bluuebunny/crossref_metadata_2025 huggingface dataset:
Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage
Authors: Zhou Ping , Qian Liping , Bieszczad Christine K. , Noelle Randolph , Binder Michael , Levy Norman B. , Craig Ruth W.
Published: October 2019

Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization.

© 1998 by The American Society of Hematology.
Murine Hematopoietic Progenitor Cells With Colony-Forming or Radioprotective Capacity Lack Expression of the β2-Integrin LFA-1
Authors: Pruijt Johannes F.M. , van Kooyk Yvette , Figdor Carl G. , Willemze Roel , Fibbe Willem E.
Published: October 2019

Recently, we have demonstrated that antibodies that block the function of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P < .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)−/Rhodamine (Rho)− sorted cells, were examined. More than 95% of the Rho− cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho− LFA-1neg or up to 600 Rho−LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.
Activation of the Human Immunodeficiency Virus-1 Long Terminal Repeat by Respiratory Burst Oxidants of Neutrophils
Authors: Klebanoff Seymour J. , Headley Catherine M.
Published: October 2019

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) introduced in association with the luciferase reporter gene into Jurkat T cells was strongly activated by a combination of human neutrophils and phorbol myristate acetate (PMA). Activation was not observed when normal neutrophils were replaced by neutrophils which lack a respiratory burst, ie, from a patient with chronic granulomatous disease (CGD), was strongly inhibited by catalase, was potentiated by vanadate, was stimulated by relatively low concentrations of azide, and was inhibited by selective inhibitors of protein kinase C (PKC). The PMA affected activation in three ways: (1) by directly activating the LTR in Jurkat LTRluc; (2) by inducing a respiratory burst in neutrophils with the formation of H2O2; and (3) by increasing the sensitivity of Jurkat LTRluc to the activating effect of H2O2. When PMA was replaced by opsonized zymosan as the neutrophil stimulus, activation of the LTR was low unless azide was added. Activation in the presence of azide was not seen when CGD neutrophils were used or when catalase was added, suggesting that azide acts by inhibiting the degradation of H2O2. These findings indicate that activation of the HIV-1 LTR in Jurkat T cells can be induced by H2O2 released by neutrophils, particularly when PKC is concomitantly activated.
Human Herpesvirus 8 Infection in Patients With POEMS Syndrome–Associated Multicentric Castleman’s Disease
Authors: Bélec Laurent , Mohamed Ali Si , Authier François-Jerôme , Hallouin Marie-Charlotte , Soe Aye Myat , Cotigny Sylvie , Gaulard Philippe , Gherardi Romain K.
Published: October 2019

The polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes (POEMS) syndrome is a rare multisystemic disorder associated with osteosclerotic myeloma and multicentric Castleman’s disease (MCD). Human herpesvirus type 8 (HHV-8) DNA sequences have been detected in lymph nodes of about 40% of human immunodeficiency virus (HIV)-negative patients with MCD, and in bone marrow stromal cells of patients with multiple myeloma. Considering these data, we investigated the presence of HHV-8 in 18 patients with POEMS syndrome (9 with MCD), by nested polymerase chain reaction (N-PCR) to detect DNA sequenses in various cells and tissues obtained by biopsy or at autopsy (13 patients, of whom 7 had MCD), and by an immunofluorescence assay to detect anti–HHV-8 IgG antibodies in blood (18 patients, of whom 9 had MCD). Detection of HHV-8 DNA was performed using three different N-PCR, targeting nonoverlapping regions in open reading frame (ORF) 25 and ORF26. Seven of 13 (54%) POEMS patients had HHV-8 DNA sequences in their tissues, as assessed by all three N-PCR, and 9 of 18 (50%) had circulating anti–HHV-8 antibodies. HHV-8 was mainly detected in the subset of POEMS patients with MCD (6 of 7 [85%] for DNA sequences; 7 of 9 [78%] for antibodies). The percentage of positive N-PCR was higher in lymph nodes than in bone marrow samples (P < .02). Sequencing of amplicons showed a homogeneous restricted variability in the ORF26 region, characteristic of the minority subgroup B defined by Zong, and responsible for isoleucine and glycine substitutions at amino acid positions 134 and 167. These findings strongly suggest an association of HHV-8 infection with POEMS syndrome-associated MCD.
Different Effect of Various Mutant MITF Encoded by mi,Mior, or Miwh Allele on Phenotype of Murine Mast Cells
Authors: Kim Dae-Ki , Morii Eiichi , Ogihara Hideki , Lee Young-Mi , Jippo Tomoko , Adachi Shiro , Maeyama Kazutaka , Kim Hyung-Min , Kitamura Yukihiko
Published: October 2019

