bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines
Authors: Geginat Jens , Lanzavecchia Antonio , Sallusto Federica
Published: May 2003

Four human CD8+ T-cell subsets, naive (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA–), effector memory (TEM, CCR7–CD45RA–), and CD45RA+ effector memory cells (TEMRA, CCR7–CD45RA+) were compared for their capacity to proliferate and differentiate in response to antigen or homeostatic cytokines. Cytokine responsiveness and interleukin-15 receptor expression were low in naive T cells and progressively increased from TCM to TEM and TEMRA. In contrast, the capacity to accumulate in response to T-cell receptor (TCR) or cytokine stimulation showed a reciprocal pattern and was associated with resistance to cell death and Bcl-2 expression. Whereas all TCR-stimulated cells acquired a CD45RA–CCR7– phenotype, cytokine-stimulated cells maintained their phenotype with the exception of TCM cells, which expressed CCR7, CD45RA, and perforin in various combinations. Single CD8+ TCM cells, but not TEM cells, could be expanded with cytokines, and the obtained clones displayed several distinct phenotypes, suggesting that TCM cells are heterogeneous. Consistently, CCR4 expression in the CD8+ TCM pool discriminated CCR4+ type 2 polarized cells (Tc2) and CCR4–CTL precursors. Finally, ex vivo bromodeoxyuridine (BrdU) incorporation experiments revealed that memory subsets have different in vivo proliferation rates, with CCR4–TCM having the highest turnover and TEMRA the lowest. These results show that human CD8+ memory T-cell subsets have different proliferation and differentiation potentials in vitro and in vivo. Furthermore, they suggest that TEMRA cells are generated from a TCM subset upon homeostatic proliferation in the absence of antigen.
Rapid recovery from T lymphopenia by CD28 superagonist therapy
Authors: Elflein Karin , Rodriguez-Palmero Marta , Kerkau Thomas , Hünig Thomas
Published: May 2003

Slow recovery of T-cell numbers and function contributes to the high incidence of life-threatening infections after cytotoxic cancer therapies. We have tested the therapeutic potential of a novel class of superagonistic CD28–specific antibodies that induce polyclonal T-cell proliferation without T-cell receptor engagement in an experimental rat model of T lymphopenia. We show that in lethally irradiated, bone marrow–reconstituted hosts, CD28 superagonist is able to dramatically accelerate repopulation by a small inoculum of mature, allotype-marked T cells. CD28-driven recovery of CD4 cells was superior to that of CD8 T cells. CD28 superagonist– expanded CD4 T cells had maintained repertoire diversity and were functional both in vitro and in vivo, suggesting that treatment with a human CD28–specific superagonist will protect T-lymphopenic patients from opportunistic infections.
Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib
Authors: Corbin Amie S. , Rosée Paul La , Stoffregen Eric P. , Druker Brian J. , Deininger Michael W.
Published: May 2003

Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.
Treatment of canine cyclic neutropenia by lentivirus-mediated G-CSF delivery
Authors: Yanay Ofer , Barry Simon C. , Katen Louis J. , Brzezinski Margaret , Flint Lisa Y. , Christensen Jeffrey , Liggitt Denny , Dale David C. , Osborne William R. A.
Published: May 2003

Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 109 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/μL, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts ± SD were 12 460 ± 4240 cells/μL, significantly increased over both pretreatment values of 3040 ± 2540 cells/μL (P < .0001) and neutrophil counts during G-CSF administration of 10 290 ± 4860 cells/μL(P < .007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias.
Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2
Authors: Liu Ji-yan , Wei Yu-quan , Yang Li , Zhao Xia , Tian Ling , Hou Jian-mei , Niu Ting , Liu Fen , Jiang Yu , Hu Bing , Wu Yang , Su Jing-mei , Lou Yan-yan , He Qiu-ming , Wen Yan-jun , Yang Jin-liang , Kan Bing , Mao Yong-qiu , Luo Feng , Peng Feng
Published: May 2003

The breaking of immune tolerance of “self-antigens” associated with angiogenesis is an attractive approach to cancer therapy by active immunity. We used vascular endothelial growth factor receptor-2 (VEGFR-2) as a model antigen to explore the feasibility of the immunotherapy with a vaccine based on a xenogeneic homologous protein. To test this concept, we prepared a quail homologous VEGFR-2 protein vaccine (qVEGFR) based on quail VEGFR-2. At the same time, a protein vaccine based on the corresponding ligand-binding domain of mouse self-VEGFR-2 (mVEGFR) was also prepared and used as a control. We found that immunotherapy with qVEGFR was effective at protective and therapeutic antitumor immunity in several solid and hematopoietic tumor models in mice. Autoantibodies against mouse VEGFR-2 (Flk-1) were identified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Anti-VEGFR antibody-producing B cells were detectable by ELISPOT. Endothelial deposition of immunoglobulins developed within tumor. VEGF-mediated endothelial cell proliferation was inhibited in vitro by immunoglobulins from qVEGFR-immunized mice. Antitumor activity was caused by the adoptive transfer of the purified immunoglobulins. Antitumor activity and production of autoantibodies against Flk-1 could be abrogated by the depletion of CD4+ T lymphocytes. Angiogenesis was apparently inhibited within the tumors, and the vascularization of alginate beads was also reduced. No marked toxicity was found in the immunized mice. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factor receptor associated with angiogenesis in a cross-reaction with single xenogeneic homologous protein.
High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PML-RARα expression
Authors: Westervelt Peter , Lane Andrew A. , Pollock Jessica L. , Oldfather Kristie , Holt Matthew S. , Zimonjic Drazen B. , Popescu Nicholas C. , DiPersio John F. , Ley Timothy J.
Published: May 2003

