bluuebunny/crossref_metadata_2025 huggingface dataset:
Up-regulation of vascular endothelial growth factor receptor Flt-1 after endothelial denudation: role of transcription factor Egr-1
Authors: Vidal Felipe , Aragonés Julián , Alfranca Arántzazu , de Landázuri Manuel O.
Published: October 2019

Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported—fms-like–tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of theflt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions −24 to −16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.
Thrombopoietin potentiates collagen receptor signaling in platelets through a phosphatidylinositol 3-kinase–dependent pathway
Authors: Pasquet Jean-max , Gross Barbara S. , Gratacap Marie-Pierre , Quek Lynn , Pasquet Sophie , Payrastre Bernard , van Willigen Gijsbert , Mountford Joanne C. , Watson Steve P.
Published: October 2019

Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cγ2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca++[Ca++]i, aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.
Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells
Authors: Russo Vincenzo , Zhou Dan , Sartirana Claudia , Rovere Patrizia , Villa Antonello , Rossini Silvano , Traversari Catia , Bordignon Claudio
Published: October 2019

In an attempt to transduce monocyte-derived dendritic cells (DCs) by a retroviral vector coding for a cell surface marker, we were confronted by the observation of high transfer of the surface molecule in the absence of vector proviral DNA in the treated cells. Indeed, DCs acquired the surface marker by a mechanism independent of the vector machinery, requiring cell-to-cell contact and involving transfer of lipids and a variety of intact membrane proteins. Most important, this property of DCs also includes acquisition of foreign human leukocyte antigen (HLA) molecules. Consequently, DCs become immunological hybrids as they display their own and foreign HLA molecules. The newly acquired HLA is fully functional because it allows recognition by allo-specific T lymphocytes and the binding and presentation of antigen peptides.
Expression of CD94/NKG2A and killer immunoglobulin-like receptors in NK cells and a subset of extranodal cytotoxic T-cell lymphomas
Authors: Haedicke Wolfgang , Ho Faith C. S. , Chott Andreas , Moretta Lorenzo , Rüdiger Thomas , Ott German , Müller-Hermelink Hans Konrad
Published: October 2019

Thirty-two natural killer (NK) and cytotoxic T-cell lymphomas and 14 noncytotoxic nodal T-cell lymphoma controls were immunostained with the use of monoclonal antibodies reactive against NK-cell receptor (NKR) molecules (CD94, NKG2A, p58.2, p58.1, p140, p70, p50.3). All NK-cell lymphomas (4 nasal/oral and 1 intestinal) expressed at least 1 NKR, the CD94/NKG2A complex. Two were positive for 1 or more killer immunoglobulin-like receptors. Of 15 extranodal cytotoxic T-cell lymphomas, 3 expressed CD94, including 2 intestinal and 1 hepatosplenic γδ T-cell lymphomas. In contrast, none of the nodal lymphomas were positive. Detection of NKRs may provide a useful tool to confirm the diagnosis of NK-cell lymphomas and to delineate a subgroup of cytotoxic T-cell lymphomas. Expression of NKRs only in extranodal cytotoxic T-cell lymphomas might reflect differences in the homing capabilities of cytotoxic T cells expressing NKRs in normal individuals and might be influenced in part by localized chronic immune reactions.
Chronic graft versus host disease is associated with long-term risk for pneumococcal infections in recipients of bone marrow transplants
Authors: Kulkarni Samar , Powles Ray , Treleaven Jennie , Riley Unell , Singhal Seema , Horton Clive , Sirohi Bhawna , Bhagwati Niyati , Meller Simon , Saso Radovan , Mehta Jayesh
Published: October 2019

