bluuebunny/crossref_metadata_2025 huggingface dataset:
Homoharringtonine therapy induces responses in patients with chronic myelogenous leukemia in late chronic phase
Authors: O'Brien S , Kantarjian H , Keating M , Beran M , Koller C , Robertson LE , Hester J , Rios MB , Andreeff M , Talpaz M
Published: October 2019

Homoharringtonine (HHT) is a plant alkaloid with potent myelosuppressive activity and little toxicity when used in a continuous infusion schedule. The antileukemic efficacy of HHT has been shown in acute myeloid leukemia, but has not been investigated in chronic myelogenous leukemia (CML). Seventy-one patients with Philadelphia chromosome-positive (Ph+) CML in late chronic phase (time from diagnosis to therapy longer than 12 months) were treated with a continuous infusion of HHT at a daily dose of 2.5 mg/m2 for 14 days for remission induction and for 7 days every month for maintenance. The median number of courses given was 6 (range, 1 to 35) and 21 patients (30%) continue on treatment. Forty-two of 58 patients (72%) evaluable for hematologic response achieved a complete hematologic remission, and 9 (16%) had a partial hematologic remission. Twenty-two of 71 patients (31%) developed a cytogenetic response; it was major (Ph+ cells less than 35%) in 11 (15%) and complete (Ph+ cells 0%) in 5 (7%). Significant myelosuppression occurred in 39% of induction courses and 9% of maintenance courses. Fever or documented infection was present in 26% of induction courses and in only 8% of maintenance courses. Nonmyelosuppressive toxicity was minimal. Homoharringtonine produced hematologic remissions in the majority of patients with advanced chronic-phase CML. Cytogenetic response occurred in some patients without an association with myelosuppression, and these responses may be prolonged. Future studies investigating homoharringtonine in combination with other active agents in CML, such as interferon, are warranted.
Retention of glycoprotein Ib/IX receptors on external surfaces of thrombin-activated platelets in suspension
Authors: White JG , Krumwiede MD , Cocking-Johnson D , Rao GH , Escolar G
Published: October 2019

The present study has evaluated the hypothesis stating that glycoprotein (GP) Ib/IX, the receptor for von Willebrand factor (vWF), is downregulated and cleared from exposed surfaces to channels of the open canalicular system (OCS) on platelets activated by thrombin in suspension. Cryosections of resting and thrombin-activated platelets fixed at intervals of 1 to 30 minutes after stimulation by thrombin and stained with antiglycocalicin antibody and protein A gold showed no decrease in the density of GPIb/IX receptors on the platelet surface or increase on linings of the OCS at any interval after stimulation by thrombin. Thin sections of platelets exposed to thrombin in suspension followed by settling onto a plastic chamber for intervals of 1 to 30 minutes revealed retention of GPIb/IX receptors on exposed surfaces detected by vWF, anti-vWF, and protein A gold throughout the 30-minute period of study. Results of this investigation indicate that GPIb/IX receptors remain on the surface of platelets activated by thrombin in suspension, are not cleared to the OCS, and retain the ability to bind vWF for at least 30 minutes.
Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction
Authors: Emmons RV , Reid DM , Cohen RL , Meng G , Young NS , Dunbar CE , Shulman NR
Published: October 2019