Transgenic mice expressing PML-RARα in early myeloid cells under control of human cathepsin G regulatory sequences all develop a myeloproliferative syndrome, but only 15% to 20% develop acute promyelocytic leukemia (APL) after a latent period of 6 to 14 months. However, this transgene is expressed at very low levels in the bone marrow cells of transgenic mice. Because the transgene includes only 6 kb of regulatory sequences from the human cathepsin G locus, we hypothesized that sequences required for high-level expression of the transgene might be located elsewhere in the cathepsin G locus and that a knock-in model might yield much higher expression levels and higher penetrance of disease. We, therefore, targeted a human PML-RARα cDNA to the 5′ untranslated region of the murine cathepsin G gene, using homologous recombination in embryonic stem cells. This model produced a high-penetrance APL phenotype, with more than 90% of knock-in mice developing APL between 6 and 16 months of age. The latent period and phenotype of APL (including a low frequency of an interstitial deletion of chromosome 2) was similar to that of the previous transgenic model. Remarkably, however, the expression level of PML-RARα in bone marrow cells or APL cells was less than 3% of that measured in the low-penetrance transgenic model. Although the explanation for this result is not yet clear, one hypothesis suggests that very low levels of PML-RARα expression in early myeloid cells may be optimal for the development of APL in mice.
ADAMTS13 gene mutation in congenital thrombotic thrombocytopenic purpura with previously reported normal VWF cleaving protease activity
Authors: Savasan Süreyya , Lee Soon-Ki , Ginsburg David , Tsai Han-Mou
Published: May 2003

Deficiency of von Willebrand factor (VWF) cleaving protease ADAMTS13 is associated with the development of thrombotic thrombocytopenic purpura (TTP). A case of congenital TTP that was previously reported to have normal ADAMTS13 activity was analyzed at the molecular level. Reanalysis of plasma VWF cleaving protease activity using a different assay revealed that the patient had less than 0.1 U/L ADAMTS13 protease activity, while the parents were both partially deficient. Sequence analysis of DNA amplified by polymerase chain reaction showed that the patient was homozygous for a novel TT deletion in exon 15 of the ADAMTS13 gene resulting in a frameshift, while both parents were heterozygous for the same mutation. Taken together with other recent reports, all the cases of hereditary TTP studied by DNA sequence analysis to date appear to be due to mutations within the ADAMTS13 gene.
Expression and function of KIR and natural cytotoxicity receptors in NK-type lymphoproliferative diseases of granular lymphocytes
Authors: Zambello Renato , Falco Michela , Chiesa Mariella Della , Trentin Livio , Carollo Davide , Castriconi Roberta , Cannas Giovanna , Carlomagno Simona , Cabrelle Anna , Lamy Thierry , Agostini Carlo , Moretta Alessandro , Semenzato Gianpietro , Vitale Massimo
Published: May 2003

Using monoclonal antibodies (mAbs) specific for different natural killer (NK) receptors, we studied the lymphocyte population from 18 patients with NK-type lymphoproliferative disease of granular lymphocytes (LDGL). The analysis of both resting and cultured NK cell populations demonstrated that these patients are frequently characterized by NK cells displaying a homogeneous staining with given anti–killer Ig-like receptor (anti-KIR) mAb (11 of 18 patients). In most patients NK cells were characterized by the CD94/NKG2A+ phenotype, whereas only a minor fraction of the cases expressed CD94/NKG2C. In 7 of these patients we could also assess the function of the various NK receptors. Remarkably those KIR molecules that, in each patient, homogeneously marked the NK cell expansion were found to display an activating function as determined by cross-linking with specific anti-KIR mAb. The KIR genotype analysis performed in 13 of 18 cases revealed that in NK-type LDGL certain activating KIRs, as well as certain infrequent KIR genotypes, were detected with higher frequencies as compared to previously analyzed healthy donors. Moreover, most KIR genotypes included multiple genes coding for activating KIRs. The analysis of non–HLA-specific triggering receptors indicated that the natural cytotoxicity receptors (NKp46, NKp30) were expressed at significantly low levels in freshly drawn NK cells from most patients analyzed. However, in most instances the expression of NKp46 and NKp30 could be up-regulated on culture in interleukin 2. Our data indicate that in NK-LDGL the expanded subset is frequently characterized by the expression of a given activating KIR, suggesting a direct role for these molecules in the pathogenetic mechanisms of this disorder.
Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571)
Authors: Wohlbold Lara , van der Kuip Heiko , Miething Cornelius , Vornlocher Hans-Peter , Knabbe Cornelius , Duyster Justus , Aulitzky Walter E.
Published: May 2003

