bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy
Authors: Ikuta Tohru , Kan Yuet Wai , Swerdlow Paul S. , Faller Douglas V. , Perrine Susan P.
Published: October 2019

The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression.

© 1998 by The American Society of Hematology.
Marginal-Zone B Cells in the Human Lymph Node and Spleen Show Somatic Hypermutations and Display Clonal Expansion
Authors: Tierens Anne , Delabie Jan , Michiels Lieve , Vandenberghe Peter , De Wolf-Peeters Chris
Published: October 2019

Splenic marginal-zone B cells, marginal-zone B cells of Peyer’s patches in the gut, and nodal marginal-zone B cells (also identified as monocytoid B cells) share a similar morphology and immunophenotype. These cells likely represent a distinct subset of B cells in humans and rodents, but their precise ontogenetic relationship as well as their origin from B cells of the germinal center is still debated. To study this, we performed a mutation analysis of the rearranged immunoglobulin variable genes (VH) of microdissected single nodal and splenic marginal-zone cells. In addition, we investigated the presence of proliferating cells and B-cell clones in the human splenic and nodal marginal zone as well as adjacent germinal centers. This was performed by immunohistochemical staining for the Ki-67 antigen and denaturing gradient gel analysis of amplified immunoglobulin heavy chain genes’ complementarity determining region 3 of microdissected cell clusters. A variable subset of nodal and splenic marginal-zone B cells showed somatic mutations in their rearranged VH genes, indicating that both virgin and memory B cells are present in the nodal and splenic marginal zone. Nodal and splenic marginal-zone B cells preferentially rearranged VH3 family genes such as DP47, DP49, DP54, and DP58. A preferential rearrangement of the same VH genes has been shown by others in the peripheral CD5− IgM+ B cells. These data suggest that the splenic and nodal marginal-zone B cells are closely related B-cell subsets. We also showed that marginal-zone B cells may cycle and that clones of B cells are frequently detected in the nodal as well as the splenic marginal zone. These clones are not related to those present in adjacent germinal centers. These data favor the hypothesis that clonal expansion occurs in the marginal zone. Whether the somatic hypermutation mechanism is activated during the clonal expansion in the marginal zone and which type of immune response triggers the clonal expansion need to be elucidated.
Inhibition of Human Umbilical Vein Endothelial Cell Proliferation by the CXC Chemokine, Platelet Factor 4 (PF4), Is Associated With Impaired Downregulation of p21Cip1/WAF1
Authors: Gentilini Grazia , Kirschbaum Nancy E. , Augustine James A. , Aster Richard H. , Visentin Gian Paolo
Published: October 2019

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E–cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21Cip1/WAF1, to the cyclin E-cdk2 complex. Analysis of total cellular p21Cip1/WAF1 showed that in the presence of PF4, p21Cip1/WAF1 levels were sustained at time points when p21Cip1/WAF1 was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21Cip1/WAF1 and provide the first evidence for interference with cell cycle mechanisms by a chemokine.
The Use of Allele-Specific Recombinant Fcγ Receptor IIIb Antigens for the Detection of Granulocyte Antibodies
Authors: Bux Juergen , Kissel Karin , Hofmann Christine , Santoso Sentot
Published: October 2019

The Fcγ receptor IIIb (FcγRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcγRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcγRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcγRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody–specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcγRIIIb-specific isoantibodies; and 12 FcγRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4°C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcγRIIIb-reactive alloantibodies and autoantibodies.
Age Is Not a Prognostic Variable With Autotransplants for Multiple Myeloma
Authors: Siegel D.S. , Desikan K.R. , Mehta J. , Singhal S. , Fassas A. , Munshi N. , Anaissie E. , Naucke S. , Ayers D. , Spoon D. , Vesole D. , Tricot G. , Barlogie B.
Published: October 2019