Thrombopoietin (TPO), the ligand for c-mpl, stimulates proliferation of committed megakaryocytic progenitors and induces maturation of megakaryocytes. To better understand factors regulating TPO levels, we measured blood levels of TPO in patients with impaired platelet production due to aplastic anemia (AA) and with platelet destructive disorders, including idiopathic thrombocytopenic purpura (ITP), posttransfusion purpura (PTP), drug purpura (DP), and X-linked thrombocytopenia (XLTP). The TPO receptor capture enzyme immunoassay (EIA) used had a detection limit of integral of approximately-150 to 200 pg/mL. TPO was undetectable in 88 of 89 normal individuals. Eighteen of 19 patients with AA and a mean platelet count (MPC) of 18,000/microliters (2,000 to 61,000/microliters) had markedly elevated TPO levels (mean, 1,467 pg/mL; range, 597 to 3,834 pg/mL). Eight AA patients who responded to immunosuppressive therapy with their MPC increasing to 140,000/microliters (92,000 to 175,000/microliters) had substantial decreases in TPO (mean, 440 pg/mL; range, 193 to 771 pg/mL). Initial TPO levels did not differ significantly between responders and nonresponders. In contrast, all 21 patients with ITP and an MPC of 16,000/microliters (1,000 to 51,000 /microliters) had undetectable TPO levels, as did 6 patients with acute PTP or DP and 2 patients with XLTP. Megakaryocyte mass, reflected in the rate of platelet production, appears to be the major determinant of TPO levels in thrombocytopenic patients rather than circulating platelet levels per se. Measurement of serum TPO may be useful in differentiating thrombocytopenias due to peripheral destruction from those due to thrombopoietic failure.
Differential regulation of leukocyte function-associated antigen-1/ intercellular adhesion molecules-1-dependent adhesion and aggregation in HL-60 cells
Authors: Katagiri K , Kinashi T , Irie S , Katagiri T
Published: October 2019

Activation of integrin and organization of cytoskeletal proteins are highly regulated in cell adhesion and aggregation. The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecules-1 (ICAM-1) mediates cell adhesion and aggregation, which facilitate leukocyte trafficking to inflamed tissues and augment effector functions. We investigated how LFA-1/ICAM-1-mediated adhesion and aggregation are regulated in HL-60 cells induced to differentiate into neutrophils by retinoic acid (RA). Uninduced HL-60 cells did not bind to ICAM-1 even with stimulation by 12–0-tetradecanoyl phorbol-13- acetate, although they express LFA-1 on the cell surface. When cultured with RA for 24 hours, HL-60 cells were able to adhere to ICAM-1 constitutively. The induction of adhesion did not accompany any change in surface density of LFA-1, indicating that the avidity of LFA-1 was increased. The change in its avidity required de novo synthesis of proteins. Although ICAM-1 was intensely expressed on RA-induced HL-60 cells, these cells did not show any cellular aggregation. The HL-60 cells transfected with the active form of Ras (Val12) exhibited LFA- 1/ICAM-1-dependent aggregation by RA stimulation without change in the avidity of LFA-1. In these Ras-transfectants, a cytoskeletal protein, paxillin, was tyrosine-phosphorylated, and the level of F-actin increased. Transforming growth factor (TGF) beta, as well as cytochalasin D, prevented both the tyrosine phosphorylation of paxillin and the aggregation without any effects on the avidity of LFA-1. Thus, an increase in the avidity of LFA-1 was not sufficient for the induction of aggregation, which required activation of Ras and reorganization of cytoskeletal proteins. These results suggest that distinct regulatory mechanisms control LFA-1/ICAM-1-dependent adhesion and aggregation in HL-60 cells differentiating into neutrophils.
p53 mutations in mantle cell lymphoma are associated with variant cytology and predict a poor prognosis
Authors: Greiner TC , Moynihan MJ , Chan WC , Lytle DM , Pedersen A , Anderson JR , Weisenburger DD
Published: October 2019

Mutations of the p53 tumor suppressor gene have been described in several subtypes of non-Hodgkin's lymphoma, but the incidence of p53 mutations in mantle cell lymphoma (MCL) is unknown. We hypothesized that cases of MCL with a variant or high-grade cytology would have a higher likelihood of p53 mutations than typical MCL. We were also interested in the prognostic significance of p53 mutations in MCL. Therefore, a series of 53 well-characterized cases of MCL with DNA from 62 tissue samples were analyzed by the polymerase chain reaction with denaturing gradient gel electrophoresis for exons 5–8 of p53. Immunoperoxidase studies with the antibody DO-7 to p53 protein were also performed on frozen sections. We found mutations of the p53 gene in 8 of the 53 cases (15%) of MCL. Missense mutations predominated, and 50% of the mutations occurred at known p53 hotspot codons. Of 21 cases with variant cytology (ie, anaplastic or blastic), 6 (28.6%) had p53 mutations as compared with only 2 of 32 cases (6.3%) with typical MCL cytology (P = .05), and p53 mutations preceded the development of variant cytology in 2 patients. Overexpression of p53 protein was observed in 6 of the 8 cases (75%) with p53 mutations and in none of the 45 wild-type cases. The median survival of the cases with mutant p53 was only 1.3 years (all died), whereas the median survival of cases with germline p53 was 5.1 years (P = .023). These results suggest that mutations of p53 may be one mechanism involved in the development of variant forms of MCL and indicate that p53 mutations in MCL predict a poor prognosis.
Clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated Fanconi anemia complementation group C polypeptide with partial activity
Authors: Yamashita T , Wu N , Kupfer G , Corless C , Joenje H , Grompe M , D'Andrea AD
Published: October 2019