Bcr-Abl proteins are effective inducers of the leukemic phenotype in chronic myeloid leukemia (CML) and distinct variants of acute lymphoblastic leukemia (ALL). Targeting bcr-abl by treatment with the selective tyrosine kinase inhibitor imatinib has proved to be highly efficient for controlling leukemic growth. However, it is unclear whether imatinib is sufficient to eradicate the disease because of primary or secondary resistance of leukemic cells. Therefore, targeting Bcr-Abl with an alternative approach is of great interest. We demonstrate that RNA interference (RNAi) with a breakpoint-specific short-interfering RNA (siRNA) is capable of decreasing Bcr-Abl protein expression and of antagonizing Bcr-Abl–induced biochemical activities. RNAi selectively inhibited Bcr-Abl–dependent cell growth. Furthermore, bcr-abl–homologous siRNA increased sensitivity to imatinib in Bcr-Abl–overexpressing cells and in a cell line expressing the imatinib-resistant Bcr-Abl kinase domain mutation His396Pro, thereby antagonizing 2 of the major mechanisms of resistance to imatinib.
Mutagenesis of GATA1 is an initiating event in Down syndrome leukemogenesis
Authors: Mundschau Gina , Gurbuxani Sandeep , Gamis Alan S. , Greene Marianne E. , Arceci Robert J. , Crispino John D.
Published: May 2003

As many as 10% of infants with Down syndrome (DS) present with transient myeloproliferative disorder (TMD) at or shortly after birth. TMD is characterized by an abundance of blasts within the peripheral blood and liver, and notably undergoes spontaneous remission in the majority of cases. TMD may be a precursor to acute megakaryoblastic leukemia (AMKL), with an estimated 30% of TMD patients developing AMKL within 3 years. We recently reported that mutations in the transcription factor GATA1 are associated with DS-AMKL. To determine whether the acquisition of GATA1 mutations is a late event restricted to acute leukemia, we analyzed GATA1 in DNA from TMD patients. Here we report that GATA1 is mutated in the TMD blasts from every infant examined. These results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD, and mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis.
CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C–dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase
Authors: Smit Martine J. , Verdijk Pauline , van der Raaij-Helmer Elisabeth M. H. , Navis Marjon , Hensbergen Paul J. , Leurs Rob , Tensen Cornelis P.
Published: May 2003

The chemokines CXCL9, 10, and 11 exert their action via CXC chemokine receptor-3 (CXCR3), a receptor highly expressed on activated T cells. These interferon γ (IFNγ)–induced chemokines are thought to be crucial in directing activated T cells to sites of inflammation. As such, they play an important role in several chronic inflammatory diseases including ulcerative colitis, multiple sclerosis, artherosclerosis, and delayed-type hypersensitivity reactions of the skin. In this study, we first demonstrate that in COS-7 cells heterologously expressing CXCR3, CXCL11 is a potent activator of the pertussis toxin (PTX)–sensitive p44/p42 mitogen-activated protein kinase (MAPK) and Akt/phosphatidylinositol 3 kinase (PI3K) pathways. Next, we show that these signal transduction pathways are also operative and PTX sensitive in primary human T cells expressing CXCR3. Importantly, abrogation of these signaling cascades by specific inhibitors did not block the migration of T cells toward CXCR3 ligands, suggesting that MAPK and Akt activation is not crucial for CXCR3-mediated chemotaxis of T cells. Finally, we demonstrate that CXCR3-targeting chemokines control T-cell migration via PTX-sensitive, phospholipase C pathways and phosphatidylinositol kinases other than class I PI3Kγ.
GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome
Authors: Hitzler Johann K. , Cheung Joseph , Li Yue , Scherer Stephen W. , Zipursky Alvin
Published: May 2003

Children with constitutional trisomy 21 (Down syndrome) have an approximately 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia. Unique to newborn infants with Down syndrome is a transient leukemia (TL), also referred to as transient myeloproliferative syndrome, that undergoes spontaneous remission in the majority of cases but in approximately 20% is followed by AMKL later in life. Recently mutations of the gene encoding the hematopoietic transcription factor GATA1 were shown to be specific for AMKL of Down syndrome. Here, we demonstrate that GATA1 mutations are present in blasts of TL and show the identical GATA1 mutation in sequential samples collected from a patient during TL and subsequent AMKL. These findings suggest a model of malignant transformation in Down syndrome AMKL in which GATA1 mutations are an early event and AMKL arises from latent TL clones following initial apparent remission.
Cappuccino, a mouse model of Hermansky-Pudlak syndrome, encodes a novel protein that is part of the pallidin-muted complex (BLOC-1)
Authors: Ciciotte Steven L. , Gwynn Babette , Moriyama Kengo , Huizing Marjan , Gahl William A. , Bonifacino Juan S. , Peters Luanne L.
Published: May 2003