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). Mutant alleles of mi,Mior, and Miwh are deletion or point mutation of the basic domain by which MITF binds DNA. The basic domain also has nuclear localization potential. In the present study, we compared the mast cell abnormalities ofMior/Mior andMiwh/Miwh mice with those ofmi/mi mice, of which many have been described by us. The number of mast cells in the skin of Mior/Miorsuckling mice was remarkably decreased from that observed inmi/mi suckling mice, but the number was normal in the skin ofMiwh/Miwh suckling mice. The decrease in skin mast cells was more severe in the mi/mi embryos than inmi/mi suckling mice, but the magnitude of the decrease was comparable between Mior/Mior embryos and Mior/Mior suckling mice. The poor mRNA expression of granzyme B and tryptophan hydroxylase genes was observed in all cultured mast cells (CMCs) derived from the spleens ofMiwh/Miwh,Mior/Mior, and mi/mi mice. However, the poor expression of mouse mast cell protease-4 (MMCP-4), MMCP-5, and MMCP-6 was observed only inMior/Mior and mi/mi CMCs. MITF encoded by Miwh mutant allele (Miwh-MITF) showed deficient but demonstratable DNA binding, but mi-MITF and Mior-MITF did not show any DNA binding ability. Although Miwh-MITF and Mior-MITF showed normal nuclear localization potential, the potential was significantly impaired in mi-MITF. The rank order of mast cell abnormality (mi/mi >Mior/Mior >Miwh/Miwh) appears to be related to the functional abnormality of MITF encoded by each mutant gene.
Myelodysplastic and Myeloproliferative Disorders of Childhood: A Study of 167 Patients
Authors: Luna-Fineman Sandra , Shannon Kevin M. , Atwater Susan K. , Davis Jeffrey , Masterson Margaret , Ortega Jorge , Sanders Jean , Steinherz Peter , Weinberg Vivian , Lange Beverly J.
Published: October 2019

Myelodysplastic syndromes (MDS) and myeloproliferative syndromes (MPS) of childhood are a heterogeneous group of clonal disorders of hematopoiesis with overlapping clinical features and inconsistent nomenclature. Although a number of genetic conditions have been associated with MDS and MPS, the overall contribution of inherited predispositions is uncertain. We report a retrospective study examining clinical features, genetic associations, and outcomes in 167 children with MDS and MPS. Of these patients, 48 had an associated constitutional disorder. One hundred one patients had adult-type myelodysplastic syndrome (A-MDS), 60 had juvenile myelomonocytic leukemia (JMML), and 6 infants with Down syndrome had a transient myeloproliferative syndrome (TMS). JMML was characterized by young age at onset and prominent hepatosplenomegaly, whereas patients with A-MDS were older and had little or no organomegaly. The most common cytogenetic abnormalities were monosomy 7 or del(7q) (53 cases); this was common both in patients with JMML and those with A-MDS. Leukemic transformation was observed in 32% of patients, usually within 2 years of diagnosis. Survival was 25% at 16 years. Favorable prognostic features at diagnosis included age less than 2 years and a hemoglobin F level of less than 10%. Older patients tended to present with an adult-type MDS that is accommodated within the French-American-British system. In contrast, infants and young children typically developed unique disorders with overlapping features of MDS and MPS. Although the type and intensity of therapy varied markedly in this study, the overall outcome was poor except in patients with TMS.
P-Glycoprotein Expression on Normal and Abnormally Expanded Natural Killer Cells and Inhibition of P-Glycoprotein Function by Cyclosporin A and Its Analogue, PSC833
Authors: Egashira Motoki , Kawamata Norihiko , Sugimoto Koichi , Kaneko Takako , Oshimi Kazuo
Published: October 2019

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by theMDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3−CD16+ or CD3−CD56+ lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% ± 0.9%, 93.4% ± 3.1% and 36.9% ± 11.7%, respectively, by 1 μmol/L CsA, and 80.2% ± 3.6%, 91.7% ± 2.6% and 32.7% ± 10.1%, respectively, by 1 μmol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.
Tumor Necrosis Factor Receptor-Associated Factor 1 Is Overexpressed in Reed-Sternberg Cells of Hodgkin’s Disease and Epstein-Barr Virus-Transformed Lymphoid Cells
Authors: Dürkop Horst , Foss Hans-Dieter , Demel Gudrun , Klotzbach Heike , Hahn Corinna , Stein Harald
Published: October 2019

The tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) is a member of the recently defined TRAF family. It takes part in the signal transduction of the TNF receptor 2 (TNFR2), the lymphotoxin-β receptor (LT-βR), CD40, CD30, and LMP1; is induced by LMP1 in vitro; and protects lymphoid cells from apoptosis. To identify the cells in which TRAF1 is active in vivo, we studied TRAF1 transcripts in normal lymphoid tissue, in Epstein-Barr virus (EBV)-induced lymphoproliferations, and in malignant lymphomas with special reference to those that overexpress the cytokine receptor CD30 and CD40 of the TNF receptor family at the single-cell level using a radioactive in situ hybridization. In normal lymphoid tissue, TRAF1 message proved to be absent from all resting B and T cells as well as from macrophages and accessory cells (follicular dendritic cells and interdigitating cells) and present in few perifollicular and intrafollicular lymphoid blasts. In contrast, there was a high and consistent TRAF1 overexpression in EBV-induced lymphoproliferations and Hodgkin’s disease. Nearly all non-Hodgkin’s lymphoma show low or no TRAF1 expression. Only some cases of diffuse large B-cell lymphoma showed a moderate to high TRAF1 signal. Several of the latter cases were EBV+. These data confirm that TRAF1 is an inducible molecule and indicates its deregulation in the mentioned disorders with the potential of a blockage of the apoptotic pathway.
Monitoring Human Blood Dendritic Cell Numbers in Normal Individuals and in Stem Cell Transplantation
Authors: Fearnley D.B. , Whyte L.F. , Carnoutsos S.A. , Cook A.H. , Hart D.N.J.
Published: October 2019

Dendritic cells (DC) originate from a bone marrow (BM) precursor and circulate via the blood to most body tissues where they fulfill a role in antigen surveillance. Little is known about DC numbers in disease, although the reported increase in tissue DC turnover due to inflammatory stimuli suggests that blood DC numbers may be altered in some clinical situations. The lack of a defined method for counting DC has limited patient studies. We therefore developed a method suitable for routine monitoring of blood DC numbers, using the CMRF44 monoclonal antibody (MoAb) and flow cytometry to identify DC. A normal range was determined from samples drawn from 103 healthy adults. The mean percentage of DC present in blood mononuclear cells (MNC) was 0.42%, and the mean absolute DC count was 10 × 106 DC/L blood. The normal ranges for DC (mean ± 1.96 standard deviation [SD]) were 0.15% to 0.70% MNC or 3 to 17 × 106 DC/L blood. This method has applications for monitoring attempts to mobilize DC into the blood to facilitate their collection for immunotherapeutic purposes and for counting blood DC in other patients. In preliminary studies, we have found a statistically significant decrease in the blood DC counts in individuals at the time of blood stem cell harvest and in patients with acute illnesses, including allogeneic bone marrow transplant (BMT) recipients with acute graft-versus-host disease (aGVHD).
A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding
Authors: Tozer Eileen Collins , Baker Elizabeth K. , Ginsberg Mark H. , Loftus Joseph C.
Published: October 2019

An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.
The 5′ Flanking Region of the Human Granzyme H Gene Directs Expression to T/Natural Killer Cell Progenitors and Lymphokine-Activated Killer Cells in Transgenic Mice
Authors: MacIvor Debra M. , Pham Christine T.N. , Ley Timothy J.
Published: October 2019

Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural killer (NK) compartment of the immune system. The gene that encodes this granzyme is located between the granzyme B and cathepsin G genes on human chromosome 14q11.2. Although the murine orthologue of human granzyme H has not yet been identified, murine granzymes C, D, E, F, and G also lie between the murine granzyme B and cathepsin G genes on murine chromosome 14; murine granzymes C, D, and F are also highly expressed in LAK cells, but minimally in cytotoxic T lymphocytes (CTL). We therefore tested whether the 5′ flanking region of human granzyme H contains the cis-acting DNA sequences necessary to target a reporter gene to the LAK/NK compartment of transgenic mice. A 1.2-kb fragment of 5′ flanking human granzyme H sequence was linked to an SV40 large T-antigen (TAg) reporter gene and used to create six transgenic founder lines. SV40 TAg was specifically expressed in the LAK cells of these mice, but not in resting T or NK cells, in CTL, or in any other tissues. Most mice eventually developed a fatal illness characterized by massive hepatosplenomegaly and disseminated organ infiltration by large malignant lymphocytes. Cell lines derived from splenic tumors were TAg+ and NK1.1+ large granular lymphocytes and displayed variable expression of CD3, CD8, and CD16. Although these cell lines contained perforin and expressed granzymes A, B, C, D, and F, they did not exhibit direct cytotoxicity. Collectively, these results suggest that the 5′ flanking sequences of the human granzyme H gene target expression to an NK/T progenitor compartment and to activated NK (LAK) cells. Mice and humans may therefore share a regulatory “program” for the transcription of NK/LAK specific granzyme genes.
Cell Adhesion Mediated Drug Resistance (CAM-DR): Role of Integrins and Resistance to Apoptosis in Human Myeloma Cell Lines
Authors: Damiano Jason S. , Cress Anne E. , Hazlehurst Lori A. , Shtil Alexander A. , Dalton William S.
Published: October 2019

Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (4β1) and VLA-5 (5β1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in 4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and 4-low phenotype. Whether VLA-4–mediated FN adhesion offers a survival advantage over VLA-5–mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.
Ibandronate Reduces Osteolytic Lesions but not Tumor Burden in a Murine Model of Myeloma Bone Disease
Authors: Dallas Sarah L. , Garrett I. Ross , Oyajobi Babatunde O. , Dallas Mark R. , Boyce Brendan F. , Bauss Frieder , Radl Jiri , Mundy Gregory R.
Published: October 2019

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.
Increased Risk of Chronic Graft-Versus-Host Disease, Obstructive Bronchiolitis, and Alopecia With Busulfan Versus Total Body Irradiation: Long-Term Results of a Randomized Trial in Allogeneic Marrow Recipients With Leukemia
Authors: Ringdén O. , Remberger M. , Ruutu T. , Nikoskelainen J. , Volin L. , Vindeløv L. , Parkkali T. , Lenhoff S. , Sallerfors B. , Mellander L. , Ljungman P. , Jacobsen N.
Published: October 2019

Leukemic patients receiving marrow from HLA-identical sibling donors were randomized to treatment with either busulfan 16 mg/kg (n = 88) or total body irradiation ([TBI] n = 79) in addition to cyclophosphamide 120 mg/kg. The patients were observed for a period of 5 to 9 years. Busulfan-treated patients had an increased risk of veno-occlusive disease (VOD) of the liver (12% v 1%,P = .01) and hemorrhagic cystitis (32% v 10%,P = .003). Acute graft-versus-host disease (GVHD) was similar in the two groups, but the 7-year cumulative incidence of chronic GVHD was 59% in the busulfan-treated group versus 47% in the TBI group (P = .05). Death from GVHD was more common in the busulfan group (22% v 3%, P < .001). Obstructive bronchiolitis occurred in 26% of the busulfan patients but in only 5% of the TBI patients (P < .01). Complete alopecia developed in 8 busulfan patients and partial alopecia in 17, versus five with partial alopecia in the TBI group (P < .001). Cataracts occurred in 5 busulfan-treated patients and 16 TBI patients (P = .02). The incidence of relapse after 7 years was 29% in both groups. Seven-year transplant-related mortality (TRM) in patients with early disease was 21% in the busulfan group and 12% in the TBI group. In patients with more advanced disease, the corresponding figures were 64% and 22%, respectively (P = .004). Leukemia-free survival (LFS) in patients with early disease was 68% in busulfan-treated patients and 66% in TBI patients. However, 7-year LFS in patients with more advanced disease was 17% in the busulfan group versus 49% in the TBI group (P < .01). In patients with chronic myeloid leukemia (CML) in first chronic phase, 7-year LFS was 72% and 83% in the two groups, respectively.
Association of the Platelet Glycoprotein Ia C807T Gene Polymorphism With Nonfatal Myocardial Infarction in Younger Patients
Authors: Santoso S. , Kunicki T.J. , Kroll H. , Haberbosch W. , Gardemann A.
Published: October 2019

