Published: October 2019

Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet- von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet- collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet- collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.
Posttranscriptional regulation of macrophage tissue factor expression by antioxidants
Authors: Brisseau GF , Dackiw AP , Cheung PY , Christie N , Rotstein OD
Published: October 2019

Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of fibrin deposition at sites of extravascular inflammation. Recent evidence suggests a role for oxidant stress in the signalling pathway of various cell types by virtue of its ability to induce DNA binding of various transcription factors, including nuclear factor kappa B and AP-1. The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism for the effect. Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the 47-kD mature glycoprotein in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did not result in an accumulation of the less mature forms of TF. When considered together, these data suggest that antioxidants exert their effects by impairing translation and/or by causing degradation of newly translated protein. The effect of antioxidants on tumor necrosis factor appeared to be species specific, with no effect on LPS-induced tumor necrosis factor in murine cells, but with inhibition in human monocytes. The posttranscriptional effect of antioxidants on TF expression data suggests a novel mechanism whereby these agents might modulate monocyte/macrophage activation.
An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age
Authors: Chang YC , Smith KD , Moore RD , Serjeant GR , Dover GJ
Published: October 2019

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).
Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation
Authors: Matthews DC , Appelbaum FR , Eary JF , Fisher DR , Durack LD , Bush SA , Hui TE , Martin PJ , Mitchell D , Press OW
Published: October 2019

In an attempt to decrease the relapse rate after bone marrow transplantation (BMT) for advanced acute leukemia, we initiated studies using 131I-labeled anti-CD45 antibody (BC8) to deliver radiation specifically to hematopoietic tissues, followed by a standard transplant preparative regimen. Biodistribution studies were performed in 23 patients using 0.5 mg/kg trace 131I-labeled BC8 antibody. The BC8 antibody was cleared rapidly from plasma with an initial disappearance half-time of 1.5 +/- 0.2 hours, presumably reflecting rapid antigen- specific binding. The mean radiation absorbed doses (cGy/mCi131I administered) were as follows: marrow, 7.1 +/- 0.8; spleen, 10.8 +/- 1.4; liver, 2.7 +/- 0.2; lungs, 2.1 +/- 0.1; kidneys, 0.7 +/- 0.1; and total body, 0.4 +/- 0.03. Patients with acute myelogenous leukemia (AML) in relapse had a higher marrow dose (11.4 cGy/mCi) than those in remission (5.2 cGy/mCi; P = .001) because of higher uptake and longer retention of radionuclide in marrow. Twenty patients were treated with a dose of 131I estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver, with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by 120 mg/kg cyclophosphamide (CY) and 12 Gy total body irradiation (TBI). Nine of 13 patients with AML or refractory anemia with excess blasts (RAEB) and two of seven with acute lymphocytic leukemia (ALL) are alive disease-free at 8 to 41 months (median, 17 months) after BMT. Toxicity has not been measurably greater than that of CY/TBI alone, and the maximum tolerated dose has not been reached. This study demonstrates that with the use of 131I-BC8 substantially greater doses of radiation can be delivered to hematopoietic tissues as compared with liver, lung, or kidney, which may improve the efficacy of marrow transplantation.