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility. Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to T), account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptides, FRP-50 (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell lines with the IVS4 + 4 A to T mutation lacked FRP-50. Overexpression of a cDNA encoding FRP-50 in an FA(C) cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FA(C) patients.
Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes
Authors: Deveaux S , Filipe A , Lemarchandel V , Ghysdael J , Romeo PH , Mignotte V
Published: October 2019

he MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans- acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.
An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect
Authors: Hayward CP , Rivard GE , Kane WH , Drouin J , Zheng S , Moore JC , Kelton JG
Published: October 2019

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta- thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.
Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line
Authors: Hill SA , Shaughnessy SG , Joshua P , Ribau J , Austin RC , Podor TJ
Published: October 2019

Type 1 plasminogen activator inhibitor (PAI-1) and its cofactor vitronectin (Vn) are stored within the alpha-granules of platelets. The two possible sources for their biosynthetic origin are endogenous synthesis in megakaryocytes or endocytosis from plasma. Using ultrastructural and confocal laser scanning microscopic (CLSM) image analysis, we observed that treatment of Dami cells, a human megakaryocytic cell line, with phorbol myristate acetate (PMA) induces the accumulation of PAI-1 and Vn in intracellular storage vacuoles that contain other alpha-granule proteins such as von Willebrand factor. To examine evidence for biosynthesis of PAI-1 and Vn by Dami cells, we immunoprecipitated PAI-1 and Vn from the conditioned media of cells biosynthetically radiolabeled with 35S-methionine in the presence or absence of PMA. In contrast to Hep G2 cells, which synthesize both PAI- 1 and Vn, only 35S-PAI-1 was recovered from PMA-treated Dami cells. Reverse transcription-PCR analysis of RNA extracted from resting and PMA-treated Dami cells confirmed that PAI-1 mRNA expression was detectable at low levels in resting cells and induced by PMA treatment. In contrast, Vn mRNA was not detected. We examined binding and internalization (endocytosis) of PAI-1 and Vn by Dami cells using biotinylated analogs (b-PAI-1 and b-Vn). Flow cytometry analysis indicated that the binding of b-Vn to Dami cells was dose-dependent, saturable, and specific for multimeric forms of Vn. Cells were incubated at 4 degrees C or 37 degrees C and endocytosis of b-Vn was shown by probing electrophoretically fractionated cell lysates with 125I-labeled streptavidin. Only cells incubated at 37 degrees C internalized b-Vn. CLSM image analysis confirmed that the b-Vn was internalized and that it colocalized with PAI-1 in storage granules. The binding of b-Vn to cells was inhibited by the presence of PAI-1, and there was no evidence of specific b-PAI-1 binding or uptake to resting or PMA-treated cells. These data suggest that accumulation of PAI-1 in Dami cell storage granules is due to endogenous synthesis and that the accumulation of Vn is due to endocytosis of serum-derived Vn.
Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand
Authors: Renard N , Lafage-Pochitaloff M , Durand I , Duvert V , Coignet L , Banchereau J , Saeland S
Published: October 2019

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti- CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.
Monoclonal Lym-1 antibody-dependent lysis of B-lymphoblastoid tumor targets by human complement and cytokinine-exposed mononuclear and neutrophilic polymorphonuclear leukocytes
Authors: Ottonello L , Morone P , Dapino P , Dallegri F
Published: October 2019