Multiple myeloma (MM) typically afflicts elderly patients with a median age of 65 years. However, while recently shown to provide superior outcome to standard treatment, high-dose therapy (HDT) has usually been limited to patients up to 65 years. Among 550 patients with MM and a minimum follow-up of 18 months, 49 aged ≥65 years were identified (median age, 67; range, 65 to 76 years). Their outcome was compared with 49 younger pair mates (median, 52; range, 37 to 64 years) selected among the remaining 501 younger patients (<65 years) matched for five previously recognized critical prognostic factors (cytogenetics, β2-microglobulin, C-reactive protein, albumin, creatinine). Nearly one half had been treated for more than 1 year with standard therapy and about one third had refractory MM. All patients received high-dose melphalan-based therapy; 76% of the younger and 65% of the older group completed a second transplant (P = .3). Sufficient peripheral blood stem cells to support two HDT cycles (CD34 > 5 × 106/kg) were available in 83% of younger and 73% of older patients (P = .2). After HDT, hematopoietic recovery to critical levels of granulocytes (>500/μL) and of platelets (>50,000/μL) proceeded at comparable rates among younger and older subjects with both first and second HDT. The frequency of extramedullary toxicities was comparable. Treatment-related mortality with the first HDT cycle was 2% in younger and 8% among older subjects, whereas no mortality was encountered with the second transplant procedure. Comparing younger/older subjects, median durations of event-free and overall survival were 2.8/1.5 years (P = .2) and 4.8/3.3 years (P = .4). Multivariate analysis showed pretransplant cytogenetics and β2-microglobulin levels as critical prognostic features for both event-free and overall survival, whereas age was insignificant for both endpoints (P = .2/.8). Thus, age is not a biologically adverse parameter for patients with MM receiving high-dose melphalan-based therapy with peripheral blood stem cell support and, hence, should not constitute an exclusion criterion for participation in what appears to be superior therapy for symptomatic MM.
Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures
Authors: Erlich Shai , Miranda Silvia R.P. , Visser Jan W.M. , Dagan Arie , Gatt Shimon , Schuchman Edward H.
Published: October 2019

The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.
Low Platelet 2β1 Levels in Type I von Willebrand Disease Correlate With Impaired Platelet Function in a High Shear Stress System
Authors: Di Paola Jorge , Federici Augusto B. , Mannucci P.M. , Canciani Maria T. , Kritzik Marcie , Kunicki Thomas J. , Nugent Diane
Published: October 2019

Platelet adhesion to collagen-coated surfaces in whole blood under flow conditions is mediated by both von Willebrand factor (vWF)-dependent recruitment of the platelet glycoprotein Ib-IX receptor complex and collagen interaction with the integrin 2β1. In type 1 von Willebrand disease (vWD), platelet adhesive functions are impaired due to the decrease in vWF levels in plasma and platelets. There are at least three alleles of the human 2 gene, distinguishable by a cluster of silent or noncoding sequence differences within a segment of the gene. Two alleles, associated with low receptor density can be distinguished by nucleotide 807C, while the third allele associated with high receptor density, expresses nucleotide 807T. Gene frequencies of these alleles in a normal population (n = 167) are 0.58 for 807C and 0.42 for 807T. We measured the frequencies of these alleles in symptomatic patients with five types of vWD (type 1, n = 78; type 2A, n = 25, type 2B, n = 14; type 2M, n = 10; and type 3, n = 20). Compared with the normal group, no significant difference in allele frequencies was observed among individuals with types 2A, 2B, 2M, or 3 vWD. However, the frequency of the 807C allele, associated with low collagen receptor density, among type 1 vWD patients (807C = .71; 807T = .29) was significantly higher than that of the normal population (P = .007). Also, in patients with vWD type 1 and borderline to normal ristocetin-cofactor (vWF:RCo) activity values, collagen receptor density correlates inversely with closure time in a high shear stress system (platelet function analyzer [PFA-100]). We propose that low platelet 2β1 density results in less efficient primary platelet adhesion and may result in increased tendency to bleed, as evidenced by the high frequency of this polymorphism in patients with type 1 vWD compared with normal individuals. In addition, this may account for the variability between patients with similar levels of vWF antigen, but strikingly different bleeding histories.
Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted Chemotherapy: A Phase I Study of an Anti-CD33 Calicheamicin Immunoconjugate
Authors: Sievers E.L. , Appelbaum F.R. , Spielberger R.T. , Forman S.J. , Flowers D. , Smith F.O. , Shannon-Dorcy K. , Berger M.S. , Bernstein I.D.
Published: October 2019

Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33+ AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3,3’-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.
Sickle Cell Anemia as a Possible State of Enhanced Anti-Apoptotic Tone: Survival Effect of Vascular Endothelial Growth Factor on Circulating and Unanchored Endothelial Cells
Authors: Solovey Anna , Gui Lizhen , Ramakrishnan Sundaram , Steinberg Martin H. , Hebbel Robert P.
Published: October 2019

