bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells
Authors: Rabiet Marie-Josèphe , Tardif Marianne , Braun Laurence , Boulay François
Published: October 2002

A tetracycline-controlled expression system was adapted to the human promyelocytic HL-60 cell line by placement of the transactivator (tTA-off) sequence under the control of the human EF-1α promoter region. Constitutively active and dominant-inhibitory forms of Cdc42 (Cdc42V12 and Cdc42N17, respectively) were conditionally expressed in this system. The expression of Cdc42V12 had no marked effect on chemoattractant-mediated superoxide production, corroborating previous results indicating that the guanosine 5′-triphosphate (GTP)–bound form of Cdc42 is ineffective in directly activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in a cell-free system. However, the N17 mutant potently inhibited chemoattractant-induced superoxide production. The expression of Cdc42N17 interfered with the GTP-loading of Rac and Ras and with the activation of the MAP-kinase pathway. A drastic reduction of chemoattractant-induced inositol-1,4,5-trisphosphate formation and calcium mobilization was observed, corroborating previous in vitro study results identifying PLCβ2 as a Rac/Cdc42 effector. Cdc42N17 was also found to inhibit the translocation of Ras-GRF2, a guanine nucleotide exchange factor for Ras and Rac but not for Cdc42. Thus, the dominant-inhibitory mutant Cdc42N17 was found to interfere at multiple levels in the signaling pathways. The pleiotropic inhibitory effects of Cdc42N17 illustrate the potential pitfalls of using dominant-inhibitory proteins to study the function of Ras-family GTPases. In this regard, a number of conclusions drawn from the use of dominant-inhibitory mutants in myeloid cells might have to be reconsidered.
Intracellular interferon-γ in circulating and marrow T cells detected by flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia
Authors: Sloand Elaine , Kim Sonnie , Maciejewski Jaroslaw P. , Tisdale John , Follmann Dean , Young Neal S.
Published: October 2002

Immunosuppressive therapy leads to meaningful hematologic improvement in most patients with aplastic anemia (AA). Failure to respond and a later relapse could be due to deficient numbers of hematopoietic stem cells, inadequate treatment of the immune process, or a nonimmunologic etiology. Interferon-γ (IFN-γ) has been implicated in the pathophysiology of hematopoietic failure in AA. On the basis of previous findings showing overexpression of IFN-γ in bone marrow (BM) and peripheral blood (PB) in this disease, we hypothesized that quantitation of IFN-γ might be applied to predict and monitor responses to immunosuppressive therapy. We measured expression of IFN-γ in lymphocytes obtained from 123 AA patients, using intracellular 2-color fluorescent staining and flow cytometry. Of 70 patients with severe AA, 36 (51%) demonstrated increased IFN-γ in circulating T cells. IFN-γ was detected in only 4 of 53 patients who had recovered from AA. IFN-γ was not found in PB lymphocytes of patients with other hematologic diseases and heavy transfusion burdens or in healthy volunteers. Among 62 AA patients who were assessed before first treatment with immunosuppressive drugs, 27 of 28 (96%) with circulating IFN-γ–containing T cells subsequently responded to therapy; in contrast, only 11 of 34 (32%) patients whose PB lacked IFN-γ lymphocytes improved to transfusion independence. IFN-γ–containing lymphocytes declined following treatment in all cases. Of 17 patients assessed during relapse, IFN-γ was present in T cells prior to the blood count decline in 13, and 12 responded to reinstitution of immunosuppressive drugs. Of 30 BMs tested prior to first treatment, 20, all in responding patients, were positive for IFN-γ, whereas the negative tests were obtained in 10 nonresponding patients. IFN-γ is increased in the PB lymphocytes of many patients with AA, and these cells decline with therapy. The presence of intracellular IFN-γ may predict response to immunosuppressive treatment and also the onset of relapse.
Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2)
Authors: Dekker Rob J. , van Soest Simone , Fontijn Ruud D. , Salamanca Sonia , de Groot Philip G. , VanBavel Ed , Pannekoek Hans , Horrevoets Anton J. G.
Published: October 2002

The endothelium expresses a large repertoire of genes under apparent transcriptional control of biomechanical forces, many of which are neither cell-type nor flow specific. We set out to identify genes that are uniquely flow responsive in human vascular endothelial cells. Transcriptional profiling using commercial DNA microarrays identified 12 of 18 000 genes that were modulated at least 5-fold after 24 hours of steady laminar flow (25 dyne/cm2). After a 7-day exposure to unidirectional pulsatile flow (19 ± 12 dyne/cm2), only 3 of 12 remained elevated at least 5-fold. A custom microarray of ∼300 vascular cell–related gene fragments was constructed, and expression analysis revealed that many flow-induced genes are also induced by at least one of the following agents: tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), transforming growth factor-β, vascular endothelial growth factor, or thrombin, indicating a more general role in adaptive or stress responses. Most flow-induced genes were also induced by TNF-α but not IL-1β, suggesting the involvement of reactive oxygen species. A limited panel of genes that are unique for flow-exposed cultures was identified, including lung Krüppel-like factor (LKLF/KLF2) and cytochrome P450 1B1 (CYP1B1). In marked contrast, both these genes were substantially repressed by TNF-α. LKLF but not CYP1B1 mRNA was detected exclusively in the vascular endothelium of healthy human aorta by in situ hybridization and appeared to be flow regulated. To date LKLF is the first endothelial transcription factor that is uniquely induced by flow and might therefore be at the molecular basis of the physiological healthy, flow-exposed state of the endothelial cell.
Molecular genetic analysis for the B3allele
Authors: Yu Lung-Chih , Twu Yuh-Ching , Chou Ming-Lun , Chang Ching-Yi , Wu Chia-Ying , Lin Marie
Published: October 2002

