bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor Cell Cytotoxicity of a Novel Metal Chelator
Authors: Torti S.V. , Torti F.M. , Whitman S.P. , Brechbiel M.W. , Park G. , Planalp R.P.
Published: October 2019

We have synthesized a novel six-coordinate metal chelator from the triamine cis-1,3,5-triaminocyclohexane by the addition of a 2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr. The experiments described here were designed to explore whether this compound exhibits potential antitumor activity. When added to MBT2 or T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic, with an IC50 of approximately 4.6 μmol/L compared with 70 μmol/L for desferioxamine. To explore the mode of action of tachpyr, several metal complexes were prepared, including Fe(II), Ca(II), Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further probe the role of Zn(II) and Cu(II) chelation in the cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were prepared through N-alkylation of tachpyr. These derivatives were unable to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and Cu(II). When added to cells, these sterically hindered tachpyr derivatives were nontoxic, consistent with a role of iron depletion in the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to proliferating cultures resulted in an early and selective inhibition of ferritin synthesis, an iron storage protein whose translation is critically dependent on intracellular iron pools. Taken together, these experiments suggest that tachpyr is a cytotoxic metal chelator that targets intracellular iron, and that the use of tachpyr in cancer therapy deserves further exploration.

© 1998 by The American Society of Hematology.
The Kinetics and Extent of Engraftment of Chronic Myelogenous Leukemia Cells in Non-Obese Diabetic/Severe Combined Immunodeficiency Mice Reflect the Phase of the Donor’s Disease: An In Vivo Model of Chronic Myelogenous Leukemia Biology
Authors: Dazzi Francesco , Capelli Debora , Hasserjian Robert , Cotter Finbarr , Corbo Margherita , Poletti Alessandro , Chinswangwatanakul Wimol , Goldman John M. , Gordon Myrtle Y.
Published: October 2019

In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0.5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor’s disease, thus providing an in vivo model of CML biology.

© 1998 by The American Society of Hematology.
Platelet Transfusion: A Dose-Response Study
Authors: Norol Françoise , Bierling Philippe , Roudot-Thoraval Françoise , Le Coeur Françoise Ferrer , Rieux Claire , Lavaux Anne , Kuentz Mathieu , Duedari Najib
Published: October 2019

Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 × 1011/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 × 1011 platelets, the high dose between 6 and 8 × 1011, and the very high dose > 8 × 1011; for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 × 1011 platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 × 109/L, respectively) than the medium dose (33 × 109/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P< .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 × 1011 per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets.The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure.

© 1998 by The American Society of Hematology.
Heparin-Induced Thrombocytopenia: New Insights Into the Impact of the FcγRIIa-R-H131 Polymorphism
Authors: Carlsson Lena E. , Santoso Sentot , Baurichter Gudrun , Kroll Hartmut , Papenberg Stephanie , Eichler Petra , Westerdaal Nomdo A.C. , Kiefel Volker , van de Winkel Jan G.J. , Greinacher Andreas
Published: October 2019

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin treatment, can be associated with new thrombotic complications. HIT antibodies activate platelets via the platelet Fcγ-receptor (FcγRIIa), which carries a functionally relevant polymorphism (FcγRIIa-R-H131). The effect of this polymorphism on the clinical manifestations of HIT is controversial. We determined prospectively the FcγRIIa-R-H131 genotypes in 389 HIT patients, in 351 patients with thrombocytopenia or thrombosis due to causes other than HIT and without detectable HIT antibodies, and in 256 healthy blood donors. For this purpose, a novel nested sequence-specific primer-polymerase chain reaction (SSP-PCR) was developed. FcγRIIa-R/R131 was found to be overrepresented in the HIT patients (27%) compared with the control groups (non-HIT patients [21%] and blood donors [20%]). In a subgroup of 122 well-characterized HIT patients, the genotype distribution in patients presenting with thrombocytopenia only was compared with that of patients who developed thromboembolic complications. The frequency of FcγRIIa-R/R131 among patients with thrombotic events was significantly elevated (37% v 17%;P = .036). Our results indicate that genotype distribution can be correlated to the clinical outcome of patients with HIT. We speculate that the reduced clearance of immune complexes in patients with the FcγRIIa-R/R131 allotype causes prolonged activation of endothelial cells and platelets, thus increasing the risk for thrombotic complications.