Lym-1 is a murine IgG2a monoclonal antibody that recognizes a polymorphic variant of HLA-DR antigens on malignant B cells, with minimal cross-reactivity with normal tissues. Because it can be safely administered in vivo, a detailed knowledge of its ability to recruit and trigger the antitumor immune effector systems is required to optimize potential serotherapeutic approaches in B-lymphoma patients. By using Raji cells as a model of B-lymphoma targets, we found that Lym- 1 activates complement-mediated lysis efficiently. Moreover, Lym-1 was capable of triggering the antibody-dependent cellular cytolysis (ADCC) by peripheral blood mononuclear cells (MNCs). On the contrary, it failed to trigger neutrophilic polymorphonuclear leukocyte (PMN)- mediated ADCC activity. In an attempt to enhance Lym-1 ADCC by MNCs and PMNs, nine biologic response modifiers were tested. MNC-mediated Lym-1 ADCC was significantly stimulated by interleukin-2 (IL-2) and unaffected by other mediators, including gamma-interferon (gamma-IFN), tumor necrosis factor a (TNFalpha), and granulocyte-macrophage colony- stimulating factor (GM-CSF). On the other hand, PMN-mediated Lym-1 ADCC was induced or significantly augmented by various cytokines, such as GM- CSF, TNFalpha, and gamma-IFN, and chemotaxins, such as formyl peptides (FMLP), complement fragment C5a, and IL-8. Both MNC- and PMN-mediated ADCC was unaffected by granulocyte colony-stimulating factor (G- CSF) and insulin-like growth factor-1 (IGF-1). Finally, only GM-CSF and TNFalpha augmented the number of PMNs actually engaged in the binding of Raji target cells. The findings presented here, in particular those showing stimulatory activity of biologic response modifiers, may inspire new attempts for developing Lym-1 antibody-based approaches to the therapy of B lymphomas.
Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia
Authors: Height SE , Swansbury GJ , Matutes E , Treleaven JG , Catovsky D , Dyer MJ
Published: October 2019

Clonal rearrangements of the Ig heavy chain (IGH) locus occur in nearly all cases of B-cell precursor acute leukemia (BCP-ALL). Some of these rearrangements may be detected by polymerase chain reaction (PCR) using VH gene framework III (FRIII) and JH consensus primers. However, about 20% of BCP-ALLs fail to amplify with this technique. To determine the causes of these PCR failures and to investigate any possible association with specific subgroups of disease, we analyzed 72 acute leukemias of defined immunophenotype and cytogenetics, comparing FRIII with VH-family leader-specific PCR methods and Southern blotting. Of 37 BCP-ALL cases, 6 (16.2%) failed totally to amplify with FRIII and JH primers. None of these cases amplified with VH leader primers. Additionally, all cases retained germline VH6 genes and 5 of 11 rearranged alleles amplified with a consensus DH primer, indicating that these rearrangements represented biallelic DH-JH recombinations. Among the 6 FRIII and VH leader PCR-negative BCP-ALL cases, there was no common immunophenotype or consistent cytogenetic abnormality, although all showed structural chromosomal abnormalities and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;q11) Philadelphia chromosome-positive [Ph+]) and IGH rearrangements (9 BCP-ALL and 4 biphenotypic cases) were also analyzed. Of 23 rearranged IGH alleles, 19 (82%) were positive by FRIII PCR, and all 4 remaining alleles were amplified by VH leader primers. Use of the leader primers in these Ph+ cases also detected 3 additional clonal rearrangements that were not anticipated from Southern blotting; such unexpected bands were not observed in 21 other Ph- cases. The additional bands represented “new” and unrelated VH rearrangements rather than VH-VH replacement events. We conclude that biallelic DHJH rearrangements occur in a subgroup of BCP-ALL; in these cases, the activation of the full VHDHJH recombination mechanism had not occurred. Therefore, these cases of BCP-ALL were arrested at an early stage of B- cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute leukemias, which may represent the transformation of multipotent hemopoietic stem cells, had undergone VHDHJH recombination. Of 9 Ph+ BCP-ALL cases, 3 also showed ongoing VHDHJH rearrangement, reflecting the persistent expression of the VHDHJH recombinase. Finally, sequence analysis of 33 rearranged VHDHJH genes showed that only 3 including 2 Ph+ BCP-ALL maintained an intact open-reading frame. Loss of the open- reading frame occurred not only because of out-of-frame VHDH and DHJH joining, but also because of VH gene mutation and deletion. These data show that most BCP-ALLs may represent the neoplastic transformation of BCPs destined to die in the bone marrow.
DNA copy number changes in diffuse large B-cell lymphoma--comparative genomic hybridization study
Authors: Monni O , Joensuu H , Franssila K , Knuutila S
Published: October 2019