Molecular genetic analysis of 14 samples from unrelated individuals with the B3 phenotype is reported here. Two different molecular changes in the blood group B gene were observed. One case was demonstrated to possess a 247G → T mutation, which predicts an Asp83Tyr alteration. The B genes of the other 13 cases were shown to have a G → A mutation at the +5 nucleotide of intron 3 (intervening sequence 3 [IVS3] + 5G → A). Reverse transcription polymerase chain reaction analysis showed that the complete exon 1–exon 7 B transcript was absent, and transcripts that skipped exon 3 were instead present in the RNA sample from the B3 individual with the IVS3 + 5G → A mutation. The result shows that the IVS3 + 5G → A mutation destroys the conserved sequence of the splice donor site and leads to the skipping of exon 3 during messenger RNA processing. TheB3 transcript without exon 3 predicts a B-transferase product that lacks 19 amino acids in the N-terminal segment.
Sequestration of Plasmodium falciparum–infected erythrocytes to chondroitin sulfate A, a receptor for maternal malaria: monoclonal antibodies against the native parasite ligand reveal pan-reactive epitopes in placental isolates
Authors: Lekana Douki Jean-Bernard , Traore Boubacar , Costa Fabio T. M. , Fusaı̈ Thierry , Pouvelle Bruno , Sterkers Yvon , Scherf Artur , Gysin Jürg
Published: October 2002

Plasmodium falciparum parasites express variant adhesion molecules on the surface of infected erythrocytes (IEs), which act as targets for natural protection. Recently it was shown that IE sequestration in the placenta is mediated by binding to chondroitin sulfate A via the duffy binding-like (DBL)–γ3 domain ofP falciparum erythrocyte membrane protein 1 (PfEMP1CSA). Conventional immunization procedures rarely result in the successful production of monoclonal antibodies (mAbs) against such conformational vaccine candidates. Here, we show that this difficulty can be overcome by rendering Balb/c mice B cells tolerant to the surface of human erythrocytes or Chinese hamster ovary (CHO) cells before injecting P falciparum IEs or transfected CHO cells expressing the chondroitin sulfate A (CSA)–binding domain (DBL-γ3) of the FCR3varCSA gene. We fused spleen cells with P3U1 cells and obtained between 20% and 60% mAbs that specifically label the surface of mature infected erythrocytes of the CSA phenotype (mIECSA) but not of other adhesive phenotypes. Surprisingly, 70.8% of the 43 mAbs analyzed in this work were IgM. All mAbs immunoprecipitated PfEMP1CSA from extracts of125I surface-labeled IECSA. Several mAbs bound efficiently to the surface of CSA-binding parasites from different geographic areas and to placental isolates from West Africa. The cross-reactive mAbs are directed against the DBL-γ3CSA, demonstrating that this domain, which mediates CSA binding, is able to induce a pan-reactive immune response. This work is an important step toward the development of a DBL-γ3–based vaccine that could protect pregnant women from pathogenesis.
Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPARα
Authors: Sethi Sanjeev , Ziouzenkova Ouliana , Ni Heyu , Wagner Denisa D. , Plutzky Jorge , Mayadas Tanya N.
Published: October 2002

Omega-3 fatty acids, which are abundant in fish oil, improve the prognosis of several chronic inflammatory diseases although the mechanism for such effects remains unclear. These fatty acids, such as eicosapentaenoic acid (EPA), are highly polyunsaturated and readily undergo oxidation. We show that oxidized, but not native unoxidized, EPA significantly inhibited human neutrophil and monocyte adhesion to endothelial cells in vitro by inhibiting endothelial adhesion receptor expression. In transcriptional coactivation assays, oxidized EPA potently activated the peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor family. In vivo, oxidized, but not native, EPA markedly reduced leukocyte rolling and adhesion to venular endothelium of lipopolysaccharide (LPS)–treated mice. This occurred via a PPARα-dependent mechanism because oxidized EPA had no such effect in LPS-treated PPARα-deficient mice. Therefore, the beneficial effects of omega-3 fatty acids may be explained by a PPARα-mediated anti-inflammatory effect of oxidized EPA.
Crucial role of timing of donor lymphocyte infusion in generating dissociated graft-versus-host and graft-versus-leukemia responses in mice receiving allogeneic bone marrow transplants
Authors: Billiau An D. , Fevery Sabine , Rutgeerts Omer , Landuyt Willy , Waer Mark
Published: October 2002