© 1998 by The American Society of Hematology.
Severe Factor VII Deficiency Due to a Mutation Disrupting an Sp1 Binding Site in the Factor VII Promoter
Authors: Carew Josephine A. , Pollak Eleanor S. , High Katherine A. , Bauer Kenneth A.
Published: October 2019

We have identified a point mutation in the promoter of the factor VII gene responsible for a severe bleeding disorder in a patient from a large French-Canadian family with known consanguinity. The proband has an extremely low plasma level of factor VII antigen and factor VII coagulant activity (<1 percent of normal) and suffers from hemarthroses and chronic arthropathy. Sequencing of the patient’s factor VII 5′ flanking region, intron/exon junctions, and coding regions showed a homozygous point mutation, a C to G transversion at position −94 relative to the translation start site. We show here that this mutation prevented binding of transcription factor Sp1 and of other nuclear proteins to this region of the factor VII promoter and resulted in a 20-fold reduction in reporter gene expression in HepG2 cells. These data underscore the importance of this region of the factor VII promoter for in vivo expression of the factor VII gene.

© 1998 by The American Society of Hematology.
Compstatin Inhibits Complement and Cellular Activation in Whole Blood in Two Models of Extracorporeal Circulation
Authors: Nilsson Bo , Larsson Rolf , Hong Jaan , Elgue Graciela , Ekdahl Kristina Nilsson , Sahu Arvind , Lambris John D.
Published: October 2019

Recently, a C3-binding cyclic synthetic peptide (Compstatin) has been identified that binds to complement component C3 and inhibits complement activation. Here we have examined the influence of Compstatin on complement activation and its indirect effects on cellular responses in whole blood in two models for extracorporeal circulation. Compstatin effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/ C3 fragments to the polymer surface. As a result of the inhibition of complement activation, the activation of polymorphonuclear leukocytes (PMNs; as assessed by the expression of CD11b) and the binding of these cells (CD16+) to the polymer surface were almost completely lost. In contrast, blood cell counts were not affected. Using surface plasmon resonance technology, we have confirmed that Compstatin exerts its inhibitory effect on complement activation by binding to native C3. These data show that complement activation, leading to activation and binding of PMNs to the biomaterial surface, can be abolished by the addition of Compstatin. The properties of Compstatin make Compstatin a promising drug for use in extracorporeal circuits to avoid bioincompatibility reactions, eg, during cardiopulmonary bypass, but also a favorable precursor peptide for the development of an anticomplement drug for oral use.

© 1998 by The American Society of Hematology.
SLP-76-Cbl-Grb2-Shc Interactions in FcγRI Signaling
Authors: Chu Julie , Liu Yenbou , Koretzky Gary A. , Durden Donald L.
Published: October 2019

SLP-76 and Cbl are complex adapter proteins that have the capacity to bind to smaller adapter proteins, such as Grb2, which subsequently binds the nucleotide exchange protein Sos in the transmission of intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated in immunoreceptor tyrosine-based activation motif (ITAM) signaling, leading to activation of Ras. However, their mechanism of action has not been determined. To date, there have been no reports of SLP-76 involvement in FcγRI-receptor signaling and no data exist for an interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence that SLP-76, Cbl, and Shc are tyrosine phosphorylated on FcγRI-receptor stimulation and are associated with the adapter protein Grb2 in γ-interferon–differentiated U937 cells (U937IF). The interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in resting U937IF cells. However, the interaction between SLP-76 and Grb2 becomes augmented twofold on FcγRI-receptor aggregation. Our results provide the first evidence for a phosphorylation-dependent interaction between SLP-76 and Shc, induced at least 10-fold on FcγRI receptor stimulation. Our data indicate that a significant portion of a multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is distinct from a trimolecular complex containing the Ras guanine nucleotide exchanger Sos, Shc, and Grb2. FcγRI-induced tyrosine phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc interaction provide the first evidence that SLP-76 and Cbl are involved in FcγRI signaling and suggest a functional significance for these interactions in FcγRI signal relay in the control of Ras in myeloid cells.