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis affecting 3 related organelles—melanosomes, platelet dense bodies, and lysosomes. Four genes causing HPS in humans (HPS1-HPS4) are known, and at least 15 nonallelic mutations cause HPS in the mouse. Where their functions are known, the HPS-associated proteins are involved in some aspect of intracellular vesicular trafficking, that is, protein sorting and vesicle docking and fusion. Biochemical and genetic evidence indicates that the HPS-associated genes encode components of at least 3 distinct protein complexes: the adaptor complex AP-3; the HPS1/HPS4 complex; and BLOC-1 (biogenesis of lysosome-related organelles complex-1), consisting of the proteins encoded at 2 mouse HPS loci, pallid (pa) and muted (mu), and at least 3 other unidentified proteins. Here, we report the cloning of the mouse HPS mutation cappuccino (cno). We show that the wild-type cno gene encodes a novel, ubiquitously expressed cytoplasmic protein that coassembles with pallidin and the muted protein in the BLOC-1 complex. Further, we identify a frameshift mutation in mutant cno/cno mice. The C-terminal 81 amino acids are replaced with 72 different amino acids in the mutant CNO protein, and its ability to interact in BLOC-1 is abolished. We performed mutation screening of patients with HPS and failed to identify any CNO defects. Notably, although defects in components of the HPS1/HPS4 and the AP-3 complexes are associated with HPS in humans, no defects in the known components of BLOC-1 have been identified in 142 patients with HPS screened to date, suggesting that BLOC-1 function may be critical in humans.
Regulation of plasminogen activation: a role for melanotransferrin (p97) in cell migration
Authors: Demeule Michel , Bertrand Yanick , Michaud-Levesque Jonathan , Jodoin Julie , Rolland Yannève , Gabathuler Reinhard , Béliveau Richard
Published: May 2003

We recently reported that human recombinant melanotransferrin (p97) presents a high transport rate across the blood-brain barrier that might involve the low-density lipoprotein receptor–related protein (LRP). We now report new interactions between p97 and another LRP ligand, the urokinase plasminogen activator (uPA) complex. By using biospecific interaction analysis, both pro-uPA and plasminogen are shown to interact with immobilized p97. Moreover, the activation of plasminogen by pro-uPA is increased by soluble p97. Because the uPA system plays a crucial role in cell migration, both in cancer and in angiogenesis, we also measured the impact of both endogenous membrane-bound and exogenous p97 on cell migration. The monoclonal antibody L235 (which recognizes a conformational epitope on p97) inhibited the migration of human microvascular endothelial cells (HMECs-1) and of human melanoma SK-MEL-28 cells, indicating that endogenous membrane-bound p97 could be associated with this process. In addition, low concentrations of exogenous p97 (10 and 100 nM) inhibited HMEC-1 and SK-MEL28 cell migration by more than 50%. These results indicate that membrane-bound and soluble p97 affect the migration capacity of endothelial and melanoma cells and suggest that p97 could be involved in the regulation of plasminogen activation by interacting with pro-uPA and plasminogen.
Myeloid cell expansion elicited by the progression of spontaneous mammary carcinomas in c-erbB-2 transgenic BALB/c mice suppresses immune reactivity
Authors: Melani Cecilia , Chiodoni Claudia , Forni Guido , Colombo Mario P.
Published: May 2003

Transgenic female mice expressing the transforming rat oncogene c-erbB-2 (HER-2/neu) under the mouse mammary tumor virus (MMTV) promoter (BALB-neuT) spontaneously develop mammary carcinomas with a progression resembling that of human breast cancer. In these mice, activating antitumor immunotherapy fails to induce T cell–mediated cytotoxicity, suggesting a suppression of the immune response. We found a direct correlation between tumor multiplicity and an increased proportion of Gr-1+ (Ly6G)/Mac-1+(CD11b)/ER-MP12+(CD31) immature myeloid cells in the peripheral blood (PB) and spleen, suggesting that tumor load profoundly affects overall BALB-neuT hematopoiesis. In fact, myeloid colony formation was increased in bone marrow (BM) and spleen. The immature myeloid cells displayed suppressive activity on host T lymphocytes, which progressively failed to respond to alloantigens and CD3 triggering, while maintaining the ability to proliferate in response to nonspecific mitogens. Transplantation of normal BM into BALB-neuT mice readily resulted in hypertrophic hematopoiesis with myeloid cell expansion. This persistent influence of the tumor was mediated through the release of vascular endothelial growth factor (VEGF) but not granulocyte-macrophage colony-stimulating factor (GM-CSF), and was down-modulated when tumor load was reduced but not when BM was transplanted. Together, the data obtained in the BALB-neuT model of naturally occurring carcinogenesis show that tumor-associated immune suppression is secondary to a more general alteration of host hematopoiesis, conditioned by tumor-secreted soluble factors.
IL-15 drives neonatal T cells to acquire CD56 and become activated effector cells
Authors: Cookson Sharon , Reen Denis
Published: May 2003