Recently, we have shown that two alleles of the glycoprotein (GP) Ia gene, designated C807 and T807, are associated with low or high platelet GPIa-IIa density and consequently with slower or faster rate of platelet adhesion to type I collagen, respectively. This polymorphism could therefore present a genetic predisposition for the development of thrombotic disease and hemostasis. We investigated the relationship of the GPIa C807T dimorphism to the risk of coronary artery disease (CAD) and myocardial infarction (MI). An allele-specific polymerase chain reaction (PCR) was developed for genotyping of C807T polymorphism. DNA samples from 2237 male patients who underwent coronary angiography on account of coronary heart disease as verified illness or presumptive diagnosis were genotyped. The odds ratio was calculated as an estimate of the relative risk by multiple logistic regression. We found a strong association between the T allele and nonfatal MI among individuals younger than the mean age of 62 years (n = 1,057; odds ratio, 1.57; P = .004). The odds ratio of MI increased for T807 carriers with decreasing age. The highest odds ratio was detected within the youngest 10% of the study sample (<49 years; n = 223; odds ratio, 2.61; P = .009). In contrast, no evidence of an association between C807T dimorphism with CAD was found. Our findings suggest that inherited platelet GP variations might have an important impact on acute thrombotic disease.
Association of the Platelet Glycoprotein Ia C807T Gene Polymorphism With Nonfatal Myocardial Infarction in Younger Patients
Authors: Santoso S. , Kunicki T.J. , Kroll H. , Haberbosch W. , Gardemann A.
Published: October 2019

Recently, we have shown that two alleles of the glycoprotein (GP) Ia gene, designated C807 and T807, are associated with low or high platelet GPIa-IIa density and consequently with slower or faster rate of platelet adhesion to type I collagen, respectively. This polymorphism could therefore present a genetic predisposition for the development of thrombotic disease and hemostasis. We investigated the relationship of the GPIa C807T dimorphism to the risk of coronary artery disease (CAD) and myocardial infarction (MI). An allele-specific polymerase chain reaction (PCR) was developed for genotyping of C807T polymorphism. DNA samples from 2237 male patients who underwent coronary angiography on account of coronary heart disease as verified illness or presumptive diagnosis were genotyped. The odds ratio was calculated as an estimate of the relative risk by multiple logistic regression. We found a strong association between the T allele and nonfatal MI among individuals younger than the mean age of 62 years (n = 1,057; odds ratio, 1.57; P = .004). The odds ratio of MI increased for T807 carriers with decreasing age. The highest odds ratio was detected within the youngest 10% of the study sample (<49 years; n = 223; odds ratio, 2.61; P = .009). In contrast, no evidence of an association between C807T dimorphism with CAD was found. Our findings suggest that inherited platelet GP variations might have an important impact on acute thrombotic disease.
Inhibition of Activated Protein C Anticoagulant Activity by Prothrombin
Authors: Smirnov Mikhail D. , Safa Omid , Esmon Naomi L. , Esmon Charles T.
Published: October 2019

In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of ≈5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.
Recipient Tumor Necrosis Factor- and Interleukin-10 Gene Polymorphisms Associate With Early Mortality and Acute Graft-Versus-Host Disease Severity in HLA-Matched Sibling Bone Marrow Transplants
Authors: Cavet James , Middleton Peter G. , Segall Miriam , Noreen Harriet , Davies Stella M. , Dickinson Anne M.
Published: October 2019

The proinflammatory cytokine tumor necrosis factor- (TNF-) is strongly implicated in graft-versus-host disease (GVHD) and other acute bone marrow transplant (BMT) complications. The antiinflammatory interleukin-10 (IL-10) antagonizes TNF- and reduces GVHD. We previously showed association of recipient TNF (TNFd) and IL-10 (IL-10−1064) gene polymorphisms with acute GVHD severity in matched sibling BMT using only cyclosporin A monotherapy. The current study tested association of GVHD with TNFd and IL-10−1064/-1082 polymorphisms in a large cohort (144 matched sibling donor/recipient pairs) given both cyclosporine A (CyA) and methotrexate (MTX) prophylaxis. Genotype results were correlated with acute and chronic GVHD and mortality. Patients homozygous for the TNFd microsatellite allele 3 had higher early mortality: 23.7% of TNFd3/d3 homozygotes died before day 30, compared with 6.80% of non-d3/d3 recipients (P = .013). Recipients possessing longer IL-10−1064 microsatellite alleles developed more severe acute GVHD: 22.3% of recipients possessing alleles 12 to 15 developed grade III to IV GVHD, versus 3.92% of those with smaller alleles (P < .01). Other recipient or donor genotypes tested did not significantly affect GVHD or mortality. We conclude that recipient TNFd and IL-10−1064 polymorphisms associate with early mortality and severe acute GVHD in matched sibling BMT with dual prophyaxis. This supports the hypothesis of genetic predisposition towards GVHD and other BMT complications other than histocompatibility antigen disparity.
The Glucocorticoid Receptor Cooperates With the Erythropoietin Receptor and c-Kit to Enhance and Sustain Proliferation of Erythroid Progenitors In Vitro
Authors: von Lindern Marieke , Zauner Wolfgang , Mellitzer Georg , Steinlein Peter , Fritsch Gerhard , Huber Klaus , Löwenberg Bob , Beug Hartmut
Published: October 2019