© 1998 by The American Society of Hematology.
Retinoic Acid Inhibits CD40 + Interleukin-4–Mediated IgE Production In Vitro
Authors: Worm M. , Krah J.M. , Manz R.A. , Henz B.M.
Published: October 2019

To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)–mediated B-cell activation, the effect of 10−12 to 10−6 mol/L RA was studied in anti-CD40 (1 μg/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4–mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% ± 5% in PBMC and 55% ± 4.4% in B cells by all-trans RA, and 58% ± 6.7% and 51% ± 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10−14mol/L for B cells and >10−10 mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10−8mol/L for all-trans RA (94% ± 1.8%) and 96% ± 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10−10 mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-γ (IFN-γ) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors , β, and γ, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor , β, and γ. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the  receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4–stimulated B cells in vitro.

© 1998 by The American Society of Hematology.
Eotaxin Modulates Myelopoiesis and Mast Cell Development From Embryonic Hematopoietic Progenitors
Authors: Quackenbush Elizabeth J. , Wershil Barry K. , Aguirre Vincent , Gutierrez-Ramos Jose-Carlos
Published: October 2019

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role in the embryonic development of the hematopoietic system has not been examined. We report here that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, such as fetal liver (FL), yolk sac (YS), and peripheral blood. We found that eotaxin acts synergistically with stem cell factor to accelerate the differentiation of embryonic mast cell progenitors, and this response can be suppressed by pertussis toxin, an inhibitor of chemokine-induced signaling through Gi protein and chemotaxis. Eotaxin promotes the differentiation of fetal mast cell progenitors into differentiated mast cells as defined by the expression of mast cell specific proteases. Furthermore, in combination with stem cell factor (SCF), it promotes the growth of Mac-1+myeloid cells from embryonic progenitors. These studies suggest that eotaxin may be involved in the growth of granulocytic progenitors and the differentiation and/or function of mast cells during embryogenesis and/or pathological conditions that induce high levels of eotaxin, such as allergic responses.

© 1998 by The American Society of Hematology.
Treatment of Non-Obese Diabetic (NOD)/Severe-Combined Immunodeficient Mice (SCID) With flt3 Ligand and Interleukin-7 Impairs the B-Lineage Commitment of Repopulating Cells After Transplantation of Human Hematopoietic Cells
Authors: Kapp Ursula , Bhatia Mickie , Bonnet Dominique , Murdoch Barbara , Dick John E.
Published: October 2019

Until recently, the identification of cellular factors that govern the developmental program of human stem cells has been difficult due to the absence of repopulation assays that detect human stem cells. The transplantation of human bone marrow (BM) or cord blood (CB) into non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice has enabled identification of primitive human cells capable of multilineage repopulation of NOD/SCID mice (termed the SCID–repopulating cell [SRC]). Here, we examined the effect of long-term in vivo treatment with various combinations of human cytokines on the developmental program of SRC. Detailed flow cytometric analysis of engrafted mice indicated that the vast majority of the human graft of untreated mice was comprised of B lymphocytes at various stages of development as well as myeloid and primitive cells; T cells were not reproducibly detected. Many studies, including murine in vitro and in vivo data and human in vitro experiments, have suggested that flt3 ligand (FL) and/or Interleukin-7 (IL-7) promotes T- and B-cell development. Unexpectedly, we found that treatment of engrafted mice with the FL/IL-7 combination did not induce human T- or B-cell development, but instead markedly reduced B-cell development with a concomitant shift in the lineage distribution towards the myeloid lineage. Effects on lineage distribution were similar in engrafted mice transplanted with highly purified cells indicating that the action of the cytokines was not via cotransplanted mature cells from CB or BM cells. These data show that the lineage development of the human graft in NOD/SCID mice can be modulated by administration of human cytokines providing a valuable tool to evaluate the in vivo action of human cytokines on human repopulating cells.