A murine model of minor histocompatibility antigen-mismatched bone marrow transplantation (BMT) was used to study the role of timing of donor lymphocyte infusion (DLI) in eliciting graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactivity. We gave DLI at weeks 3 and 12 after BMT and related its ability to induce a GVL effect with (1) evolution of T cell chimeric status and (2) the extent to which DLI could elicit lymphohematopoietic GVH (LHGVH) reactivity. All mice remained free of GVH disease, but only week 3 DLI chimeras exhibited a significant GVL response when challenged with host-type leukemia cells. In these week 3 DLI chimeras, host-reactive T cells were found to proliferate in vivo (5- [and-6]-carboxyfluorescein diacetate, succinimidyl esther [CFSE]-labeled DLI inocula, TCR-Vβ6+ T-cell frequency) and T-cell chimerism rapidly converted from mixed into complete donor type, indicating the occurrence of LHGVH reactivity. In week 12 chimeras, DLI elicited none of the activities noted at week 3. Yet, in both instances, splenocytes, recovered following DLI, generated an equally strong antihost proliferative response in a mixed lymphocyte reaction, thereby arguing against a decisive role of regulatory cells. The lack of in vivo LHGVH reactivity after week 12 DLI was associated with a substantially increased level of pre-existing host-type T-cell chimerism. We conclude that elicitation of a GVL effect may require LHGVH reactivity and that the reason why timing of DLI was critical for obtaining LHGVH reactivity and the desired GVL effect may lie in the evolution of chimeric status. A possible direct involvement of residual host-type antigen-presenting cells in eliciting LHGVH reactivity after DLI should be studied using models that allow chimerism analysis in non–T-cell lineages.
Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1low mice)
Authors: Vannucchi Alessandro Maria , Bianchi Lucia , Cellai Cristina , Paoletti Francesco , Rana Rosa Alba , Lorenzini Rodolfo , Migliaccio Giovanni , Migliaccio Anna Rita
Published: October 2002

The phenotype induced by the GATA-1low (neoδHS) mutation is here further characterized by analyzing the hemopoietic system during the aging (up to 20 months) of a GATA-1lowcolony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct = 45.9 ± 4.0) until 12 months but became anemic from 15 months on (Hct = 30.9 ± 3.9;P < .05). Anemia was associated with several markers of myelofibrosis such as the presence of tear-drop poikilocytes and progenitor cells in the blood, collagen fibers in the marrow and in the spleen, and hemopoietic foci in the liver. Semiquantitative reverse transcription–polymerase chain reaction showed that growth factor genes implicated in the development of myelofibrosis (such as osteocalcin, transforming growth factor-β1, platelet-derived growth factor, and vascular endothelial growth factor) were all expressed in the marrow from the mutants at higher levels than in corresponding normal tissues. The GATA-1low mutants experienced a slow progression of the disease because the final exitus was not observed until at least 15 months with a probability of survival more favorable than that of W/Wv mice concurrently kept in the animal facility (P < .001, by Kaplan-Meier analysis). In conclusion, impaired GATA-1 expression may contribute to the development of myelofibrosis, and the GATA-1low mutants may represent a suitable animal model for the human disease that may shed light on its pathogenesis.
Antigen-bearing immature dendritic cells induce peptide-specific CD8+ regulatory T cells in vivo in humans
Authors: Dhodapkar Madhav V. , Steinman Ralph M.
Published: October 2002

Regulatory T cells (TRs) can suppress the function of other effector T cells in the setting of autoimmunity, transplantation, and resistance to tumors. The mechanism for the induction of TRs has not been defined. We previously reported that an injection of immature dendritic cells (DCs) pulsed with influenza matrix peptide (MP) led 7 days later to antigen-specific silencing of effector T-cell function in the blood of 2 healthy human subjects. Here, we found that interferon-γ–producing effectors return by 6 months. Importantly, in mixing experiments, CD8+ T cells from the sample obtained 7 days after injection could suppress MP-specific effectors obtained before injection and those in recovery samples. This suppression or regulation was specific for the immunizing peptide (MP) and cell-dose dependent, and it required contact between the 2 samples. These data show the capacity of immature DCs to induce antigen-specific regulatory CD8+ T cells in humans.
Fludarabine induces apoptosis, activation, and allogenicity in human endothelial and epithelial cells: protective effect of defibrotide
Authors: Eissner Günther , Multhoff Gabriele , Gerbitz Armin , Kirchner Silvia , Bauer Sonja , Haffner Silvia , Sondermann Daniela , Andreesen Reinhard , Holler Ernst
Published: October 2002

Fludarabine is a nonmyeloablative immunosuppressant increasingly used as a component of alternative conditioning regimens before allogeneic bone marrow transplantation. It is expected to reduce conditioning-related toxicity and proinflammatory activation of the host tissues. However, in our in vitro study, we provide evidence that 2-fluoroadenine 9-β-d-arabinofuranoside (F-Ara) as the active metabolized form of fludarabine damages human microvascular endothelial cells (HMECs) and dermal and alveolar epithelial cell lines after 48 hours of culture when it is used in pharmacologically relevant concentrations (range, 10 μg/mL-1 μg/mL). In addition, flow cytometric analyses revealed a significant up-regulation of intercellular adhesion molecule 1 and major histocompatibility complex (MHC) class I molecules by F-Ara, suggesting a proinflammatory activation of HMECs. Cytotoxicity assays demonstrated that target HMECs pretreated with F-Ara (10 μg/mL) showed increased lysis by allogeneic MHC class I-restricted cytotoxic T lymphocytes from healthy human donors. We conclude that, beside its immunosuppressive activities, F-Ara can be harmful for target tissues of transplantation-related complications and can even stimulate allogeneic immune responses. We identified the pharmaceutical compound defibrotide as protective against F-Ara– induced apoptosis and alloactivation, importantly, without affecting the antileukemic effect of F-Ara. This observation argues for a potential clinical usage of defibrotide in pretransplantation conditioning.
Therapy for chronic graft-versus-host disease: a randomized trial comparing cyclosporine plus prednisone versus prednisone alone
Authors: Koc Sibel , Leisenring Wendy , Flowers Mary E. D. , Anasetti Claudio , Deeg H. Joachim , Nash Richard A. , Sanders Jean E. , Witherspoon Robert P. , Storb Rainer , Appelbaum Frederick R. , Martin Paul J.
Published: October 2002