Incidences of and risk factors for Streptococcus pneumoniaesepsis (SPS) after hematopoietic stem cell transplantation were analyzed in 1329 patients treated at a single center between 1973 and 1997. SPS developed in 31 patients a median of 10 months after transplantation (range, 3 to 187 months). The infection was fatal in 7 patients. The probability of SPS developing at 5 and 10 years was 4% and 6%, respectively. Age, sex, diagnosis, and graft versus host disease (GVHD) prophylaxis did not influence the development of SPS. Allogeneic transplantation (10-year probability, 7% vs 3% for nonallogeneic transplants; P = .03) and chronic GVHD (10-year probability, 14% vs 4%; P = .002) were associated with significantly higher risk for SPS. All the episodes of SPS were seen in patients who had undergone allograft or total body irradiation (TBI) (31 of 1202 vs 0 of 127;P = .07). Eight patients were taking regular penicillin prophylaxis at the time of SPS, whereas 23 were not taking any prophylaxis. None of the 7 patients with fatal infections was taking prophylaxis for Pneumococcus. Pneumococcal bacteremia was associated with higher incidences of mortality (6 of 15 vs 1 of 16;P = .04). We conclude that there is a significant long-term risk for pneumococcal infection in patients who have undergone allograft transplantation, especially those with chronic GVHD. Patients who have undergone autograft transplantation after TBI-containing regimens also appear to be at increased risk. These patients should receive lifelong pneumococcus prophylaxis. Consistent with increasing resistance to penicillin, penicillin prophylaxis does not universally prevent SPS, though it may protect against fatal infections. Further studies are required to determine the optimum prophylactic strategy in patients at risk.
A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease
Authors: van Oosterhout Ypke V. J. M. , van Emst Liesbeth , Schattenberg Anton V. M. B. , Tax Wil J. M. , Ruiter Dirk J. , Spits Hergen , Nagengast Fokke M. , Masereeuw Roos , Evers Sabine , de Witte Theo , Preijers Frank W. M. B.
Published: October 2019

This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10−8 mol/L (about 1.8 μg/mL). Because most natural killer (NK) cells are CD7+, NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m2 IT combination, administered intravenously at 48-hour intervals. The T1/2 was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells.
Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation
Authors: Kirby Suzanne , Walton William , Smithies Oliver
Published: October 2019

In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM.
CD40-ligand stimulates myelopoiesis by regulating flt3-ligand and thrombopoietin production in bone marrow stromal cells
Authors: Solanilla Anne , Déchanet Julie , El Andaloussi Abdel , Dupouy Moryse , Godard François , Chabrol Jerome , Charbord Pierre , Reiffers Josy , Nurden Alan T. , Weksler Babette , Moreau Jean-François , Ripoche Jean
Published: October 2019

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell–dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis.
Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1
Authors: Sheng Nijing , Fairbanks Michael B. , Heinrikson Robert L. , Canziani Gabriela , Chaiken Irwin M. , Mosser David M. , Zhang Hong , Colman Robert W.
Published: October 2019

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1–specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a Kd = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (kon and koff, respectively) were determined, and they yielded a dissociation constant (Kd) of 3.2×10−9mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1–mediated cell–extracellular matrix and cell–cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury.
Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation
Authors: Ajzenberg Nadine , Ribba Anne-Sophie , Rastegar-Lari Ghassem , Meyer Dominique , Baruch Dominique
Published: October 2019

The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.
Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2b bone marrow cell allografts
Authors: Raziuddin Arati , Bennett Michael , Winkler-Pickett Robin , Ortaldo John R. , Longo Dan L. , Murphy William J.
Published: October 2019

Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, Dd, binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2b targets in vitro. We therefore examined the ability of these subsets to reject H2b bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A+ NK cells in BALB/c or B10.D2 mice (both H2d) had no effect on the rejection of H2b BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2k) mice to reject H2ballografts. Although depletion of either Ly-49A+ or Ly-49G2+ NK cells alone had no effect on the ability of B10.D2 mice to reject H2b BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F1 hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A+ NK cells appear to play a role in the rejection H2b bone marrow allografts, but, in most strains of mice studied, Ly-49G2+ NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated.
TEL-JAK2 transgenic mice develop T-cell leukemia
Authors: Carron Clémence , Cormier Françoise , Janin Anne , Lacronique Virginie , Giovannini Marco , Daniel Marie-Thérèse , Bernard Olivier , Ghysdael Jacques
Published: October 2019

We previously reported a fusion between TEL and JAK2in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor–independent proliferation to the interleukin-3–dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2–induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EμSR enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRβ locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells.
Longitudinal monitoring of immune reconstitution by CDR3 size spectratyping after T-cell–depleted allogeneic bone marrow transplant and the effect of donor lymphocyte infusions on T-cell repertoire
Authors: Verfuerth Stephanie , Peggs Karl , Vyas Paulomi , Barnett Lorna , O'Reilly Richard J. , Mackinnon Stephen
Published: October 2019