© 1998 by The American Society of Hematology.
The Induction of Nitric Oxide by Interleukin-12 and Tumor Necrosis Factor- in Human Natural Killer Cells: Relationship With the Regulation of Lytic Activity
Authors: Salvucci Ombretta , Kolb Jean Pierre , Dugas Bernard , Dugas Nathalie , Chouaib Salem
Published: October 2019

We have investigated the interleukin-12 (IL-12) and tumor necrosis factor- (TNF)-induced regulation of human natural killer (NK) cell function and their relationship with nitric oxide (NO) generation. We demonstrate that both cytokines were efficient to trigger the transcription of the inducible nitric oxide synthase (iNOS) mRNA, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis and intracytoplasmic fluorescence showed that iNOS protein was also induced by both cytokines. However, our data indicate that NO does not play a significant role in the effector phase of the cytotoxic activity mediated by NK-stimulated cells, inasmuch as the lytic activity was not affected in the presence of specific NO synthase inhibitors. When aminoguanidine (AMG), an inhibitor of iNOS, was added during the afferent phase of NK stimulation with IL-12 and TNF, a subsequent increase in the lytic potential of the effector cells towards the NK-sensitive target cells (K562) and lymphokine-activated killer (LAK) target cells (Daudi) was observed. Conversely, the addition of chemical NO donors during the afferent step resulted in a dose-dependent inhibition of the NK and LAK cytotoxicity. Our data suggest that the enhancement of NK-cell cytotoxic activity resulting from iNOS inhibition may be correlated, at least in part, to an increase in interferon-γ production and granzyme B expression.

© 1998 by The American Society of Hematology.
Activation of the Small GTPase Rap1 in Human Neutrophils
Authors: M’Rabet Laura , Coffer Paul , Zwartkruis Fried , Franke Barbara , Segal Anthony W. , Koenderman Leo , Bos Johannes L.
Published: October 2019

The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b558 or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca2+ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca2+ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca2+ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation.

© 1998 by The American Society of Hematology.
A Novel Mutation in the Coding Sequence of the FY*B Allele of the Duffy Chemokine Receptor Gene Is Associated With an Altered Erythrocyte Phenotype
Authors: Parasol Niva , Reid Marion , Rios Maria , Castilho Lilian , Harari Ilana , Kosower Nechama S.
Published: October 2019

The Duffy blood group system is of clinical and biological significance. Antibodies to Duffy antigens are responsible for some cases of transfusion incompatibility and newborn hemolytic disease. The Duffy protein is a receptor for the Plasmodium vivaxerythrocyte-binding protein and is also a receptor for various chemokines (thus renamed Duffy Antigen Receptor for Chemokines [DARC]). The two Duffy polymorphic antigens, Fya and Fyb (coded by the FY*A and FY*B alleles), are present on erythrocyte membranes. The Fy(a−b−) phenotype is the predominant one in populations of black people and also occurs in other populations, including some non-Ashkenazi Jewish groups. The Fy(a−b−) phenotype has been associated with a mutation in the FY*B promoter at the GATA box that abolishes the expression of erythrocyte Duffy protein. We describe here a novel mutation, present in the FY*B coding sequence (271C → T), that is associated with some Fy(b−) phenotypes among non-Ashkenazi Jews and among Brazilian blacks. The mutation is present in Fy(b−) individuals, who have wild-type FY*B GATA and carry the previously described 304G → A substitution. The 271C → T and 304G → A can be identified by restriction enzyme–generated restriction fragment length polymorphisms. The 271C → T substitution represents a considerable change in chemical nature (Arg91 → Cys), one which may affect the antigenic determinants of DARC, and thus be of clinical significance. The mutation may have implications for some physiological roles of DARC and be of interest in malaria research and in studies of population genetics.
Deficient Major Histocompatibility Complex Class II Antigen Presentation in a Subset of Hodgkin's Disease Tumor Cells
Authors: Bosshart Herbert , Jarrett Ruth F.
Published: October 2019

Hodgkin's disease is a common malignancy of the lymphoid system. Although the scarce Hodgkin and Reed-Sternberg (HRS) tumor cells in involved tissue synthesize major histocompatibility complex (MHC) class II and costimulatory molecules such as CD40 or CD86, it is unclear whether these tumor cells are operational antigen-presenting cells (APC). We developed an immunofluorescence-based assay to determine the number of MHC class II molecules present on the surface of single living HRS cells. We found that in fresh Hodgkin's disease lymph node biopsies, a subset of HRS cells express a substantial number of surface MHC class II molecules that are occupied by MHC class II–associated invariant chain peptides (CLIP), indicating deficient loading of MHC class II molecules with antigenic peptides. Cultured Hodgkin's disease–derived (HD) cell lines, however, were found to express few MHC class II molecules carrying CLIP peptides on the cell surface and were shown to generate sodium dodecyl sulphate (SDS)-stable MHC class II αβ dimers. In addition to showing deficient MHC class II antigen presentation in a subset of HRS cells, our results show that the widely used HD-cell lines are not ideal in vitro models for the disease. The disruption of MHC class II–restricted antigen presentation in HRS cells could represent a key mechanism by which these tumor cells escape immune surveillance.
BCL-6 Gene Mutations in Posttransplantation Lymphoproliferative Disorders Predict Response to Therapy and Clinical Outcome
Authors: Cesarman Ethel , Chadburn Amy , Liu Yi-Fang , Migliazza Anna , Dalla-Favera Riccardo , Knowles Daniel M.
Published: October 2019