Results of previous studies have suggested that transplantation-related mortality among patients with chronic graft-versus-host disease (GVHD) may be reduced by combined treatment with cyclosporine (CSP) and prednisone rather than by prednisone alone. In a randomized trial, we assessed the efficacy of cyclosporine plus prednisone versus prednisone alone as initial therapy for chronic GHVD among patients whose platelet counts were higher than 100 000/μL. Prednisone was administered initially at a dose of 1.0 mg/kg per day orally, followed by a prolonged taper, and cyclosporine was administered at 6 mg/kg orally twice daily every other day. The cumulative incidence of transplantation-related mortality at 5 years from enrollment was 17% (95% CI, 0.11-0.23) in the CSP plus prednisone arm and 13% (95% CI, 0.08-0.19) in the prednisone arm. The hazards of transplantation-related mortality, overall mortality, recurrent malignancy, secondary therapy, and discontinuation of all immunosuppressive therapy were not significantly different between the 2 arms, but survival without recurrent malignancy was lower in the 2-drug arm (P = .03). Avascular necrosis developed in 18 (13%) of the 142 patients in the CSP plus prednisone arm and in 32 (22%) of the 145 patients in the prednisone arm (P = .04). Treatment with CSP plus prednisone may reduce the risk for steroid-related toxicity, but results of the current study do not substantiate the hypothesis that the administration of CSP reduces transplantation-related mortality among patients with chronic GVHD.
Acute myeloid leukemia and myelodysplastic syndrome in children treated for cancer: comparison with primary presentation
Authors: Barnard Dorothy R. , Lange Beverley , Alonzo Todd A. , Buckley Jonathan , Kobrinsky J. Nathan , Gold Stuart , Neudorf Steven , Sanders Jean , Burden Laura , Woods William G.
Published: October 2002

There has not been a reported series of children with therapy-induced myelodysplastic syndrome/acute myeloid leukemia (tMDS/tAML) who were treated systematically. This paper describes 24 children with tMDS/tAML who were assigned randomly to standard- or intensive-timing induction on protocol CCG 2891. Presenting features and outcomes of those children were compared with those of 960 patients with de novo MDS (62 patients) or AML (898 patients). Children with tMDS/tAML were older at presentation (P = .015), had lower white blood cell counts (P = .01), and were more likely to have MDS (21% vs 7%) (P = .02) and trisomy 8 (P = .06). Fewer had hepatomegaly (P = .02), splenomegaly (P = .03), hepatosplenomegaly (P = .02), or classic AML translocations [t(8;21), t(15;17), 16q22; P = .02]. They had a poorer induction rate (50% vs 72%,P = .016), overall survival (26% vs 47% at 3 years,P = .007), and event-free survival (21% vs 39% at 3 years, P =.023). Disease-free survival after achieving remission was similar (45% vs 53%, P = .868). Children with tMDS/tAML who received intensive-timing induction had better outcomes than those who received standard-timing induction (overall survival 32% vs 0%, P = .54). In this study, the latency period to development of tMDS/tAML was the same for presumed alkylator-induced as for topoisomerase-induced myeloid leukemia. The findings of this study confirm that most children with tMDS/tAML have disease resistant to current therapies. Standard-timing induction appears less effective for this population.
Identification of CD8α+CD11c− lineage phenotype-negative cells in the spleen as committed precursor of CD8α+ dendritic cells
Authors: Wang Yong , Zhang Yanyun , Yoneyama Hiroyuki , Onai Nobuyuki , Sato Taku , Matsushima Kouji
Published: October 2002