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% ± 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% ± 15% (P < .001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P = .001) higher in plasma of sickle donors (120.1 ± 81.4 pg/mL) than that of normal donors (37.6 ± 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r = .612,P = .001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of vβ3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.
Expression and Functional Characterization of the β-Isoform of the Folate Receptor on CD34+ Cells
Authors: Reddy Joseph A. , Haneline Laura S. , Srour Edward F. , Antony Asok C. , Clapp D. Wade , Low Philip S.
Published: October 2019

We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34+ cells express both the β and γ isoforms of FR. The  isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-), the abundance of FR-β on CD34+ cell surfaces was relatively low (≈8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34+ cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34+ cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function.
Red Blood Cells Inhibit Apoptosis of Human Neutrophils
Authors: Aoshiba Kazutetsu , Nakajima Yuri , Yasui Shuji , Tamaoki Jun , Nagai Atsushi
Published: October 2019

Oxidative stress has been implicated in the triggering of apoptosis in neutrophils. Because red blood cells (RBCs) are well known to scavenge oxidants including H2O2, we tested the hypothesis that RBCs inhibit apoptosis of neutrophils by reducing intracellular oxidative stress. Apoptosis of neutrophils was evaluated by light microscopy and DNA gel electrophoresis. We found that coculture with RBCs protected against neutrophil apoptosis. Neither physical contact between RBCs and neutrophils nor the cellular integrity of RBCs was required to protect against neutrophil apoptosis. Neutrophil apoptosis was promoted by exogenous H2O2 but suppressed by catalase, indicating a role for H2O2 as a mediator of apoptosis. The protective effect of RBCs against apoptosis was due to catalase and glutathione metabolism because blocking of these antioxidant systems in RBCs attenuated the protective effect of RBCs. These results suggest that neutrophils are protected against apoptosis in the circulation.
Granulocyte Colony-Stimulating Factor–Mobilized Allogeneic Stem Cell Transplantation Maintains Graft-Versus-Leukemia Effects Through a Perforin-Dependent Pathway While Preventing Graft-Versus-Host Disease
Authors: Pan Luying , Teshima Takanori , Hill Geoffrey R. , Bungard David , Brinson Yani S. , Reddy Vijay S. , Cooke Kenneth R. , Ferrara James L.M.
Published: October 2019

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)–mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF–mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 → B6D2F1). B6 mice (H-2b) were injected subcutaneously with human G-CSF (100 μg/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v0%, P < .001). Systemic levels of lipopolysaccharide and tumor necrosis factor- were markedly reduced in recipients of allogeneic G-CSF–mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2d) at the time of transplantation, all surviving recipients of G-CSF–mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell–depleted (TCD) splenocytes from G-CSF–mobilized B6 donors died of leukemia. When splenocytes from G-CSF–mobilized perforin-deficient (pfp−/−) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF–mobilized PBSCT. These data illustrate that G-CSF–mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.
Purified Photoproducts of Merocyanine 540 Trigger Cytochrome C Release and Caspase 8-Dependent Apoptosis in Human Leukemia and Melanoma Cells
Authors: Pervaiz Shazib , Seyed Mohamed A. , Hirpara Jayshreekumari L. , Clément Marie-Véronique , Loh Kok W.
Published: October 2019

If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non–receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (▵ψm), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial ▵ψm are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.
Presenting White Blood Cell Count and Kinetics of Molecular Remission Predict Prognosis in Acute Promyelocytic Leukemia Treated With All-Trans Retinoic Acid: Result of the Randomized MRC Trial
Authors: Burnett Alan K. , Grimwade David , Solomon Ellen , Wheatley Keith , Goldstone Anthony H.
Published: October 2019