We studied DNA copy number changes in diffuse large B-cell lymphoma using comparative genomic hybridization analysis on 20 primary tumors and on 12 recurrent tumors excised after chemotherapy or radiotherapy. Twenty-nine (91%) of the cases showed abnormal copy number karyotypes. Chromosomal regions at X (41%), 1q (38%), 7 (31%), 3 (24%), 6p (21%), 11 (21%), 12 (21%), and 18 (21%) were most frequently gained, and the most common losses involved 6q (38%), X (21%), 1p (14%), and 8p (10%). High-level amplifications were observed at 6p23-ter, 10p12–14, 17p1l.2, 18q21-ter, and Xq22-ter, all but 18q appearing only in the recurrent tumors. Gains (median, 2; range, 0 to 10) were more frequent than losses (median, 1; range, 0 to 7; P = .0004). The median number of aberrations found in the recurrent tumors (6.5) was greater than that in the primary tumors (2; P = .01). The copy number changes found in the recurrent tumors were more random than those found in the primary tumors, which were mainly located in the most frequently affected regions. Our findings are in line with those observed using conventional cytogenetic analysis, but especially novel high-level amplifications were detected. Southern blot analysis showed BCL2 amplification, but not translocation t(14;18)(q32;q21), in cases in which a gain at 18q was detected by comparative genomic hybridization, which strongly suggests that, in addition to translocation, gene amplification is another mechanism for the overexpression of the BCL2 protein.
Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation
Authors: Alemany M , Concord E , Garin J , Vincon M , Giles A , Marguerie G , Gulino D
Published: October 2019

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274–368) and rIII-13 (beta 3 274–403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274–368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.
Internalization of bound fibrinogen modulates platelet aggregation
Authors: Wencel-Drake JD , Boudignon-Proudhon C , Dieter MG , Criss AB , Parise LV
Published: October 2019

In agonist-stimulated platelets, the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) is converted from an inactive to an active fibrinogen receptor, thereby mediating platelet aggregation. With time after agonist addition, at least two events occur: fibrinogen becomes irreversibly bound to the platelet and, when stirring is delayed, platelets lose the ability to aggregate despite the presence of maximally bound fibrinogen. Because we previously identified an actively internalized pool of alpha IIb, beta 3 in platelets, we explored the possibility that both of these events might result from the internalization of fibrinogen bound to active alpha IIb beta 3. Under conditions of irreversible fibrinogen binding, fluorescence microscopy showed that biotinylated fibrinogen is rapidly internalized by activated platelets to a surface-inaccessible, intracellular pool. Flow cytometric analysis showed that the observed loss in accessibility to extracellular probes immediately precedes a loss in ability to the platelets to aggregate. Moreover, prevention of irreversible fibrinogen binding results in a prevention of internalization and a retention of aggregation capacity. Thus, the internalization of fibrinogen from the activated platelet surface appears to contribute not only to the irreversible phase of fibrinogen binding, but also to the downregulation of platelet adhesiveness. Fibrinogen internalization is therefore likely to represent a fundamental regulatory mechanism that modulates platelet function.
B-chronic lymphocytic leukemias can undergo isotype switching in vivo and can be induced to differentiate and switch in vitro [see comments]
Authors: Malisan F , Fluckiger AC , Ho S , Guret C , Banchereau J , Martinez-Valdez H
Published: October 2019