CD8α+ dendritic cells (DCs) represent a functionally distinct DC subset in vivo, which plays a critical role in initiating various cellular immune responses. However, the committed precursor of CD8α+ DCs remains to be identified. We reported here that murine splenic CD8α+CD11c− lineage phenotype (Lin)− cells could differentiate into CD8α+DCs in vivo after intravenous transplantation. Immunohistochemistry staining showed that donor-derived DCs mainly located in T-cell areas of the spleen. Functionally, these CD8α+CD11c−Lin− cell–derived DCs were capable of stimulating allogenic T-cell response, as well as secreting bioactive interleukin 12 p70 and interferon γ. Freshly isolated CD8α+CD11c−Lin− cells expressed CC chemokine receptor (CCR)2, CCR5, and CCR7 messenger RNA, whereas CD8α+ DCs derived from CD8α+CD11c−Lin− cells further obtained the expression of CCR6 and macrophage-derived chemokine. Flow cytometry analysis showed that CD8α+CD11c−Lin− cells were identified in bone marrow and lymph nodes. Moreover, transplanted splenic CD8α+CD11c−Lin− cells could also home to thymus and lymph nodes and were capable of developing into CD8α+ DCs in these locations. However, CD8α+CD11c−Lin−cells failed to differentiate into CD8α− DCs, T cells, natural killer cells, or other myeloid lineage cells in irradiated chimeras. Taken together, all these findings suggest that CD8α+CD11c−Lin− cells are a committed precursor of CD8α+ DCs.
Strategy for the treatment of acute myelogenous leukemia based on folate receptor β–targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid
Authors: Pan Xing Q. , Zheng Xuan , Shi Guangfeng , Wang Huaqing , Ratnam Manohar , Lee Robert J.
Published: October 2002

Up-regulation of folate receptor (FR) type-β in acute myelogenous leukemia (AML) by all-trans retinoic acid (ATRA) and its restricted normal tissue distribution makes it a potential target for therapeutic intervention. The FR-β in peripheral blood granulocytes was unable to bind folate and appeared to have a variant GPI membrane anchor, evident from its insensitivity to phosphatidylinositol-specific phospholipase C but not nitrous acid. Granulocyte FR-β lacked mutations, and neither deglycosylation nor detergent solubilization restored folate binding. The posttranslational modification causing its nonfunctionality was evidently absent in FR-β from AML cells from patient marrow, which bound folate. From flow cytometric analysis of 78 AML bone marrow specimens of different subtypes, 68% expressed FR-β, most of which were also CD34+. In model cell lines that are FR (−) (KG-1a, L1210, and Chinese hamster ovary [CHO]) or FR (+) (KG-1, L1210 JF, and recombinant CHO-FR–β), selective FR-mediated binding and cytotoxicity was obtained using folate-coated liposomes encapsulating fluorescent calcein (f-L-calcein) and doxorubicin (f-L-DOX), respectively, which could be blocked by 1 mM free folic acid. In the FR-β–expressing KG-1 human AML cells, treatment with ATRA further increased this specificity. In mouse ascites leukemia models generated using L1210JF or KG-1 cells, increased median survival times were obtained with f-L-DOX treatment compared to nontargeted L-DOX. In the KG-1 model, ATRA treatment increased the cure rate with f-L-DOX from 10% to 60%. The above combined data from our 2 laboratories further support the feasibility and potential usefulness of selective ATRA-facilitated liposomal drug delivery in FR-β (+) AMLs.
ATM mutations are rare in familial chronic lymphocytic leukemia
Authors: Yuille Martin R. , Condie Alison , Hudson Chantelle D. , Bradshaw Paul S. , Stone Elaine M. , Matutes Estella , Catovsky Daniel , Houlston Richard S.
Published: October 2002

It is now recognized that a subset of B-cell chronic lymphocytic leukemia (CLL) is familial. The genetic basis of familial CLL is poorly understood, but recently germ line mutations in the Ataxia Telangiectasia (ATM) gene have been proposed to confer susceptibility to CLL. The evidence for this notion is, however, not unequivocal. To examine this proposition further we have screened the ATM gene for mutations in CLLs from 61 individuals in 29 families. Truncating ATM mutations, including a knownATM mutation, were detected in 2 affected individuals, but the mutations did not cosegregate with CLL in the families. In addition, 3 novel ATM missense mutations were detected. Common ATM missense mutations were not overrepresented. The data support previous observations that ATM mutation is associated with B-CLL. However, ATM mutations do not account for familial clustering of the disease.
Monoclonal B lymphocytes with the characteristics of “indolent” chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts
Authors: Rawstron Andy C. , Green Michael J. , Kuzmicki Anita , Kennedy Ben , Fenton James A. L. , Evans Paul A. S. , O'Connor Sheila J. M. , Richards Stephen J. , Morgan Gareth J. , Jack Andrew S. , Hillmen Peter
Published: October 2002

Molecular and cellular markers associated with malignant disease are frequently identified in healthy individuals. The relationship between these markers and clinical disease is not clear, except where a neoplastic cell population can be identified as in myeloma/monoclonal gammopathies of undetermined significance (MGUS). We have used the distinctive phenotype of chronic lymphocytic leukemia (CLL) cells to determine whether low levels of these cells can be identified in individuals with normal complete blood counts. CLL cells were identified by 4-color flow cytometric analysis of CD19/CD5/CD79b/CD20 expression in 910 outpatients over 40 years old. These outpatients were age- and sex-matched to the general population with normal hematologic parameters and no evident history of malignant disease. CLL phenotype cells were detectable in 3.5% of individuals at low level (median, 0.013; range, 0.002- 1.458 × 109 cells/L), and represented a minority of B lymphocytes (median, 11%; range, 3%-95%). Monoclonality was demonstrated by immunoglobulin light-chain restriction in all cases with CLL phenotype cells present and confirmed in a subset of cases by consensus-primer IgH-polymerase chain reaction. As in clinical disease, CLL phenotype cells were detected with a higher frequency in men (male-to-female ratio, 1.9:1) and elderly individuals (2.1% of 40- to 59-year-olds versus 5.0% of 60- to 89-year-olds, P = .01). The neoplastic cells were identical to good-prognosis CLL, being CD5+23+20wk79bwk11a−22wksIgwkCD38−, and where assessed had a high degree (4.8%-6.6%) of IgH somatic hypermutation. The monoclonal CLL phenotype cells present in otherwise healthy individuals may represent a very early stage of indolent CLL and should be useful in elucidating the mechanisms of leukemogenesis.
An animal model for human cellular immunotherapy: specific eradication of human acute lymphoblastic leukemia by cytotoxic T lymphocytes in NOD/scid mice
Authors: Nijmeijer Bart A. , Willemze Roel , Falkenburg J. H. Frederik
Published: October 2002