All-trans retinoic acid (ATRA) is an essential component of the treatment of acute promyelocytic leukemia (APL), but the optimal timing and duration remain to be determined. Molecular characterization of this disease can refine the diagnosis and could be potentially useful in monitoring response to treatment. Patients defined morphologically to have APL were randomized to receive a 5-day course of ATRA before commencing chemotherapy or to receive daily ATRA commencing with chemotherapy and continuing until complete remission (CR). The chemotherapy was that used in current MRC Leukaemia Trials. Outcome comparisons were by intention to treat with additional analysis for relevant risk factors. Patients were characterized by molecular techniques for the fusion products of the t(15;17) and monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) during and after treatment. Two hundred thirty-nine patients were randomized. Treatment with extended ATRA resulted in a superior remission rate (87% v 70%, P < .001), due to fewer early and induction deaths (12% v 23%, P = .02), and less resistant disease (2% v 7%, P = .03), which was associated with a significantly more rapid recovery of neutrophils and platelets. Extended ATRA reduced relapse risk (20%v 36% at 4 years, P = .04) and resulted in superior survival (71% v 52% at 4 years, P = .005). Presenting white blood cell count (WBC) was a key determinant of outcome. The 70% of patients who presented with a WBC less than 10 × 109/L had a better CR (85% v62%, P = .0001) and reduced relapse risk (22% v42%, P = .002) and superior survival (69%v 43%, P < .0001). Within the low count group, extended ATRA resulted in a better CR (94% v 76%, P= .001), reduced relapse risk (13% v 35%, P = .04), and improved survival (80% v 57%, P = .0009). There was no evidence of benefit in patients presenting with a higher WBC (>10 × 109/L). Molecular monitoring after the third chemotherapy course had a correlation with risk of relapse. The relapse risk was 57% if the RT-PCR was positive versus 27% if the RT-PCR was negative (P = .006). APL patients who present with a low WBC derive substantial benefit from combining ATRA with induction chemotherapy until remission is achieved, whereas patients with a higher WBC did not benefit. Molecular characterization of disease can improve diagnostic precision and a positive RT-PCR after consolidation identifies patients at a higher risk of relapse.
Identification of HLA-A2–Restricted T-Cell Epitopes Derived From the MUC1 Tumor Antigen for Broadly Applicable Vaccine Therapies
Authors: Brossart Peter , Heinrich Kathrin S. , Stuhler Gernot , Behnke Lars , Reichardt Volker L. , Stevanovic Stefan , Muhm Alexandra , Rammensee Hans-Georg , Kanz Lothar , Brugger Wolfram
Published: October 2019

The tumor-associated antigen MUC1 is overexpressed on various hematological and epithelial malignancies and is therefore a suitable candidate for broadly applicable vaccine therapies. It was demonstrated that major histocompatibility complex (MHC)-unrestricted cytotoxic T cells can recognize epitopes of the MUC1 protein core localized in the tandem repeat domain. There is increasing evidence now that MHC-restricted T cells can also be induced after immunization with the MUC1 protein or segments of the core tandem repeat. Using a computer analysis of the MUC1 amino acid sequence, we identified two novel peptides with a high binding probability to the HLA-A2 molecule. One of the peptides is derived from the tandem repeat region and the other is derived from the leader sequence of the MUC1 protein, suggesting that, in contrast to previous reports, the MUC1-directed immune responses are not limited to the extracellular tandem repeat domain. Cytotoxic T cells (CTL) were generated from several healthy donors by primary in vitro immunization using peptide-pulsed dendritic cells. The addition of a Pan-HLA-DR binding peptide PADRE as a T-helper epitope during the in vitro priming resulted in an increased cytotoxic activity of the MUC1-specific CTL and a higher production of cytokines such as interleukin-12 and interferon-γ in the cell cultures, demonstrating the importance of CD4 cells for an efficient CTL priming. The peptide induced CTL lysed tumors endogenously expressing MUC1 in an antigen-specific and HLA-A2–restricted fashion, including breast and pancreatic tumor cells as well as renal cell carcinoma cells, showing that these peptides are shared among many tumors. The use of MUC1-derived peptides could provide a broadly applicable approach for the development of dendritic cell-based vaccination therapies.
Activated Dendritic Cells From Bone Marrow Cells of Mice Receiving Cytokine-Expressing Tumor Cells Are Associated With the Enhanced Survival of Mice Bearing Syngeneic Tumors
Authors: Fujii Shin-ichiro , Hamada Hirofumi , Fujimoto Koji , Shimomura Taizo , Kawakita Makoto
Published: October 2019

Dendritic cells (DCs), which phagocytose antigens and subsequently proliferate and migrate, may be the most powerful antigen-presenting cells that activate naive T cells. To determine their role in the immune response to tumors, we used WEHI-3B murine leukemia cells transduced with adenovirus vectors expressing cytokines. We found that mixtures of irradiated cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) plus those expressing interleukin-4 (IL-4) or tumor necrosis factor  (TNF) protected mice against WEHI-3B–induced leukemias. When bone marrow mononuclear cells (BMMNCs) obtained from mice that had been injected with irradiated, cytokine-expressing tumor cells were injected into tumor-bearing mice, the survival of the latter was significantly prolonged; the longest survival was observed in mice receiving BMMNCs containing an increased number of DCs from animals injected with a mixture of tumor cells expressing GM-CSF with those expressing IL-4. Assay for antileukemic effects in spleen of the latter animals showed specific antitumor cytotoxicity against WEHI-3B, suggesting that DCs from donor mice activate specific T cells in the tumor-bearing recipients. These results suggest that the infusion of syngeneic BMMNCs stimulated with cytokine-expressing tumor cells may be effective in treating certain types of tumors.
Sialoadhesin-Positive Host Macrophages Play an Essential Role in Graft-Versus-Leukemia Reactivity in Mice
Authors: Müerköster Susanne , Rocha Marian , Crocker Paul R. , Schirrmacher Volker , Umansky Victor
Published: October 2019