B-chronic lymphocytic leukemias (B-CLL) represent expanded clones of resting B lymphocytes that mostly express surface IgM (sIgM). The present study shows that B-CLL cells, freshly isolated from two patients, were sIgM+, sIgG-, and sIgA- but expressed IgG and IgA transcripts. cDNA cloning and sequencing showed that the VDJ segments associated to gamma and alpha heavy chain transcripts are identical to those from mu transcripts, thus showing that B lymphocytes giving rise to CLL cells have undergone isotype switching in vivo. Stimulation of these B-CLL cells through surface CD40 in the presence of interleukin- 10 induced them to secrete IgG and IgA, proving that they can also differentiate into Ig-secreting cells. Finally, CD40-stimulated B-CLL cells were induced to switch towards IgE in response to interleukin-4, as shown by the presence of specific VDJ-C epsilon transcripts and the secretion of IgE. Therefore, B-CLL cells tested herein can undergo isotype switching in vivo and can be induced to undergo further isotype switching and differentiation in vitro.
Mechanisms of retinoid resistance in leukemic cells: possible role of cytochrome P450 and P-glycoprotein
Authors: Kizaki M , Ueno H , Yamazoe Y , Shimada M , Takayama N , Muto A , Matsushita H , Nakajima H , Morikawa M , Koeffler HP , Ikeda Y
Published: October 2019

Retinoic acid (RA) regulates the differentiation and proliferation of a wide variety of different cell types and all-trans RA induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). However, clinical resistance to retinoids may develop and poses a serious problem for differentiation-inducing therapy. We studied the effects of RA in combination with a cytochrome P450 inhibitor (clotrimazole) and a P-glycoprotein antagonist (verapamil) on cell growth and differentiation of RA-resistant HL-60 cells and fresh RA-resistant leukemic cells from two APL patients. RA-resistant HL-60 cells and APL cells differentiated to mature granulocytes when cultured with all-trans RA and either clotrimazole and verapamil but not with either of the agents alone. These findings were confirmed in these cells by their increased expression of CD11b antigen and migration- inhibitory factor-related protein-8/14 mRNAs and decreased levels of c- myc mRNA. These combinations also markedly decreased the number of viable cells and inhibited cellular proliferation. After isolation of microsomes, measurements showed that levels of cytochrome P450 activities in both wild-type and RA-resistant HL-60 cells were almost comparable. Moreover, expression of the CYP1A1-type cytochrome P450 gene could not be detected in either cell type. However, RA-resistant HL-60 cells and APL cells, but not RA-sensitive HL-60 cells and APL cells, expressed multidrug-resistance-1 gene transcripts. Taken together, acquired resistance to RA may be explained in part by drug metabolism in leukemic cells. Possible mechanisms for accelerated clearance of RA include the induction of non-CYP1A1 cytochrome P450 enzymes and P-glycoprotein.
p59fyn-p125FAK cooperation in development of CD4+CD8+ thymocytes
Authors: Kanazawa S , Ilic D , Hashiyama M , Noumura T , Yamamoto T , Suda T , Aizawa S
Published: October 2019

p59fyn is an Src family nonreceptor tyrosine kinase that has been suggested to play an important role in T-cell development and function. p125FAK is a unique nonreceptor tyrosine kinase and has been known to respond to integrin-extracellular matrix interactions. To examine their roles in thymocytes, heterozygous fak mutation was introduced into homozygous Fyn deficiency. The double mutation, but neither Fyn deficiency nor FAK heterozygosity alone, displayed impaired development of CD4+CD8+ thymocytes with atrophy of the thymic cortex, suggesting a unique cooperation between p59fyn and p125FAK in CD4+CD8+ T-cell development.
Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system
Authors: Spivak JL , Avedissian LS , Pierce JH , Williams D , Hankins WD , Jensen RA
Published: October 2019

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.
A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells
Authors: Bazil V , Brandt J , Chen S , Roeding M , Luens K , Tsukamoto A , Hoffman R
Published: October 2019

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker- negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.
Polymerization of recombinant hemoglobin F gamma E6V and hemoglobin F gamma E6V, gamma Q87T alone, and in mixtures with hemoglobin S
Authors: Adachi K , Pang J , Konitzer P , Surrey S
Published: October 2019