Adoptive immunotherapy using in vitro–generated donor-derived cytotoxic T lymphocytes (CTLs) can be effective in the treatment of relapsed leukemia after allogeneic transplantation. To determine effector cell characteristics that result in optimal in vivo antileukemic efficacy, we developed an animal model for human CTL therapy. Nonobese diabetic/severe combined immunodeficiency (NOD/scid) mice were inoculated with either of 2 primary human acute lymphoblastic leukemia (ALL), denoted as SK and OF. Anti-SK and anti-OF CTLs were generated in vitro by repeated stimulation of donor peripheral blood mononuclear cells with either SK or OF cells. Both CTL lines displayed HLA-restricted reactivity against the original targets and non-major histocompatibility class (MHC)–restricted cross-reactivity in vitro. The CTLs were administered intravenously weekly for 3 consecutive weeks to mice engrafted with either SK or OF leukemia. In 3 of 8 SK-engrafted and anti-SK–treated mice, complete remissions were achieved in blood, spleen, and bone marrow. In the remaining 5 animals partial remissions were observed. In 4 of 4 OF-engrafted anti-OF–treated mice partial remissions were observed. The antileukemic effect of specific CTLs was exerted immediately after administration and correlated with the degree of HLA disparity of the donor-patient combination. In cross-combination–treated animals, no effect on leukemic progression was observed indicating that in vivo antileukemic reactivity is mediated by MHC-restricted effector cells. The CTLs, however, displayed an impaired in vivo proliferative capacity. Ex vivo analysis showed decreased reactivity as compared to the moment of infusion. We therefore conclude that the model can be used to explore the requirements for optimal in vivo efficacy of in vitro– generated CTLs.
Inhibition of the von Willebrand (VWF)–collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons
Authors: Wu Dongmei , Vanhoorelbeke Karen , Cauwenberghs Nancy , Meiring Muriel , Depraetere Hilde , Kotze Harry F. , Deckmyn Hans
Published: October 2002

The interaction between collagen, von Willebrand factor (VWF), and glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a new kind of antithrombotic strategy and determine for the first time experimental in vivo data concerning the importance of the collagen-VWF interaction. We used a modified Folts model to study the antithrombotic efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured in the femoral artery. Administering a dose of 100, 300, and 600 μg/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs, respectively. When 100 μg/kg 82D6A3 was infused into the baboons, 80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex vivo inhibition of VWF binding to collagen, with no prolongation of the bleeding time. The bleeding time was also not significantly prolonged when the CFRs were abolished at doses of 300 μg/kg and 600 μg/kg. At these doses 100% of VWF was occupied by the antibody and 100% ex vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a high affinity for VWF; after 48 hours still 68% VWF (300μg/kg) was occupied with a pharmacologic effect up to 5 hours after administration (80%-100% occupancy). In conclusion, these results clearly indicate that the VWF-collagen interaction is important in vivo in thrombosis under high shear conditions and thus might be a new target for preventing arterial thrombosis.
Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function
Authors: Kunz Gabriella , Öhlin Ann-Kristin , Adami Antonella , Zöller Bengt , Svensson Peter , Lane David A.
Published: October 2002

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% ± 5%, P < .001), Pro477Ser (76.8% ± 1%, P < .001), Pro483Leu (82.1% ± 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (Km) for wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P < .0001) Km, 2967 ±  283 nM, and 2342 ± 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.
Epstein-Barr virus inhibits the development of dendritic cells by promoting apoptosis of their monocyte precursors in the presence of granulocyte macrophage–colony-stimulating factor and interleukin-4
Authors: Li LiQi , Liu Daorong , Hutt-Fletcher Lindsey , Morgan Andrew , Masucci Maria G. , Levitsky Victor
Published: October 2002