Although erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 105- to 106-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34+ blast cells, and from bone marrow depleted from CD34+ cells.
Overexpression of Wild-Type Retinoic Acid Receptor  (RAR) Recapitulates Retinoic Acid-Sensitive Transformation of Primary Myeloid Progenitors by Acute Promyelocytic Leukemia RAR-Fusion Genes
Authors: Du Changchun , Redner Robert L. , Cooke Michael P. , Lavau Catherine
Published: October 2019

Retinoic acid receptor  (RAR) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RAR to different partner genes creating the chimeric proteins: PML-RAR, PLZF-RAR, and NPM-RAR. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RAR are resistant. We have used retroviruses to express PML-RAR, PLZF-RAR, NPM-RAR, RAR403 (a dominant negative mutant of RAR), and wild-type RAR in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RAR pathway because overexpression of RARβ, RARγ, or RXR did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RAR, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RAR. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RAR-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RAR-fusion proteins results from their nature to behave as unliganded RAR in the presence of physiological concentrations of retinoic acid.
Increased Clearance Explains Lower Plasma Levels of Tissue-Type Plasminogen Activator by Estradiol: Evidence for Potently Enhanced Mannose Receptor Expression in Mice
Authors: Lansink Mirian , Jong Miek , Bijsterbosch Martin , Bekkers Marian , Toet Karin , Havekes Louis , Emeis Jef , Kooistra Teake
Published: October 2019

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0.32 mL/min in controls; P < .01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL · min in treated animals v 31.9 ng/mL · min in controls; P < .05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P < .001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P < .005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.
Sickle Cell Acute Chest Syndrome: Pathogenesis and Rationale for Treatment
Authors: Stuart Marie J. , Setty B.N. Yamaja
Published: October 2019

Acute chest syndrome (ACS) is a leading cause of death in sickle cell disease (SCD). Our previous work showed that hypoxia enhances the ability of sickle erythrocytes to adhere to human microvessel endothelium via interaction between very late activation antigen-4 (VLA4) expressed on sickle erythrocytes and the endothelial adhesion molecule vascular cell adhesion molecule-1 (VCAM-1). Additionally, hypoxia has been shown to decrease the production of nitric oxide (NO) which inhibits VCAM-1 upregulation. Based on these observations, we hypothesize that during ACS, the rapidly progressive clinical course that can occur is caused by initial hypoxia-induced pulmonary endothelial VCAM-1 upregulation that is not counterbalanced by production of cytoprotective mediators, including NO, resulting in intrapulmonary adhesion. We assessed plasma NO metabolites and soluble VCAM-1 in 36 patients with SCD and 23 age-matched controls. Patients with SCD were evaluated at baseline (n = 36), in vaso-occlusive crisis (VOC; n = 12), and during ACS (n = 7). We observed marked upregulation of VCAM-1 during ACS (1,290 ± 451 ng per mL; mean ± 1 SD) with values significantly higher than controls (P < .0001) or patients either in steady state or VOC (P < .01). NO metabolites were concomitantly decreased during ACS (9.2 ± 1.5 nmol/mL) with values lower than controls (22.2 ± 5.5), patients during steady state (21.4 ± 5.5), or VOC (14.2 ± 1.2) (P< .0001). Additionally, the ratio of soluble VCAM-1 to NO metabolites during ACS (132.9 ± 46.5) was significantly higher when compared with controls (P < .0001) or patients either in steady state or VOC (P < .0001). Although hypoxia enhanced in vitro sickle erythrocyte-pulmonary microvessel adhesion, NO donors inhibited this process with concomitant inhibition of VCAM-1. We suggest that in ACS there is pathologic over expression of endothelial VCAM-1. Our investigations also provide a rationale for the therapeutic use in ACS of cytoprotective modulators including NO and dexamethasone, which potentially exert their efficacy by an inhibitory effect on VCAM-1 and concomitant inhibition of sickle erythrocyte-endothelial adhesion.
Interleukin-1β and Tumor Necrosis Factor- Stimulate DNA Binding of Hypoxia-Inducible Factor-1
Authors: Hellwig-Bürgel Thomas , Rutkowski Karen , Metzen Eric , Fandrey Joachim , Jelkmann Wolfgang
Published: October 2019