Posttransplantation lymphoproliferative disorders (PT-LPDs) represent a heterogeneous group of Epstein-Barr virus–associated lymphoid proliferations that arise in immunosuppressed transplant recipients. Some of these lesions regress after a reduction in immunosuppressive therapy, whereas some progress despite aggressive therapy. Morphological, immunophenotypic, and immunogenotypic criteria have not been useful in predicting clinical outcome. Although structural alterations in oncogenes and/or tumor suppressor genes identified in some PT-LPDs correlate with a poor clinical outcome, the presence of these alterations has not been a consistently useful predictor of lesion regression after reduction of immunosuppression. We examined 57 PT-LPD lesions obtained from 36 solid organ transplant recipients for the presence of mutations in the BCL-6 proto-oncogene using single-strand conformation polymorphism and sequence analysis, followed by correlation with histopathologic classification and clinical outcome, which was known in 33 patients. BCL-6 gene mutations were identified in 44% of the specimens and in 44% of the patients; none were identified in the cases classified as plasmacytic hyperplasia. However, mutations were present in 43% of the polymorphic lesions and 90% of the PT-LPDs diagnosed as non-Hodgkin's lymphoma or multiple myeloma. BCL-6 gene mutations predicted shorter survival and refractoriness to reduced immunosuppression and/or surgical excision. Our results suggest that the BCL-6 gene structure is a reliable indicator for the division of PT-LPDs into the biological categories of hyperplasia and malignant lymphoma, of which only the former can regress on immune reconstitution. The presence of BCL-6 gene mutations may be a useful clinical marker to determine whether reduction in immunosuppression should be attempted or more aggressive therapy should be instituted.
The Importance of Diagnostic Cytogenetics on Outcome in AML: Analysis of 1,612 Patients Entered Into the MRC AML 10 Trial
Authors: Grimwade David , Walker Helen , Oliver Fiona , Wheatley Keith , Harrison Christine , Harrison Georgina , Rees John , Hann Ian , Stevens Richard , Burnett Alan , Goldstone Anthony
Published: October 2019

Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, −5, del(5q), −7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.
MCP-1, not MIP-1α, Is the Endogenous Chemokine That Cooperates With TGF-β to Inhibit the Cycling of Primitive Normal but not Leukemic (CML) Progenitors in Long-Term Human Marrow Cultures
Authors: Cashman J.D. , Eaves C.J. , Sarris A.H. , Eaves A.C.
Published: October 2019

The long-term culture (LTC) system has been useful for analyzing mechanisms by which stromal cells regulate the proliferative activity of primitive normal, but not chronic myeloid leukemia (CML), hematopoietic progenitor cells. In previous studies, we identified two endogenous inhibitors in this system. One is transforming growth factor-β (TGF-β), which is equally active on primitive normal and CML progenitors. The other we now show to be monocyte chemoattractant protein-1 (MCP-1). Thus, MCP-1, when added to LTC, blocked the activation of primitive normal progenitors but did not arrest the cycling of primitive CML progenitors. Moreover, the endogenous inhibitory activity of LTC stromal layers could be overcome by the addition of neutralizing antibodies to MCP-1, but not to macrophage inflammatory protein-1α (MIP-1α). However, neither of these antibodies antagonized the inhibitory activity of NAc-Ser-Asp-Lys-Pro (AcSDKP) on primitive normal but not CML progenitor cycling in this system. Moreover, none of six other -C-C- or -C-X-C- chemokines, previously shown to inhibit primitive normal human CFC proliferation in semisolid assays, were found to act as negative regulators when added to normal LTC. These results provide further support for the concept that primitive CML progenitor cell proliferation is deregulated when these cells are exposed to limiting concentrations of multiple inhibitors, only some of which have differential actions on normal and Ph+/BCR-ABL+ cells.
Transgenic Mice Expressing Human Fetal Globin Are Protected From Malaria by a Novel Mechanism
Authors: Shear Hannah L. , Grinberg Leonid , Gilman John , Fabry Mary E. , Stamatoyannopoulos George , Goldberg Daniel E. , Nagel Ronald L.
Published: October 2019