Expression of one or more natural killer (NK) receptors on T cells may correlate with effector function. This study investigated the frequency of neonatal NK receptor–positive (NKR+) T cells and their expansionary properties with interleukin-2 (IL-2), IL-7, or IL-15. While cord blood contains significantly decreased frequencies of NKR+ T cells compared with adult blood, newborn CD56+CD3+ cells could be expanded 200-fold during culture with IL-15. By depleting CD56+ cells, we were able to determine that this expansion was due to a subpopulation of T cells acquiring CD56 expression. Moreover, CD56 acquisition was associated with a distinct CD8+CD25+ interferon γ–positive (IFN-γ+) phenotype. This property could therefore be exploited during bone marrow reconstitution and may partially account for the resilience of the newborn to infection.
Transgenic mice overexpressing erythropoietin adapt to excessive erythrocytosis by regulating blood viscosity
Authors: Vogel Johannes , Kiessling Isabel , Heinicke Katja , Stallmach Thomas , Ossent Pete , Vogel Olga , Aulmann Michael , Frietsch Thomas , Schmid-Schönbein Holger , Kuschinsky Wolfgang , Gassmann Max
Published: May 2003

Severe elevation of red blood cell number is often associated with hypertension and thromboembolism resulting in severe cardiovascular complications. However, some individuals such as high altitude dwellers cope well with an increased hematocrit level. We analyzed adaptive mechanisms to excessive erythrocytosis in our transgenic (tg) mice that, due to hypoxia-independent erythropoietin (Epo) overexpression, reached hematocrit values of 0.8 to 0.9 without alteration of blood pressure, heart rate, or cardiac output. Extramedullar erythropoiesis occurred in the tg spleen, leading to splenomegaly. Upon splenectomy, hematocrit values in tg mice decreased from 0.89 to 0.62. Tg mice showed doubled reticulocyte counts and an increased mean corpuscular volume. In tg mice, plasma volume was not elevated whereas blood volume was up to 25% of the body weight compared with 8% in wild-type (wt) siblings. Although plasma viscosity did not differ between tg and wt mice, tg whole-blood viscosity increased to a lower degree (4-fold) than expected from corresponding hemoconcentrated wt blood (8-fold). This moderate increase in viscosity is explicable by the up to 3-fold higher elongation of tg erythrocytes at physiologic shear rates. Apart from the nitric oxide–mediated vasodilation we reported earlier, adaptation to high hematocrit levels in tg mice involves regulated elevation of blood viscosity by increasing erythrocyte flexibility.
Heme and lipid peroxides in hemoglobin-modified low-density lipoprotein mediate cell survival and adaptation to oxidative stress
Authors: Asatryan Liana , Ziouzenkova Ouliana , Duncan Roger , Sevanian Alex
Published: May 2003

Low-density lipoprotein (LDL) oxidation mediated by a variety of catalysts in atherosclerotic lesions plays a crucial role in the genesis and evolution of atherosclerotic plaques. In this study we focused on oxidative properties of hemoglobin (Hb)–modified LDL because Hb is present in atherosclerotic lesions. Under low oxygen tensions Hb was previously found to modify apolipoprotein B100 with covalent binding of Hb fragments and formation of electronegative LDL particles (LDL–). Here we show that HbLDL is highly susceptible to oxidation, but is not cytotoxic to vascular cells, as was found for LDL– isolated from human plasma. HbLDL and LDL– have similar levels of oxidized lipid products and low uptake rates; however, the virtual absence of HbLDL-induced toxicity depends on a marked adaptive oxidative stress response. This was evidenced by a time- and dose-dependent induction of heme oxygenase (HO-1). Cell survival was significantly decreased in the presence of HO-1 inhibitor, tin protoporphyrin (SnPPIX). HO-1 induction by HbLDL increased resistance of cells to toxic doses of hemin or t-BuOOH. The high sensitivity to oxidation and HO-1 induction was largely dependent on lipid hydroperoxides and heme associated with HbLDL. Reduction of pre-existing lipid peroxides using ebselen delayed HbLDL kinetics and inhibited HO-1 induction. Moreover, heme inactivation or its degradation inhibited HO-1 induction and provided an additive inhibitory effect to ebselen. We conclude that Hb-catalyzed reactions may modulate vascular cell survival and oxidative stress adaptation due to the presence of peroxides and heme, thus providing a possible mechanism for the evolution of atherosclerotic and hemorrhagic lesions.
Mechanisms for T-cell selective cytotoxicity of arabinosylguanine
Authors: Rodriguez Carlos O. , Stellrecht Christine M. , Gandhi Varsha
Published: May 2003