The rate of transcription of several genes encoding proteins involved in O2 and energy homeostasis is controlled by hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of  and β subunits. HIF-1 is considered the primarytrans-acting factor for the erythropoietin (EPO) and vascular endothelial growth factor (VEGF) genes. Since EPO gene expression is inhibited by the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor- (TNF-), while no such effect has been reported with respect to the VEGF gene, we investigated the effects of IL-1β and TNF- on the activation of the HIF-1 DNA-binding complex and the amount of HIF-1 protein in human hepatoma cells in culture. Under normoxic conditions, both cytokines caused a moderate activation of HIF-1 DNA binding. In hypoxia, cytokines strongly increased HIF-1 activity compared with the effect of hypoxia alone. Only IL-1β increased HIF-1 protein levels. In transient transfection experiments, HIF-1–driven reporter gene expression was augmented by cytokines only under hypoxic conditions. In contrast to their effect on EPO synthesis, neither IL-1β nor TNF- decreased VEGF production. The mRNA levels of HIF-1 and VEGF were unaffected. Thus, cytokine-induced inhibition of EPO production is not mediated by impairment of HIF-1 function. We propose that HIF-1 may be involved in modulating gene expression during inflammation.
Suppression of Interleukin-12 Production by Human Monocytes After Preincubation With Lipopolysaccharide
Authors: Wittmann Miriam , Larsson Vivi-Ann , Schmidt Petra , Begemann Gabriele , Kapp Alexander , Werfel Thomas
Published: October 2019

Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-γ (IFN-γ) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-γ– or GM-CSF–primed human monocytes can be completely suppressed by preincubation with LPS (fromEscherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E2 (PGE2), tumor necrosis factor- (TNF-), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-γ–induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.
Human Immunodeficiency Virus-1 (HIV-1)-Tat Protein Promotes Migration of Acquired Immunodeficiency Syndrome–Related Lymphoma Cells and Enhances Their Adhesion to Endothelial Cells
Authors: Chirivi Renato G.S. , Taraboletti Giulia , Bani Maria Rosa , Barra Luca , Piccinini Giampiero , Giacca Mauro , Bussolino Federico , Giavazzi Raffaella
Published: October 2019

Human immunodeficiency virus-1 (HIV-1)-Tat, the transactivating gene product of HIV-1, has been shown to interact with different cell types, inducing gene expression, altering their growth and migratory behavior. In this study we examined whether Tat might affect functions of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin’s lymphoma (NHL), relevant to the in vivo dissemination. Our results show that Tat significantly augmented the motility of the two AIDS-related Burkitt’s lymphoma cell lines (AS283 and PA682PB) and AIDS-primary effusion lymphoma cell line (HBL-6-AIDS-PEL). Mutations in RGD or basic domain of Tat (KGE-MBP and LxI-MBP, respectively) sharply reduced migration compared with wild type, suggesting that both domains are required for migration. In contrast, a Tat protein mutation outside the active domains (NH2-TAT-GST) did not reduce lymphoma cell migration. The treatment of lymphoma cells with Tat did not influence their adhesion to matrix proteins or to human vascular endothelial cells, but endothelial cells treated with Tat became more adhesive to lymphoma cells. Flow cytometric analysis showed that treatment of endothelial cells with Tat induced the cell surface expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and E-selectin and increased the expression of intercellular adhesion molecule-1 (ICAM-1). Only antibodies against VCAM-1 on endothelial cells or against the VLA-4 integrin expressed on AS283 cells inhibited the increment of adhesion, indicating the relevance of this pathway in the adhesion of lymphoma cells to vascular endothelium. In our work, we show for the first time that Tat can enhance the migration of lymphoma cells and their adhesion to endothelial cells, two processes that may contribute to the malignant behavior of NHL in patients with AIDS.