Studies in vitro by Pasvol et al (Nature, 270:171, 1977) have indicated that the growth of Plasmodium falciparum in cells containing fetal hemoglobin (HbF = α2γ2) is retarded, but invasion is increased, at least in newborn cells. Normal neonates switch from about 80% HbF at birth to a few percent at the end of the first year of life. Carriers of β-thalassemia trait exhibit a delay in the normal HbF switch-off, which might partially explain the protection observed in populations with this gene. To study this hypothesis in vivo, we used transgenic (γ) mice expressing human Aγ and Gγ chains resulting in 40% to 60% α2Mγ2 hemoglobin, infected with rodent malaria. Two species of rodent malaria were studied.P chabaudi adami causes a nonlethal infection, mainly in mature red blood cells (RBC). P yoelii 17XNL is a nonlethal infection, invading primarily reticulocytes, whereas P yoelii 17XL is a lethal variant of P yoelii 17XNL and causes death of mice in approximately 1 to 2 weeks. Data indicate that this strain may cause a syndrome resembling cerebral malaria caused by P falciparum (Am J Trop Med Hyg, 50:512, 1994). In γ transgenic mice infected with P chabaudi adami, the parasitemia rose more quickly (in agreement with Pasvol) than in control mice, but was cleared more rapidly. In mice infected with P yoelii 17XNL, a clear reduction in parasitemia was observed. Interestingly, splenectomy before this infection, did not reverse protection. The most striking effect was in lethal P yoelii17XL infection. Control mice died between 11 to 13 days, whereas γ mice cleared the infection by day 22 and survived, a phenomenon also observed in splenectomized animals. These results suggest that HbF does indeed have a protective effect in vivo, which is not mediated by the spleen. In terms of mechanisms, light microscopy showed that intraerythrocytic parasites develop slowly in HbF erythrocytes, and electron microscopy showed that hemozoin formation was defective in transgenic mice. Finally, digestion studies of HbF by recombinant plasmepsin II demonstrated that HbF is digested only half as well as hemoglobin A (HbA). We conclude that HbF provides protection from P falciparum malaria by the retardation of parasite growth. The mechanism involves resistance to digestion by malarial hemoglobinases based on the data presented and with the well-known properties of HbF as a super stable tetramer. In addition, the resistance of normal neonates for malaria can now be explained by a double mechanism: increased malaria invasion rates, reported in neonatal RBC, will direct parasites to fetal cells, as well as F cells, and less to the ≈20% of HbA containing RBC, amplifying the antimalarial effects of HbF.
Human Integrin β3 Gene Expression: Evidence for a Megakaryocytic Cell-Specific cis-Acting Element
Authors: Jin Ying , Wilhide Calvin C. , Dang Chi , Li Lu , Li Su-Xia , Villa-Garcia Manuel , Bray Paul F.
Published: October 2019

The human integrin β3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for β3 expression, a number of upstream regulatory regions in the β3 gene were identified. (1) The three regions from −1159 to −584, −290 to −146, and −126 to −115 demonstrated positive, negative, and negative activity, respectively. (2) The region from −115 to +29 of the β3 gene was sufficient for cell-specific activity. Deletion of the sequence from −115 to −89 produced a 6- to 40-fold reduction in reporter gene activity in β3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in β3-expressing endothelial and melanoma cell lines, and 1.3- and 2.8-fold reduction, respectively, in non–β3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions −113 to −108) is a megakaryocytic cell line-specificcis-acting element. (3) The region from −89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position −70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5′ region of the β3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment β3 gene expression.