Nelarabine, prodrug of arabinosylguanine (ara-G), has demonstrated T-lymphoblastic antileukemic activity in cell lines and in the clinic. To investigate the mechanism for lineage-specific toxicity, the effects of ara-G were compared in CEM (T-lymphoblast), Raji (B-lymphoblast), and ML-1 (myeloid) cell lines. CEM cells were the most sensitive to ara-G–induced apoptosis and accumulated the highest levels of ara-G triphosphate (ara-GTP). However, compared with myeloid and B-lineage cell lines, CEM cells incorporated fewer ara-G molecules—which were at internucleotide positions in all 3 cell lines— into DNA. Ara-G induced an S-phase arrest in both Raji and ML-1, while in CEM the S-phase cells decreased with a concomitant increase in the sub-G1 population. Within 3 hours of ara-G treatment, the levels of soluble Fas ligand (sFasL) in the medium increased significantly in CEM cultures. In parallel, an induction of FasL gene expression was observed by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Pretreatment of CEM cells with a Fas antagonistic antibody inhibited ara-G–mediated cell death. These results demonstrate that high ara-GTP accumulation in T cells results in an S phase–dependent apoptosis induced by ara-G incorporation into DNA, which may lead to a T cell–specific signal for the induction and liberation of sFasL. Subsequently, the sFasL induces an apoptotic response in neighboring non–S-phase cells. In contrast, myeloid and B cells accumulated lower levels of ara-GTP and arrested in S phase, blocking any apoptotic signaling.
BAALC expression predicts clinical outcome of de novo acute myeloid leukemia patients with normal cytogenetics: a Cancer and Leukemia Group B Study
Authors: Baldus Claudia D. , Tanner Stephan M. , Ruppert Amy S. , Whitman Susan P. , Archer Kellie J. , Marcucci Guido , Caligiuri Michael A. , Carroll Andrew J. , Vardiman James W. , Powell Bayard L. , Allen Steven L. , Moore Joseph O. , Larson Richard A. , Kolitz Jonathan E. , de la Chapelle Albert , Bloomfield Clara D.
Published: May 2003

Cytogenetic aberrations are important prognostic factors in acute myeloid leukemia (AML). Of adults with de novo AML, 45% lack cytogenetic abnormalities, and identification of predictive molecular markers might improve therapy. We studied the prognostic impact of BAALC (Brain And Acute Leukemia, Cytoplasmic), a novel gene involved in leukemia, in 86 de novo AML patients with normal cytogenetics who were uniformly treated on Cancer and Leukemia Group B 9621. BAALC expression was determined by comparative real-time reverse transcriptase–polymerase chain reaction in pretreatment blood samples, and patients were dichotomized at BAALC's median expression into low and high expressers. Low expressers had higher white counts (P = .03) and more frequent French-American-British M5 morphology (P = .007). Compared to low expressers, high BAALC expressers showed significantly inferior overall survival (OS; median, 1.7 vs 5.8 years, P = .02), event-free survival (EFS; median, 0.8 vs 4.9 years, P = .03), and disease-free survival (DFS; median, 1.4 vs 7.3 years, P = .03). Multivariable analysis confirmed high BAALC expression as an independent risk factor. For high BAALC expressers the hazard ratio of an event for OS, EFS, and DFS was respectively 2.7, 2.6, and 2.2. We conclude that high BAALC expression predicts an adverse prognosis and may define an important risk factor in AML with normal cytogenetics.
Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) has minimal effect on markers of coagulation, fibrinolysis, and inflammation in acute human endotoxemia
Authors: Derhaschnig Ulla , Reiter Rosemarie , Knöbl Paul , Baumgartner Magdalena , Keen Priska , Jilma Bernd
Published: May 2003

Inflammatory and procoagulant host responses are closely related in sepsis. The protein C pathway serves as a regulatory pathway with anti-inflammatory and anticoagulant properties. Recently, recombinant human activated protein C (rhAPC) was shown to reduce mortality in severe sepsis. Nevertheless, the effects of rhAPC in humans are still ill defined. The infusion of low endotoxin doses into humans provides a standardized model to study inflammatory and hemostatic mechanisms. Thus, we investigated whether rhAPC acts as an anticoagulant or anti-inflammatory drug in human endotoxemia. There were 24 volunteers randomized to receive either 24 μg/kg per hour rhAPC or placebo intravenously for 8 hours. Lipopolysaccharide (LPS, 2 ng/kg) was administered 2 hours after starting the infusions. rhAPC decreased basal tissue factor (TF)–mRNA expression, and thrombin formation and action. In contrast, rhAPC did not significantly blunt LPS-induced thrombin generation. Consistently, rhAPC did not reduce LPS-induced levels of TF-mRNA or D-dimer and had no effect on fibrinolytic activity or inflammation. Finally, endogenous APC formation was enhanced during endotoxemia and appeared to be associated with inflammation rather than thrombin formation. In conclusion, even low-grade endotoxemia induces significant protein C activation. Infusion of rhAPC decreases “spontaneous” activation of coagulation but does not blunt LPS-induced, TF-mediated coagulation in healthy volunteers, which is in contrast to a number of anticoagulants.
Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions
Authors: Rufer Nathalie , Zippelius Alfred , Batard Pascal , Pittet Mikaël J. , Kurth Isabel , Corthesy Patricia , Cerottini Jean-Charles , Leyvraz Serge , Roosnek Eddy , Nabholz Markus , Romero Pedro
Published: May 2003

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7–27+28+ and RA+CCR7–27+28–, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon γ, and tumor necrosis factor α. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.
Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane
Authors: Forbes John R. , Gros Philippe
Published: May 2003