Epstein-Barr virus (EBV) is a tumorigenic human herpesvirus that persists for life in healthy immunocompetent carriers. The viral strategies that prevent its clearance and allow reactivation in the face of persistent immunity are not well understood. Here we demonstrate that EBV infection of monocytes inhibits their development into dendritic cells (DCs), leading to an abnormal cellular response to granulocyte macrophage–colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and to apoptotic death. This proapoptotic activity was not affected by UV inactivation and was neutralized by EBV antibody-positive human sera, indicating that binding of the virus to monocytes is sufficient to alter their response to the cytokines. Experiments with the relevant blocking antibodies or with mutated EBV strains lacking either the EBV envelope glycoprotein gp42 or gp85 demonstrated that interaction of the trimolecular gp25–gp42–gp85 complex with the monocyte membrane is required for the effect. Our data provide the first evidence that EBV can prevent the development of DCs through a mechanism that appears to bypass the requirement for viral gene expression, and they suggest a new strategy for interference with the function of DCs during the initiation and maintenance of virus-specific immune responses.
Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients
Authors: Fonseca Rafael , Blood Emily A. , Oken Martin M. , Kyle Robert A. , Dewald Gordon W. , Bailey Richard J. , Van Wier Scott A. , Henderson Kimberly J. , Hoyer James D. , Harrington David , Kay Neil E. , Van Ness Brian , Greipp Philip R.
Published: October 2002

The t(11;14)(q13;q32) results in up-regulation of cyclin D1 and is the most common translocation detected in multiple myeloma, where it is also associated with a lymphoplasmacytic morphology. We performed an interphase fluorescent in situ hybridization (FISH) study to determine the clinical and biologic significance of the abnormality when testing a large cohort of myeloma patients. Bone marrow slides from multiple myeloma patients entered into the Eastern Cooperative Oncology Group phase III clinical trial E9486 and associated laboratory correlative study E9487 were analyzed using interphase FISH combined with immune-fluorescent (cytoplasmic immunoglobulin–FISH) detection of clonal plasma cells. We used FISH probes that hybridize to the 14q32 and 11q13 chromosomal loci. The t(11;14)(q13;q32) was correlated with known biologic and prognostic factors. Of 336 evaluable patients, 53 (16%) had abnormal FISH patterns compatible with the t(11;14)(q13;q32). These patients appeared to be more likely to have a serum monoclonal protein of less than 10 g/L (1 g/dL) (28% vs 15%, P = .029) and a lower plasma cell labeling index (P = .09). More strikingly, patients were less likely to be hyperdiploid by DNA content analysis (n = 251, 14% vs 62%, P < .001). Patients with the t(11;14)(q13;q32) appeared to have better survival and response to treatment, although this did not reach statistical significance. Multiple myeloma with the t(11;14)(q13;q32) is a unique subset of patients, not only characterized by cyclin D1 up-regulation and a lymphoplasmacytic morphology, but is also more frequently associated with small serum monoclonal proteins and is much less likely to be hyperdiploid. These patients do not have a worsened prognosis as previously thought.
Interleukin-21 is a growth and survival factor for human myeloma cells
Authors: Brenne Anne-Tove , Baade Ro Torstein , Waage Anders , Sundan Anders , Borset Magne , Hjorth-Hansen Henrik
Published: October 2002

Interleukin-21 (IL-21) is a recently cloned cytokine with homology to IL-2, IL-4, and IL-15. In this study we examined the effects of IL-21 on human myeloma cells. We found that IL-21 induced proliferation and inhibited apoptosis of the IL-6–dependent human myeloma cell lines ANBL-6, IH-1, and OH-2. The potency of IL-21 was close to that of IL-6 in the OH-2 cell line. Neutralizing antibodies to IL-6 or the IL-6 receptor transducer chain (gp130) did not affect IL-21–induced DNA synthesis, indicating that IL-21–induced proliferation was not mediated through these proteins. Tumor necrosis factor (TNF), another stimulator of myeloma cell growth, up-regulated the expression level of IL-21 receptor (IL-21R), and combinations of TNF and IL-21 gave synergistic effects on myeloma cell proliferation. Furthermore, 4 of 9 purified samples of primary myeloma cells showed a significant increase in DNA synthesis on stimulation of the cells by IL-21. By Western blot analysis, we demonstrated that the intracellular signaling pathways of IL-21 in myeloma cells involved phosphorylation of Jak1, Stat3, and Erk1/2 (p44/42 mitogen-activated protein kinase). IL-21 is a novel growth and survival factor in multiple myeloma and may represent a target for future therapy.
Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation
Authors: Holtz Melissa S. , Slovak Marilyn L. , Zhang Feiyu , Sawyers Charles L. , Forman Stephen J. , Bhatia Ravi
Published: October 2002

Imatinib mesylate (STI571) is a promising new treatment for chronic myelogenous leukemia (CML). The effect of imatinib mesylate on primitive malignant progenitors in CML has not been evaluated, and it is not clear whether suppression of progenitor growth represents inhibition of increased proliferation, induction of apoptosis, or both. We demonstrated here that in vitro exposure to concentrations of imatinib mesylate usually achieved in patients (1-2 μM) for 96 hours inhibited BCR/ABL-positive primitive progenitors (6-week long-term culture–initiating cells [LTCICs]) as well as committed progenitors (colony-forming cells [CFCs]). No suppression of normal LTCICs and significantly less suppression of normal CFCs were observed. A higher concentration of imatinib mesylate (5 μM) did not significantly increase suppression of CML or normal LTCICs but did increase suppression of CML CFCs, and to a lesser extent, normal CFCs. Analysis of cell division using the fluorescent dye carboxyfluorescein diacetate succinimidyl ester indicated that imatinib mesylate (1-2 μM) inhibits cycling of CML primitive (CD34+CD38−) and committed (CD34+CD38+) progenitors to a much greater extent than normal cells. Conversely, treatment with 1 to 2 μM imatinib mesylate did not significantly increase the percentage of cells undergoing apoptosis. Although a higher concentration of imatinib mesylate (5 μM) led to an increase in apoptosis of CML cells, apoptosis also increased in normal samples. In summary, at clinically relevant concentrations, imatinib mesylate selectively suppresses CML primitive progenitors by reversing abnormally increased proliferation but does not significantly increase apoptosis. These results suggest that inhibition of Bcr-Abl tyrosine kinase by imatinib mesylate restores normal hematopoiesis by removing the proliferative advantage of CML progenitors but that elimination of all CML progenitors may not occur.
Paradoxical secondary polycythemia in von Hippel-Lindau patients treated with anti–vascular endothelial growth factor receptor therapy
Authors: Richard Stéphane , Croisille Laure , Yvart Jeannine , Casadeval Nicole , Eschwège Pascal , Aghakhani Nozar , David Philippe , Gaudric Alain , Scigalla Paul , Hermine Olivier
Published: October 2002