© 1998 by The American Society of Hematology.
Prolonged Phenotypic, Functional, and Molecular Change in Group I Burkitt Lymphoma Cells on Short-Term Exposure to CD40 Ligand
Authors: Baker Matthew P. , Eliopoulos Aristides G. , Young Lawrence S. , Armitage Richard J. , Gregory Christopher D. , Gordon John
Published: October 2019

Group I Burkitt lymphoma (BL) cell lines (L3055, Elijah, Louckes, BL2, and BL29) retaining the original biopsy phenotype were found to undergo prolonged phenotypic, functional, and molecular change on short-term exposure to soluble recombinant CD40L trimer. Sensitivity to, extent of, and duration of change appeared to reflect passage number in that the earliest passaged lines, L3055 and BL29, were generally the most susceptible. Culture of group I BL lines with CD40L resulted in significant growth arrest (without apoptosis) that, for L3055 cells, was sustained for 7 to 9 days after 72 hours of exposure. This was accompanied by the formation of large homotypic aggregates together with gross changes in individual cell morphology and a concomitant upregulation of CD54 (ICAM-1). Three of the five group I BL lines exhibited rapid downregulation of the hallmark CD77 surface antigen, which, for L3055 cells, was maintained for at least 12 days after 72 hours of incubation with CD40L. With the exception of BL2, all group I BL lines were induced to express CD40 homodimers on CD40-stimulation, whereas only monomers were detected in unstimulated cells. Experiments using CD40-transfected Rat-1 fibroblasts showed that the ability to signal for dimer formation required Thr234of CD40. For L3055 and BL29 cells, an initial 72 hours of exposure to CD40L resulted in the maintenance of homodimers for up to 14 and 10 days, respectively. There was a close correlation between the induction and duration of CD40 homodimers and the appearance of Bcl-2. For L3055 cells, which are sensitive to apoptosis-induction on BCR-engagement, exposure to CD40L for 72 hours was found to provide considerable protection from anti-IgM, which was still significant to 20 days. The implications of such sustained effects on relatively short-term exposure of tumor B cells to CD40L are discussed.

© 1998 by The American Society of Hematology.
Altered Expression and Activity of Topoisomerases During All-Trans Retinoic Acid-Induced Differentiation of HL-60 Cells
Authors: Aoyama Masako , Grabowski Dale R. , Isaacs Richard J. , Krivacic Kim A. , Rybicki Lisa A. , Bukowski Ronald M. , Ganapathi Mahrukh K. , Hickson Ian D. , Ganapathi Ram
Published: October 2019

Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.

© 1998 by The American Society of Hematology.
Serum Transferrin Receptor and Transferrin Receptor-Ferritin Index Identify Healthy Subjects With Subclinical Iron Deficits
Authors: Suominen Pauli , Punnonen Kari , Rajamäki Allan , Irjala Kerttu
Published: October 2019

Despite the established utility of serum transferrin receptor (sTfR), serum ferritin, and the sTfR/log ferritin ratio (TfR-F Index) in the diagnosis of iron deficiency (ID) anemia, the numeric values of these parameters, which are indicative of subclinical ID, remain to be clearly defined. In this study, 65 apparently healthy nonanemic adults (22 men and 43 women) were treated with 3 months of oral iron supplementation to evaluate its effect on parameters reflecting iron status and to determine the prevalence of subclinical iron deficiency in apparently healthy adults. Significant supplementation-induced changes were observed in sTfR, ferritin, and TfR-F Index values in women, whereas in men, none of the studied parameters showed any significant change. Iron-deficient erythropoiesis (IDE) was not observed in men, but was found in 17 women (40%). Although individuals with a compromised iron status may be represented in substantial numbers in conventional reference populations, they can be readily identified using sTfR, ferritin, and TfR-F Index determinations.

© 1998 by The American Society of Hematology.
Constitutive and Interleukin-7/Interleukin-15 Stimulated DNA Binding of Myc, Jun, and Novel Myc-Like Proteins in Cutaneous T-Cell Lymphoma Cells
Authors: Qin J-Z. , Dummer R. , Burg G. , Döbbeling U.
Published: October 2019