Delayed immune reconstitution after allogeneic bone marrow transplantation (BMT) with associated infection is a major cause of morbidity and mortality. We used third complementarity region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire reconstitution in 19 patients over a median time of 40 months after T-cell–depleted allogeneic BMT for chronic myeloid leukemia (CML). Furthermore, the effect of donor lymphocyte infusions (DLI) for the treatment of relapse in 18 of the 19 patients was analyzed. All BMT recipients had irregular spectratypes in the first 3- to -6 months after transplant. These evolved to more normal patterns by 12 months after transplant and continued to improve thereafter. In approximately a third of the patients, it took 2 to 3 years for all spectratypes to normalize, whereas in the other two thirds, some abnormal spectratypes persisted even after several years. In 9 patients, there was no immediate change in the CDR3 size profiles after DLI. In 3 patients, spectratypes improved slightly after DLI, whereas in 6 patients, spectratypes became more restricted and irregular. Overall, T-cell spectratypes in BMT patients were characterized by instability over time and in patients with graft-versus-host disease (GVHD), this was even more exaggerated. Several factors, such as pre-BMT conditioning, T-cell depletion of the donor marrow, loss of thymic function in adults, exposure to infectious agents, GVHD, and immunosuppressive treatment, are likely contributors to the delay in T-cell–repertoire reconstitution.
A novel syndrome of radiation-associated acute myeloid leukemia involving AML1 gene translocations
Authors: Hromas Robert , Shopnick Rinah , Jumean Hani George , Bowers Charles , Varella-Garcia Marileila , Richkind Kathleen
Published: October 2019

AML1 is a transcriptional activator that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. We recently identified 3 patients in whom pancytopenia developed almost 50 years after high-level radiation exposure from nuclear explosions during or after World War II. In all 3 patients, acute myeloid leukemia (AML) eventually developed that had similar characteristics and clinical courses. Cytogenetics from the 3 patients revealed a t(1;21)(p36;q22), a t(18;21)(q21;q22), and a t(19;21)(q13.4;q22). By fluorescent in situ hybridization (FISH), all 3 translocations disrupted the AML1 gene. Two of theseAML1 translocations, the t(18;21) and the t(19;21), have not been reported previously. It is possible that the AML1 gene is a target for radiation-induced AML.
Selective elimination of leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific for WT1
Authors: Gao Liquan , Bellantuono Ilaria , Elsässer Annika , Marley Stephen B. , Gordon Myrtle Y. , Goldman John M. , Stauss Hans J.
Published: October 2019

Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34+ progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201– restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34+ progenitor cells isolated from patients with chronic myeloid leukemia (CML), whereas colony formation by normal CD34+ progenitor cells is unaffected. Thus, the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo.
Splenectomy in myelofibrosis with myeloid metaplasia: a single-institution experience with 223 patients
Authors: Tefferi Ayalew , Mesa Ruben A. , Nagorney David M. , Schroeder Georgene , Silverstein Murray N.
Published: October 2019

In a 20-year period, 223 patients (median age, 64.8 years) with myelofibrosis with myeloid metaplasia (MMM) had therapeutic splenectomy at our institution. Primary indications for surgery were transfusion-dependent anemia (45.3%), symptomatic splenomegaly (39.0%), portal hypertension (10.8%), and severe thrombocytopenia (4.9%). Operative mortality and morbidity rates were 9% and 31%, respectively. The 203 survivors of surgery had a median postsplenectomy survival time (PSS) of 27 months (range, 0-155). Among preoperative variables, thrombocytopenia (platelet count less than 100 × 109/L) and nonhypercellular bone marrow were identified as independent risk factors for decreased PSS. Durable remissions in constitutional symptoms, transfusion-dependent anemia, portal hypertension, and severe thrombocytopenia were achieved in 67%, 23%, 50%, and 0% of the patients, respectively. Histologic or cytogenetic features of bone marrow obtained before splenectomy did not predict a response in cytopenias. After splenectomy, substantial enlargement of the liver and marked thrombocytosis occurred in 16.1% and 22.0% of the patients, respectively. The thrombocytosis was associated with an increased risk of perioperative thrombosis and decreased PSS. The rate of blast transformation (BT) was 16.3%, and the risk of BT was higher in the presence of increased spleen mass and preoperative thrombocytopenia. However, the PSS of patients with BT was not significantly different from that of patients without BT. We conclude that presplenectomy thrombocytopenia in MMM may be a surrogate for advanced disease and is associated with an increased risk of BT and inferior PSS. However, the development of BT after splenectomy may not affect overall survival and does not undermine the palliative role of the procedure for the other indications.
Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell–depleted stem cell transplantation
Authors: Broers Annoek E. C. , van der Holt Ron , van Esser Joost W. J. , Gratama Jan-Willem , Henzen-Logmans Sonja , Kuenen-Boumeester Vibeke , Löwenberg Bob , Cornelissen Jan J.
Published: October 2019

We evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 × 109/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P = .01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell–depleted allogeneic stem cell transplantation.
Phenotypic and functional characteristics of hematopoietic cell lineages in CD69-deficient mice
Authors: Lauzurica Pilar , Sancho David , Torres Miguel , Albella Beatriz , Marazuela Mónica , Merino Teresa , Bueren Juan A. , Martı́nez-A Carlos , Sánchez-Madrid Francisco
Published: October 2019

AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca++ flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220hiIgMneg bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T-dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors.
A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication
Authors: Kuwahara Kazuhiko , Yoshida Mikoto , Kondo Eisaku , Sakata Atsuko , Watanabe Yuko , Abe Eiji , Kouno Yoshihiko , Tomiyasu Shinjirou , Fujimura Satoru , Tokuhisa Takeshi , Kimura Hiroshi , Ezaki Taichi , Sakaguchi Nobuo
Published: October 2019

Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-μ and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.
Bidirectional induction of the cognate receptor-ligand α4/VCAM-1 pair defines a novel mechanism of tumor intravasation
Authors: Bogetto Laura , Gabriele Elena , Cariati Roberta , Dolcetti Riccardo , Spessotto Paola , Doglioni Claudio , Boiocchi Mauro , Perris Roberto , Colombatti Alfonso
Published: October 2019

Engagement of cell surface adhesion receptors with extracellular constituents and with cellular counter-receptors is crucial for the extravasation of blood-borne neoplastic cells and their seeding at distant sites; however, the early events of tumor dissemination—ie, the intravasation step(s)—have been largely neglected. A role for the 4β7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To clarify the mechanisms of early aggressivity, the behavior of highly and poorly leukemogenic cell lines were compared in vitro. Cocultivation of physically separated leukemic cells with resting endothelial cells resulted in the up-regulation of VCAM-1 expression. NQ22 cells expressed mRNA of different cytokines that up-regulate VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and they migrated more efficiently through an activated endothelial cell layer. With the use of neutralizing antibodies against interferon-γ, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor (TNF)-, it was determined that TNF- is one of the soluble factors released by NQ22 cells involved in the up-regulation of VCAM-1. The finding that vascular cells within the early local growth were strongly positive for VCAM-1 indicated that NQ22 cells could activate endothelial cells also in vivo. Finally, cocultivation of preleukemic 4−NQ22 cells with TNF--activated endothelial cells induced the expression of 4 integrins on the former cells. Reciprocal up-regulation and engagement of 4/VCAM-1 pairs determined the sequential transmigration and intravasation steps, and similar mechanisms might affect the aggressivity of human T lymphoblastic lymphomas.
Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease
Authors: Anderson Larry D. , Savary Cherylyn A. , Mullen Craig A.
Published: October 2019