Mutations in the Nramp1 gene (Slc11a1) cause susceptibility to infection by intracellular pathogens. The Nramp1 protein is expressed at the phagosomal membrane of macrophages and neutrophils and is a paralog of the Nramp2 (Slc11a2) iron transporter. The Nramp1 transport mechanism at the phagosomal membrane has remained controversial. An Nramp1 protein modified by insertion of a hemagglutinin epitope into the predicted TM7/8 loop was expressed at the plasma membrane of Chinese hamster ovary cells as demonstrated by immunofluorescence and surface biotinylation. Experiments in Nramp1HA transfectants using the metal-sensitive fluorophors calcein and Fura2 showed that Nramp1HA can mediate Fe2+, Mn2+, and Co2+ uptake. Similar results were obtained in transport studies using radioisotopic 55Fe2+ and 54Mn2+. Nramp1HA transport was dependent on time, temperature, and acidic pH, occurring down the proton gradient. These experiments suggest that Nramp1HA may be a more efficient transporter of Mn2+ compared to Fe2+ and a more efficient Mn2+ transporter than Nramp2HA. The membrane topology and transport properties of Nramp1HA and Nramp2HA were indistinguishable, suggesting that Nramp1 divalent-metal transport at the phagosomal membrane is mechanistically similar to that of Nramp2 at the membrane of acidified endosomes. These results clarify the mechanism by which Nramp1 contributes to phagocyte defenses against infections.
The HIF family member EPAS1/HIF-2α is required for normal hematopoiesis in mice
Authors: Scortegagna Marzia , Morris Margaret A. , Oktay Yavuz , Bennett Michael , Garcia Joseph A.
Published: May 2003

Hypoxic stress plays a role in pathophysiologic states such as myocardial infarction and cerebral vascular events as well as in normal physiologic conditions including development and hematopoiesis. Members of the hypoxia inducible factor (HIF) family function as transcriptional regulators of genes involved in the hypoxic response. After generating adult mice that globally lack endothelial PAS domain protein 1 (EPAS1, also known as HIF-2α/HRF/HLF/MOP3), the second member of the HIF family, characterization of the hematopoietic cell population indicated that the loss of EPAS1/HIF-2α resulted in pancytopenia. Using bone marrow reconstitution experiments of lethally irradiated hosts, we have defined the extent and site of hematopoietic impairment in the EPAS1/HIF-2α null mice. These data suggest a critical role for EPAS1/HIF-2α in maintaining a functional microenvironment in the bone marrow for effective hematopoiesis.
Mechanisms of donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation
Authors: Quezada Sergio A. , Fuller Bruce , Jarvinen Lamis Z. , Gonzalez Mercedes , Blazar Bruce R. , Rudensky Alexander Y. , Strom Terry B. , Noelle Randolph J.
Published: May 2003

Induction of transplantation tolerance to alloantigens without general immunosuppression remains an enduring challenge. Injecting a donor-specific transfusion (DST) of spleen cells together with blocking αCD154 antibody prior to graft transplantation is an effective way to induce long-lived graft acceptance. Using a novel T-cell receptor (TCR) transgenic (Tg) model of CD4+ T-cell–mediated rejection, this study sheds new insights into the cellular basis for enhanced graft survival induced by DST and αCD154. The study shows that DST and αCD154 induce an early, robust, abortive expansion of the Tg T cells that results in profound anergy. This is contrasted with the more delayed, regional, productive response elicited by an allogeneic graft. Studies show that the induction of tolerance to the allograft induced by DST is mediated by indirect presentation by host antigen-presenting cells. Based on these observations, we conclude that DST and αCD154 preemptively tolerize the alloreactive T-cell compartment to prohibit subsequent responses to the immunogenic allograft.
High-dose radioimmunotherapy versus conventional high-dose therapy and autologous hematopoietic stem cell transplantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort analysis
Authors: Gopal Ajay K. , Gooley Theodore A. , Maloney David G. , Petersdorf Stephen H. , Eary Janet F. , Rajendran Joseph G. , Bush Sharon A. , Durack Lawrence D. , Golden Jane , Martin Paul J. , Matthews Dana C. , Appelbaum Frederick R. , Bernstein Irwin D. , Press Oliver W.
Published: May 2003

We performed a multivariable comparison of 125 consecutive patients with follicular lymphoma (FL) treated at our centers with either high-dose radioimmunotherapy (HD-RIT) using 131I-anti-CD20 (n = 27) or conventional high-dose therapy (C-HDT) (n = 98) and autologous hematopoietic stem cell transplantation. The groups were similar, although more patients treated with HD-RIT had an elevated pretransplantation level of lactate dehydrogenase (41% versus 20%, P = .03) and elevated international prognostic score (41% versus 19%, P = .02). Patients treated with HD-RIT received individualized therapeutic doses of 131I-tositumomab (median, 19.7 GBq [531 mCi]) to deliver 17 to 31 Gy (median, 27 Gy) to critical organs. Patients treated with C-HDT received total body irradiation plus chemotherapy (70%) or chemotherapy alone (30%). Patients treated with HD-RIT experienced improved overall survival (OS) (unadjusted hazard ratio [HR] for death = 0.4 [95% confidence interval (95% CI), 0.2-0.9], P = .02; adjusted HR, 0.3, P = .004) and progression-free survival (PFS) (unadjusted HR = .6 [95% C.I., 0.3-1.0], P = .06; adjusted HR, 0.5, P = .03) versus patients treated with C-HDT. The estimated 5-year OS and PFS were 67% and 48%, respectively, for HD-RIT and 53% and 29%, respectively, for C-HDT. One hundred-day treatment-related mortality was 3.7% in the HD-RIT group and 11% in the C-HDT group. The probability of secondary myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) was estimated to be .076 at 8 years in the HD-RIT group and .086 at 7 years in the C-HDT group. HD-RIT may improve outcomes versus C-HDT in patients with relapsed FL. (Blood. 2003;102:2351-2357)
Disease-associated dendritic cells respond to disease-specific antigens through the common heat shock protein receptor
Authors: Stebbing Justin , Gazzard Brian , Portsmouth Simon , Gotch Frances , Kim Louise , Bower Mark , Mandalia Sundhiya , Binder Robert , Srivastava Pramod , Patterson Steve
Published: May 2003