Members of the Myc and Jun/Fos gene families have been found to be expressed in late stages of cutaneous T-cell lymphoma (CTCL) and may be responsible for the transition from low-grade to high-grade tumors. The composition of these complexes is an important parameter, as the different homo- and heterodimeric jun and myc complexes can have gene transcription activating or suppressing activities. We determined the composition of the jun and myc DNA-binding complexes in three CTCL cell lines and malignant cells of seven Sézary patients by electrophoretic mobility shift assays (EMSAs) and “supershift” assays in which specific antibodies against the different members of the tested gene families were included in the binding reactions. Complexes containing JunD were found in three cell lines and two patients. The three cell lines and one patient contained also c-Myc/Max heterodimers. Because c-Myc/Max heterodimers are strong gene transcription activators and are necessary for cell-cycle progression, they may play a role in the progression of CTCL. JunD may also promote cell-cycle progression and influence the expression of cell death survival genes. Interleukin-7 (IL-7) and IL-15, which have been identified as growth factors for CTCL cells, stimulated the DNA binding of JunD and two novel c-Myc recognition site (E-box) binding proteins, but not the DNA binding of c-Myc/Max heterodimers.
Two Candidate Downstream Target Genes for E2A-HLF
Authors: Kurosawa Hidemitsu , Goi Kumiko , Inukai Takeshi , Inaba Toshiya , Chang Kun-San , Shinjyo Tetsuharu , Rakestraw Karen M. , Naeve Clayton W. , Look A. Thomas
Published: October 2019

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation, is thought to drive the leukemic transformation of early B-cell precursors by repressing an evolutionarily conserved apoptotic pathway. To test this hypothesis, we sought to identify downstream targets of E2A-HLF in t(17;19)+ pro-B leukemia cells (UOC-B1) that had been transfected with a zinc-inducible vector encoding a dominant-negative suppressor (E2A-HLF[dn]) of the oncoprotein. Representational difference analysis of mRNAs from E2A-HLF(dn)+ UOC-B1 cells grown with (E2A-HLF inactive) or without (E2A-HLF active) the addition of zinc yielded several differentially expressed cDNA fragments that were individually subcloned. Two of the clones, designated F-5 and G-4, hybridized with mRNAs that were upregulated by E2A-HLF. Levels of both transcripts declined sharply within 8 to 12 hours after suppression of E2A-HLF DNA-binding activity, becoming undetectable after 96 hours. The F-5 cDNA was identified as a portion of ANNEXIN VIII, whose product was expressed in promyelocytic leukemia cells and UOC-B1 cells, but not in other leukemic cell lines. A novel full-length cDNA cloned with the G-4 fragment encoded a protein that we have named SRPUL (sushi-repeat protein upregulated in leukemia). It is normally expressed in heart, ovary, and placenta, but could not be detected in leukemic cell lines other than UOC-B1. Neither protein prevented apoptosis in interleukin-3–dependent murine pro-B cells, suggesting that they have paraneoplastic roles in leukemias that express E2A-HLF, perhaps in the disseminated intravascular coagulopathy and hypercalcemia that characterize these cases.
CC Chemokine Receptors, CCR-1 and CCR-3, Are Potentially Involved in Antigen-Presenting Cell Function of Human Peripheral Blood Monocyte-Derived Dendritic Cells
Authors: Sato Katsuaki , Kawasaki Hiroshi , Nagayama Hitomi , Serizawa Ryo , Ikeda Junji , Morimoto Chikao , Yasunaga Kunio , Yamaji Noboru , Tadokoro Kenji , Juji Takeo , Takahashi Tsuneo A.
Published: October 2019

We examined the potential involvement of two CC chemokine receptors (CCRs), CCR-1 and CCR-3, in the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)–generated human peripheral blood monocyte-derived immature dendritic cells (DCs). Flow cytometric analysis showed that CCR-1, CCR-3, CCR-5, and CXC chemokine receptor (CXCR)-4 were expressed on the cell surface of monocyte-derived DCs. Treatment with a monoclonal antibody (MoAb) to either CCR-1 or CCR-3 but not MoAbs to CCR-5 and CXCR-4 abolished chemotactic migration of monocyte-derived DCs. The DCs treated with either the anti–CCR-1 MoAb or anti–CCR-3 MoAb were less efficient than untreated DCs in proliferation of allogeneic T cells (TCs) and TC-derived secretion of interferon-γ (IFN-γ). The homotypic aggregation of DCs and heterotypic aggregation of DCs with TCs were suppressed by the anti–CCR-1 MoAb or anti–CCR-3 MoAb. These results indicate that CCR-1 and CCR-3 specifically regulate interaction of TCs and DCs in the process of antigen presentation.