Allogeneic bone marrow transplantation (BMT) induces 2 closely associated immune responses: graft-versus-tumor (GVT) activity and graft-versus-host disease (GVHD). We have previously shown that pretransplant immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine increases both GVT activity and lethal GVHD because of the priming of donor T cells against putative minor histocompatibility antigens (mHAgs) on the tumor vaccine cells. The work reported here tested the hypothesis that tumor cell vaccination after BMT would produce an increase in GVT activity without exacerbating GVHD. C3H.SW donor bone marrow and splenocytes were transplanted into major histocompatibility complex-matched, mHAg-mismatched C57BL/6 recipients. One month after BMT, recipients were immunized against either a C57BL/6 myeloid leukemia (C1498) or fibrosarcoma (205). Immunized recipients had a significant increase in survival and protection against tumor growth in both tumor models, and significant tumor protection was seen even in recipients with preexisting micrometastatic cancer before immunization. Alloreactivity appeared to contribute to the in vitro anti-tumor cytolytic activity, but in vivo immunity was tumor specific, and no exacerbation of GVHD was observed. Although the immunodominant mHAg B6dom1 was shown to be expressed by all B6 tumors tested and was largely responsible for the alloreactivity resulting from tumor immunization of donors, the in vitro alloreactivity of immune recipients was more restricted and was not mediated by recognition of B6dom1. In conclusion, post-transplant tumor immunization of allogeneic BMT recipients against either a leukemia or a solid tumor can increase GVT activity and survival without exacerbating GVHD.
Blockade of CD134 (OX40)-CD134L interaction ameliorates lethal acute graft-versus-host disease in a murine model of allogeneic bone marrow transplantation
Authors: Tsukada Nobuhiro , Akiba Hisaya , Kobata Tetsuji , Aizawa Yoshifusa , Yagita Hideo , Okumura Ko
Published: October 2019

Expression of CD134 (OX40) on activated CD4+ T cells has been observed in acute graft-versus-host disease (GVHD) after human and rat allogeneic bone marrow transplantation (BMT). We investigated the role of interaction between CD134 and CD134 ligand (CD134L) in a murine model of acute GVHD by using a newly established monoclonal antibody (mAb) against murine CD134L. Acute GVHD was induced by transfer of bone marrow cells and spleen cells into lethally irradiated recipients in a parent (C57BL/6) to first filial generation (C57BL/6 crossed with DBA/2) BMT. Administration of anti-CD134L mAb significantly reduced the lethality of acute GVHD and other manifestations of the disease, such as loss of body weight, hunched posture, diarrhea, and patchy alopecia. The survival rate 80 days after BMT in mice treated with the mAb was about 70%, whereas all mice treated with control antibodies died within 43 days. Histologic examinations revealed that inflammatory changes in target organs such as the liver, gut, and skin were also ameliorated in mice treated with the mAb compared with control mice. An in vitro assay of T-cell proliferation showed a marked hyporesponsiveness to host alloantigen in samples from mice treated with anti-CD134L mAb. In addition, low levels of interferon γ and transiently elevated levels of interleukin 4 and IgE in serum samples were found in mice treated with anti-CD134L mAb. These results suggest that CD134-CD134L interactions have an important role in the pathogenesis of acute GVHD.
Genetic control of hematopoietic stem cell frequency in mice is mostly cell autonomous
Authors: Müller-Sieburg Christa E. , Cho Rebecca H. , Sieburg Hans B. , Kupriyanov Sergey , Riblet Roy
Published: October 2019

Previously we reported that the size of the stem cell compartment (measured as LTC-IC) is 11-fold greater in DBA/2 than in C57BL/6 mice, and we identified genes that regulate the size of the stem cell pool. To determine whether stem cell intrinsic or extrinsic events account for these differences, we created chimeras by aggregating morulae from the strains C57BL/6 and DBA/2. In these chimeras stem cells of both genotypes are exposed to a common mixed environment. Thus, an equalization of stem cell frequencies is expected if stem cell extrinsic effects dominate. Conversely, the parental ratio of LTC-IC should be preserved if the regulation is stem cell autonomous. For each chimera, individual LTC-IC were genotyped on the clonal levels by analyzing their progeny. We found that most of the difference that regulates the size of the stem cell compartment was intrinsic.
Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells
Authors: Arpinati Mario , Green Cherie L. , Heimfeld Shelly , Heuser Jill E. , Anasetti Claudio
Published: October 2019