The most abundant intracellular proteins, heat shock proteins (HSPs), serve as molecular chaperones for regulatory and maturation pathways. Diverse families of HSPs have been shown to bind antigenic peptides and to play major roles in innate and adaptive immune responses through the common HSP receptor, CD91. HIV-1+ patients with Kaposi sarcoma (KS) were matched for CD4 count and HIV-1 RNA viral load to HIV-1+ patients without Kaposi sarcoma (and negative for Kaposisarcoma–associated herpesvirus). We then investigated the pathways used by tumor lysates, viral lysates, and viral particles in their activation. In particular, we observed immune responses after HSP depletion using antitumor antibiotics and blockade of the common HSP receptor, CD91. Despite the impaired functional capacity of dendritic cells (DCs) derived from patients with KS, DCs retain the ability to prime the adaptive arm of the immune system through the common HSP receptor, leading to phenotypic activation and stimulation of tetramer-positive CD8+ cytotoxic T cells. We also show that interferon-producing plasmacytoid DCs are selectively depleted in KS-positive compared with matched KS-negative HIV-1–infected patients. Functionally impaired DCs can effectively cross-present immune responses through the common HSP receptor. These results have important implications for the etiopathogenesis of KS and for the development and design of any compounds, including vaccines, derived from cellular lysates.
ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma
Authors: De Paepe Pascale , Baens Mathijs , van Krieken Han , Verhasselt Bruno , Stul Michel , Simons Annet , Poppe Bruce , Laureys Geneviève , Brons Paul , Vandenberghe Peter , Speleman Frank , Praet Marleen , De Wolf-Peeters Chris , Marynen Peter , Wlodarska Iwona
Published: May 2003

We present 3 cases of large B-cell lymphoma (LBCL) with a granular cytoplasmic staining for anaplastic lymphoma kinase (ALK). All of the cases showed striking similarities in morphology and immunohistochemical profile characterized by a massive monomorphic proliferation of CD20-/CD138+ plasmablast-like cells. In one of the cases, initially diagnosed as a null-type anaplastic large cell lymphoma (ALCL), the B-cell phenotype became evident only at recurrence. Fluorescent in situ hybridization (FISH) and molecular studies led to the detection of a CLTC-ALK rearrangement in all 3 cases, without any evidence of full-length ALK receptor expression. The associated t(2;17)(p23;q23) was demonstrated in the karyotype of 2 cases. Although a similar CLTC-ALK aberration was previously identified in ALK-positive T-/null cell ALCL and inflammatory myofibroblastic tumor, its association with ALK-positive LBCL seems to be specific and intriguing. (Blood. 2003;102:2638-2641)
Signaling through JAM-1 and αvβ3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and αvβ3 complex
Authors: Naik Meghna U. , Mousa Shaker A. , Parkos Charles A. , Naik Ulhas P.
Published: May 2003

Growth factor–induced neovascularization has received a great deal of attention because it is fundamental to the growth and metastasis of solid tumors. This multistep process requires extensive signaling through growth factor receptors and integrins. Among the integrins involved in this process, integrin αvβ3 is specific to basic fibroblast growth factor (bFGF)–induced angiogenesis. Here we show that junctional adhesion molecule 1/A (JAM-1/A) and αvβ3 form a complex in the absence of bFGF. JAM-1, which is normally localized at the cell-cell junctions of quiescent endothelial cells, redistributes to the cell surface on bFGF treatment. Blockage of the extracellular domain of JAM-1 inhibits bFGF-induced endothelial cell morphology, proliferation, and angiogenesis. Additionally, mutation in the JAM-1 cytoplasmic domain blocks bFGF-induced mitogen-activated protein (MAP) kinase activation and ablates its ability to induce endothelial cell tube formation, suggesting that signaling through JAM-1 is key to bFGF-induced signaling. Immunoprecipitation analysis suggests that bFGF signaling dissociates the JAM-1/ αvβ3 complex, allowing for signaling through JAM-1 and αvβ3. In addition, blockage of either JAM-1 or αvβ3 inhibits bFGF-induced MAP kinase activation. Thus, our results suggest that signaling through JAM-1 and αvβ3 is necessary for bFGF-induced angiogenesis.