We recently established an effective immune T-cell–mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell–mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell–mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen β-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous β-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.
Human Immunoglobulin A Receptor (FcRI, CD89) Function in Transgenic Mice Requires Both FcR γ Chain and CR3 (CD11b/CD18)
Authors: van Egmond Marjolein , van Vuuren A.J. Hanneke , Morton H. Craig , van Spriel Annemiek B. , Shen Li , Hofhuis Frans M.A. , Saito Takashi , Mayadas Tanya N. , Verbeek J. Sjef , van de Winkel Jan G.J.
Published: October 2019

Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR γ chain or the β2 integrin CR3 (CD11b/CD18) in FcRI biological function, FcRI transgenic mice were crossed with either FcR γ chain −/− or CR3 −/− mice. In contrast to in vitro data, FcR γ chain was essential for surface expression of hFcRI in vivo. Functional studies in hFcRI/ γ−/−mice were, therefore, limited. In vitro studies showed FcR γ chain to be necessary for phagocytosis. Neither hFcRI expression nor phagocytosis, triggered via hFcRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcRI transgenic/ CR3−/− mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcRI-mediated antibody-dependent cellular cytotoxicity (ADCC).
Lineage-Restricted Expression of Protein Kinase C Isoforms in Hematopoiesis
Authors: Bassini Alessandra , Zauli Giorgio , Migliaccio Giovanni , Migliaccio Anna Rita , Pascuccio Massimiliano , Pierpaoli Sabina , Guidotti Lia , Capitani Silvano , Vitale Marco
Published: October 2019

The pattern of expression of several protein kinase C (PKC) isoforms (, βΙ, δ, ɛ, η, and ζ) during the course of hematopoietic development was investigated using primary human CD34+ hematopoietic cells and stable cell lines subcloned from the growth factor-dependent 32D murine hematopoietic cell line. Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC-ɛ in primary human CD34+ hematopoietic cells resulted in a twofold increase in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells showed a lower level of bulk PKC catalytic activity, lacked the expression of ɛ and η PKC isoforms, and showed a weak or absent upregulation of the remaining isoforms, except βΙ, upon readdition of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1 cell lines with mast cell, granulo-macrophagic, and granulocytic phenotype, respectively, expressed all the PKC isoforms investigated, but showed distinct responses to growth factor readdition. (4) 32D Epo 1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D Epo1, expressed the ɛ isoform only when cultured with IL-3. On the other hand, when cultured in Epo, 32D Epo1.1 cells lacked the expression of both ɛ and η PKC isoforms, similarly to 32D Epo1. (5) All 32D cell lines expressed the mRNA for PKC-ɛ, indicating that the downmodulation of the ɛ isoform occurred at a posttranscriptional level. In conclusion, the PKC isoform expression during hematopoiesis appears to be lineage-specific and, at least partially, related to the growth factor response.
Both Stat3-Activation and Stat3-Independent BCL2 Downregulation Are Important for Interleukin-6–Induced Apoptosis of 1A9-M Cells
Authors: Oritani Kenji , Tomiyama Yoshiaki , Kincade Paul W. , Aoyama Keisuke , Yokota Takafumi , Matsumura Itaru , Kanakura Yuzuru , Nakajima Koichi , Hirano Toshio , Matsuzawa Yuji
Published: October 2019