To further understand determinants for Hemoglobin (Hb) S polymerization, as well as the inhibitory mechanism of Hb F on Hb S polymerization, Hb F variants containing Val-gamma 6 (Hb F gamma E6V) or Val-gamma 6, Thr-gamma 87 (Hb F gamma E6V, gamma Q87T) were expressed in yeast. The oxy form of Hb F gamma E6V was about 10-fold less stable to mechanical agitation than native oxy Hb F, which is similar to stability differences comparing oxy Hb S and oxy Hb A. Deoxy Hb F gamma E6V showed approximately 20-fold decreased solubility compared with native deoxy Hb F in high phosphate buffer and formed gels like deoxy Hb S in low phosphate buffer, indicating that the Val- gamma 6 substitution decreases solubility of Hb F like Val-beta 6 in deoxy Hb S. Oversaturated deoxy Hb F gamma E6V polymerized without a delay time in low and high phosphate buffers, in contrast to deoxy Hb S, which is accompanied by a distinct delay time before polymerization. Deoxy Hb F gamma E6V, gamma Q87T also polymerized without a delay time like deoxy Hb F gamma E6V. These results suggest that deoxy Hb F gamma E6V gamma Q87T polymers are different from those of deoxy Hb S, and that contact sites differ from those of deoxy Hb S, even though both have the same primary donor (A3) and acceptor sites in the EF helix. These results also suggest that other amino acids in addition to beta 6 Val and amino acids in the F helix are critical for nucleation- controlled polymerization of deoxy Hb S. 1:1 mixtures of deoxy Hb S and either Hb F variant polymerized with a delay time when the concentrations for the Hb S/Hb F gamma E6V and Hb S/Hb F gamma E6V, gamma Q87T mixtures were about 2- and 1.5-fold, respectively, higher than that for Hb S. Logarithmic plots of delay time versus concentration for Hb S/Hb F gamma E6V mixtures showed the same straight line as the line for Hb S/Hb S beta T87Q mixtures, but values for Hb S/Hb F gamma E6V, gamma Q87T mixtures were intermediate between those for Hb S and Hb S/Hb F gamma E6V mixtures. A 1:1 mixture of deoxy Hb A and Hb F gamma E6V, gamma Q87T also polymerized, but exhibited biphasic kinetics, when the concentration was increased to more than 3.5-fold higher than that required for Hb S polymer formation. These results suggest that Gin-gamma 87 is a critical amino acid for exclusion of FS hybrids (alpha 2 beta S gamma) from nuclei formation with Hb S. Our findings also show that Val-gamma 6 in hybrids that form in mixtures of the Hb F variants with either Hb S or Hb A interacts with the hydrophobic acceptor pocket on the EF helix of an adjacent tetramer containing Thr-beta 87.
Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb
Authors: Melotti P , Calabretta B
Published: October 2019

To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5′ flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.
Eosinophils express a functional receptor for interferon alpha: inhibitory role of interferon alpha on the release of mediators
Authors: Aldebert D , Lamkhioued B , Desaint C , Gounni AS , Goldman M , Capron A , Prin L , Capron M
Published: October 2019

Recent reports describe the beneficial use of alpha interferon (IFNalpha) for the treatment of idiopathic hypereosinophilic syndrome (HES) unresponsive to conventional therapy. A clinical improvement associated with a rapid decrease of peripheral blood eosinophilia suggested possible direct effects of IFNalpha on eosinophils through the presence of IFNalpha receptors (IFNalphaR). Reverse transcriptase- polymerase chain reaction (RT-PCR) and cytochemistry were used respectively to detect the presence and define the distribution of IFNalphaR on enriched eosinophil preparations purified from blood cells. IFNalphaR was found on eosinophils collected from patients with various eosinophilic disorders. In addition, IFNalpha inhibited the release of eosinophil granule proteins such as eosinophil cationic protein (ECP), neurotoxin (EDN, or interleukin-5 (IL-5). Moreover, antiparasite cytotoxicity was also strongly reduced in a dose-dependent manner by IFNalpha. These results provide the first evidence that human eosinophils express a functional receptor for IFNalpha and represent a potential basis for the beneficial effects of IFNalpha in patients with hypereosinophilic syndromes.
Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow
Authors: Delfino DV , Patrene KD , Lu J , Deleo A , Deleo R , Herberman RB , Boggs SS
Published: October 2019