Peripheral blood stem cells (PBSC) obtained from granulocyte-colony stimulating factor (G-CSF)-mobilized donors are increasingly used for allogeneic transplantation. Despite a 10-fold higher dose of transplanted T cells, acute graft-versus-host disease (GVHD) does not develop in higher proportion in recipients of PBSC than in recipients of marrow. T cells from G-CSF-treated experimental animals preferentially produce IL-4 and IL-10, cytokines characteristic of Th2 responses, which are associated with diminished GVHD-inducing ability. We hypothesized that G-CSF-mobilized PBSC contain antigen-presenting cells, which prime T-lymphocytes to produce Th2 cytokines. Two distinct lineages of dendritic cells (DC) have been described in humans, DC1 and DC2, according to their ability to induce naive T-cell differentiation to Th1 and Th2 effector cells, respectively. We have used multicolor microfluorometry to enumerate DC1 and DC2 in the peripheral blood of normal donors. G-CSF treatment with 10 to 16 μg/kg per day for 5 days increased peripheral blood DC2 counts from a median of 4.9 × 106/L to 24.8 × 106/L (P = .0009), whereas DC1 counts did not change. Purified DC1, from either untreated or G-CSF treated donors, induced the proliferation of allogeneic naive T cells, but fresh DC2 were poor stimulators. Tumor necrosis factor- (TNF-)-activated DC1 induced allogeneic naive T cells to produce IFN-γ, which is typical of Th1 responses, whereas TNF--activated DC2 induced allogeneic naive T cells to produce IL-4 and IL-10, which are typical of Th2 responses. PBSC transplants contained higher doses of DC2 than marrow transplants (median, 2.4 × 106/kg versus 0.5 × 106/kg) (P = .006), whereas the dose of DC1 was comparable. Thus, it is conceivable that transplantation of G-CSF-stimulated PBSC does not result in overwhelming acute GVHD because the graft contains predominantly Th2-inducing DC. Adoptive transfer of purified DC2 may be exploited to induce immune deviation after transplantation of hematopoietic stem cells or organ allografts.
Development of lentiviral vectors for gene therapy for human diseases
Authors: Buchschacher Gary L. , Wong-Staal Flossie
Published: October 2019

Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy.
Granulocyte-macrophage colony-stimulating factor stimulation results in phosphorylation of cAMP response element-binding protein through activation of pp90RSK
Authors: Kwon Evelyn M. , Raines Maribeth A. , Blenis John , Sakamoto Kathleen M.
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates several kinases and transcription factors through interaction with a heterodimeric receptor complex. We previously demonstrated that phosphorylation of the cyclic adenosine monophosphate (cAMP) response element-binding protein, CREB, occurs through a protein kinase A-independent pathway and is required for GM-CSF–induced transcriptional activation of the immediate early gene, early growth response-1 (egr-1). Recent reports indicate that receptor tyrosine kinases can induce CREB phosphorylation through activation of pp90RSK. We performed immune complex kinase assays in the human myeloid leukemic cell line, TF-1, which revealed that GM-CSF induced pp90RSK activation and phosphorylation of CREB within 5 minutes of stimulation. Transfection with the kinase-defective pp90RSK expression plasmid demonstrated a statistically significant decrease in transcriptional activation of a −116 CAT/egr-1 promoter construct in response to GM-CSF. Furthermore, activation of pp90RSK, CREB and egr-1in GM-CSF–treated cells was inhibited by the presence of the inhibitor, PD98059. In this study, we report that GM-CSF induces CREB phosphorylation and egr-1 transcription by activating pp90RSK through an MEK-dependent signaling pathway.
Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation
Authors: Sobocka Malgorzata B. , Sobocki Tomasz , Banerjee Probal , Weiss Cipora , Rushbrook Julie I. , Norin Allen J. , Hartwig John , Salifu Moro O. , Markell Mariana S. , Babinska Anna , Ehrlich Yigal H. , Kornecki Elizabeth
Published: October 2019

This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcγRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcγRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation.
Functional comparison of thymic B cells and dendritic cells in vivo
Authors: Kleindienst Petra , Chretien Isabelle , Winkler Thomas , Brocker Thomas
Published: October 2019

In this report we present a transgenic mouse model in which we targeted gene expression specifically to B-lymphocytes. Using the human CD19 promoter, we expressed major histocompatibility complex class II I-E molecules specifically on B cells of all tissues, but not on other cell types. If only B cells expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. A comparison of the frequencies of I-E reactive Vβ5+ and Vβ11+ T cells shows that I-E expression on thymic B cells is sufficient to negatively select I-E reactive CD4+ T cells partially, but not CD8+ T cells. Thus partial negative but no positive selection events can be induced by B-lymphocytes in vivo.
Abundance of cyclin B1 regulates γ-radiation–induced apoptosis
Authors: Porter Lisa A. , Singh Gurmit , Lee Jonathan M.
Published: October 2019

γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.