A unique subclone of a bone marrow-derived stromal cell line, BMS2.4, produces soluble factors that inhibit proliferation of several types of hematopoietic cell lines. An understanding of these molecules may be informative about negative regulatory circuits that can potentially limit blood cell formation. We used expression cloning to identify interleukin-6 (IL-6) as one factor that suppressed growth of a pre-B–cell variant line, 1A9-M. Moreover, IL-6 induced macrophage-differentiation and apoptosis of 1A9-M cells. During this process, IL-6 downregulated expression of BCL2 in 1A9-M cells and stimulated BCL-XL expression, but had no effect on p53, Bax, or Bak gene expression. Mechanisms for transduction of IL-6–induced signals were then evaluated in IL-6–stimulated 1A9-M cells. Whereas the signal transducer and activator of transcription 3 (Stat3) was phosphorylated and activated, there was no effect on either Stat1 or Stat5. The importance of BCL2 and Stat3 on IL-6–induced macrophage-differentiation and apoptosis was studied with 1A9-M cells expressing human BCL2 or a dominant-negative form of Stat3, respectively. IL-6–induced apoptosis, but not macrophage-differentiation, was blocked by continuously expressed BCL2. A dominant-negative form of Stat3 inhibited both macrophage-differentiation and apoptosis induced by IL-6. However, diminished Stat3 activity did not prevent IL-6–induced downregulation of the BCL2 gene. Therefore, activation of Stat3 is essential for IL-6–induced macrophage-differentiation and programmed cell death in this model. Whereas overexpression of BCL2 abrogates the apoptotic response, Stat3-independent signals appear to downregulate expression of the BCL2 gene.
Anionic Polysaccharides Inhibit Adhesion of Sickle Erythrocytes to the Vascular Endothelium and Result in Improved Hemodynamic Behavior
Authors: Barabino G.A. , Liu X.D. , Ewenstein B.M. , Kaul D.K.
Published: October 2019

The abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.
Nonimmunoglobulin Gene Hypermutation in Germinal Center B Cells
Authors: Peng Huai-Zheng , Du Ming-Qing , Koulis Athanasios , Aiello Antonella , Dogan Ahmet , Pan Lang-Xing , Isaacson Peter G.
Published: October 2019

Somatic hypermutation is the most critical mechanism underlying the diversification of Ig genes. Although mutation occurs specifically in B cells during the germinal center reaction, it remains a matter of debate whether the mutation machinery also targets non-Ig genes. We have studied mutations in the 5′ noncoding region of the Bcl6 gene in different subtypes of lymphomas. We found frequent hypermutation in follicular lymphoma (25 of 59 = 42%) (germinal center cell origin) and mucosa-associated lymphoid tissue (MALT) lymphoma (19 of 45 = 42%) (postgerminal center), but only occasionally in mantle cell lymphoma (1 of 21 = 4.8%) (pregerminal center). Most mutations were outside the motifs potentially important for transcription, suggesting they were not important in lymphomagenesis but may, like Ig mutation, represent an inherent feature of the lymphoma precursor cells. Therefore, we investigated their normal cell counterparts microdissected from a reactive tonsil. Bcl6 mutation was found in 13 of 24 (54%) clones from the germinal centre but only in 1 of 24 (4%) clones from the naive B cells of the mantle zone. The frequency, distribution, and nature of these mutations were similar to those resulting from the Ig hypermutation process. The results show unequivocal evidence of non-Ig gene hypermutation in germinal center B cells and provide fresh insights into the process of hypermutation and lymphomagenesis.
Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor
Authors: Arrighi Jean-François , Hauser Conrad , Chapuis Bernard , Zubler Rudolf H. , Kindler Vincent
Published: October 2019

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.
Interaction Between Terminal Complement Proteins C5b-7 and Anionic Phospholipids
Authors: Liu Clive , Marshall Patricia , Schreibman Ian , Vu Ann , Gai Weiming , Whitlow Michael
Published: October 2019

We have recently shown that C5b-6 binds to the erythrocyte membrane via an ionic interaction with sialic acid before the addition of C7 and subsequent membrane insertion. In this study we assessed the role of anionic lipids in the binding of the terminal complement proteins to the membrane and the efficiency of subsequent hemolysis. Human erythrocytes were modified by insertion of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl phosphatidylethanolamine (DPPE), or dipalmitoyl phosphatidic acid (DPPA). Lipid incorporation and the hemolytic assays were done in the presence of 100 μmol/L sodium orthovanadate to prevent enzymatic redistribution of lipid. We found that the neutral lipids, DPPC and DPPE, did not affect C5b-7 uptake or hemolysis by C5b-9. In contrast, the two acidic phospholipids, DPPS and DPPA, caused a dose-dependent increase in both lysis and C5b-7 uptake. We conclude that the presence of anionic lipids on the exterior face of the membrane increases C5b-7 uptake and subsequent hemolysis. It is known that sickle cell erythrocytes have increased exposure of phosphatidylserine on their external face and are abnormally sensitive to lysis by C5b-9. The data presented here provide a plausible mechanism for this increased sensitivity.