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK- LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the “lymphoid” gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5- fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.
Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5
Authors: Poirel H , Rack K , Delabesse E , Radford-Weiss I , Troussard X , Debert C , Leboeuf D , Bastard C , Picard F , Veil-Buzyn A , Flandrin G , Bernard O , Macintyre E
Published: October 2019

To determine the incidence of MLL rearrangement in acute myeloid leukemia (AML) French-American-British (FAB) type M1 and to evaluate optimal screening strategies for the characterization of such abnormalities, we analyzed specimens from 41 patients with AML by Southern blotting with two MLL genomic probes and compared the capacities of reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH) to identify the types of rearrangement found in AML M1 with those observed in AML M5. MLL rearrangement was found in 6 of 29 (20%) AML M1 and 6 of 10 AML M5 cases. RT-PCR characterization of 11 cases showed four MLL self- fusions, four MLL-AF6, two MLL-AF9, including a novel AF9 breakpoint, and one uncharacterized t(11:19). Only 5 of 10 MLL-rearranged cases tested demonstrated karyotypic 11q23 abnormalities. FISH analysis of nine cases with an MLL-specific yeast artificial chromosome (YAC) confirmed the cytogenetic abnormalities in two cases, clarified them in one, and did not detect six cases, including three MLL self-fusions, one case with a probable MLL-rearranged subclone not represented karyotypically, and twoMLL-AF6. A whole chromosome 11 paint detected one of these MLL-AF6, and an AF6 cosmid demonstrated that the other was probably due to insertion of a submicroscopic portion of chromosome 6, including part of AF6, into an apparently normal chromosome 11. We conclude that MLL rearrangements are common in adult AML M1, that MLL self-fusion and MLL-AF6 are the most frequent types of abnormalities, and that RT-PCR is preferable to 11q23 FISH analysis for their characterization.
Comparison of genomic DNA and cDNA for detection of residual disease after treatment of chronic myeloid leukemia with allogeneic bone marrow transplantation
Authors: Zhang JG , Lin F , Chase A , Goldman JM , Cross NC
Published: October 2019

To test whether patients in remission after allogeneic bone marrow transplantation (BMT) possess a pool of chronic myeloid leukemia (CML) cells that do not express BCR-ABL mRNA, we have compared the results and sensitivity of amplification of BCR-ABL from genomic DNA with conventional reverse transcription-polymerase chain reaction (RT-PCR). Bubble PCR was used to amplify the genomic BCR-ABL translocation breakpoints from chronic-phase DNA of 10 patients with CML who subsequently underwent BMT. After cloning and sequencing of the amplification products, patient-specific ABL primers were synthesized and tested for both specificity and sensitivity in nested or heminested combinations with a variety of primers derived from the major breakpoint cluster region of the BCR gene. In all cases, combinations of primers were selected that enabled the detection of chronic-phase DNA from a specific patient at up to a 10(5)x dilution into DNA from a normal individual. Patterns of residual disease obtained by serial RT- PCR and DNA-PCR analyses of blood and bone marrow samples obtained after BMT were similar for most patients, including one treated for relapse by infusion of donor leukocytes. Of the 24 samples for direct comparison of RT-PCR and DNA-PCR, results were concordant in 19 (79%) cases. Five results were discordant. In two instances, RT-PCR was positive, while PCR from genomic DNA was negative; this discrepancy might have arisen due to the slightly greater sensitivity of RT-PCR compared with DNA-PCR. In three samples from three patients, two of whom had been transplanted in the accelerated phase, PCR from genomic DNA was positive while RT-PCR was negative; this could mean that some CML cells in these samples had a reduced or absent capacity to express BCR-ABL mRNA post-transplant. Of these three patients, one subsequently relapsed; and two are in remission at 21 and 24 months after the discordant result. Thus, the finding of a single DNA-PCR- positive, RT- PCR-negative results does not necessarily predict relapse. Because the great majority of samples (79%) gave concordant results with the two assays, we believe that patients in remission do not generally harbor a substantial pool of CML cells that do not express BCR-ABL mRNA.