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome caused by germline mutations in the VHL tumor-suppressor gene. Central nervous system (CNS) and retinal hemangioblastomas are highly vascular tumors that are hallmarks of the disease. These tumors overexpress vascular endothelial growth factor (VEGF) and represent a potential target for anti-angiogenic drugs. We observed, after 3 to 4 months of treatment, secondary paradoxical polycythemia in 3 VHL patients with CNS or retinal hemangioblastomas treated by the anti-VEGF receptor SU5416. Hematocrit was normal before the beginning of the trial, and no progression of hemangioblastomas was observed. Polycythemia vera and all known causes of secondary polycythemia were also ruled out. Polycythemia has never been reported in current SU5416 trials for advanced malignancies and could express a specific action on red blood cell precursors occurring only in the absence of a functional VHL gene. These findings could also affect the inclusion of VHL patients with pre-existing polycythemia in future anti-VEGF receptor trials.
Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-α plus fibronectin-activated peripheral blood eosinophils
Authors: Esnault Stéphane , Malter James S.
Published: October 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critical for promoting the long-term survival of lung- or airway-based eosinophils. Previously, we have shown that fibronectin and tumor necrosis factor α induced autocrine production of GM-CSF that markedly enhanced eosinophil survival. Cytokine release was preceded by and dependent on messenger RNA (mRNA) stabilization. Here, we show that mitogen-activated protein kinase (MAPK) activation is responsible for GM-CSF mRNA stabilization in peripheral blood eosinophils (pbeos). Activation of extracellular signal-regulated kinase (ERK) but not p38 correlated with GM-CSF mRNA stability. Although ERK inhibition completely prevented GM-CSF mRNA stabilization, p38 inhibition had a partial effect. To establish which MAPK was crucial, we transduced pbeos with dominant-active TatMEK1(E) or TatMKK3b(E) proteins that selectively phosphorylate ERK or p38, respectively. These studies showed that ERK but not p38 was sufficient for GM-CSF mRNA stabilization. These data are in contradistinction to the c-Jun NH2-termainal kinase–mediated regulation of interleukin 2 and 3 mRNAs and suggest unique regulatory features for GM-CSF mRNA in eosinophils.
Defective T-helper cell function after T-cell–depleting therapy affecting naive and memory populations
Authors: Heitger Andreas , Winklehner Patricia , Obexer Petra , Eder Johannes , Zelle-Rieser Claudia , Kropshofer Gabriele , Thurnher Martin , Holter Wolfgang
Published: October 2002

Impaired T-cell function after T-cell– depleting (TCD) therapy has been hypothesized to be related to a transient predominance of extrathymically expanding memory T cells. To test whether after TCD therapy the naive T-helper cell population is functionally intact, the in vitro immune response of CD4+CD45RA+ (naive) and of CD4+CD45RA− (memory) cells to polyclonal mitogens (immobilized anti-CD3, phytohemagglutinin) was analyzed by flow cytometry in 22 pediatric patients after high-dose chemotherapy (including 5 after autologous and 5 after allogeneic stem cell support). At 1 to 3 months after TCD therapy, patient samples showing decreased lymphoproliferative responses also showed a reduced induction of the early activation marker CD69 by CD4+ T cells from 4 to 72 hours after stimulation even when supplemented with exogenous interleukin-2. This defect affected CD4+CD45RA− cells, but, strikingly, also CD4+CD45RA+ cells, including samples in which CD4+CD45RA+ cells were more than 90/μL, thus indicating ongoing thymopoiesis. Histogram analyses showed the median peak channel of CD69 in control CD4+CD45RA+cells rising 98-fold (median) but only 28-fold in patient cells (P < .0001). Apoptosis as detected by annexin V staining was increased in resting patient CD4+ T cells (25% versus 6%) and also affected CD4+CD45RA+ cells (12% versus 5%, P < .01). When peripheral blood mononuclear cells (PBMCs) were enriched for T cells, stimulatory responses of CD4+ cells and of CD4+CD45RA+ cells markedly improved. Thus, after TCD therapy suppressor factors contained in the non–T-cell fraction of PBMCs may affect T-helper cells irrespective of their naive or memory phenotype thus extending T-cell dysfunction to the presumably thymus-dependently regenerated T cells.