bluuebunny/crossref_metadata_2025 huggingface dataset:
Autologous Epstein-Barr virus (EBV)–specific cytotoxic T cells for the treatment of persistent active EBV infection
Authors: Savoldo Barbara , Huls M. Helen , Liu Zhensheng , Okamura Takayuki , Volk Hans-Dieter , Reinke Petra , Sabat Robert , Babel Nina , Jones James F. , Webster-Cyriaque Jennifer , Gee Adrian P. , Brenner Malcolm K. , Heslop Helen E. , Rooney Cliona M.
Published: November 2002

Chronic active Epstein-Barr virus (CAEBV) infection syndrome is a heterogeneous EBV-related disorder characterized by chronic fatigue, fever, lymphadenopathy, and/or hepatosplenomegaly, associated with abnormal patterns of antibody to EBV. CAEBV can range from disabling mild/moderate forms to rapidly lethal disorders. Even patients with mild/moderate disease frequently suffer adverse effects from long-term anti-inflammatory agents and have a quality of life that progressively deteriorates. It is still unknown why these individuals are unable to produce an effective immune response to control EBV, and no effective treatment is currently available. Since ex vivo–expanded EBV-specific cytotoxic T lymphocytes (EBV-CTLs) can safely restore EBV-specific cellular immune responses in immunodeficient patients, we assessed the possibility that adoptive immunotherapy might also effectively treat CAEBV infection. Following stimulation with irradiated EBV-transformed lymphoblastoid cell lines (LCLs), EBV-CTLs were successfully generated from 8 of 8 patients with the mild/moderate form of CAEBV infection. These CTLs were predominantly CD3+ CD8+ cells and produced specific killing of the autologous LCLs. There were 5 patients with 1- to 12-year histories of disease who were treated with 1 to 4 injections of EBV-CTLs. Following infusion, there was resolution of fatigue and malaise, disappearance of fever, and regression of lymphadenopathy and splenomegaly. The pattern and titers of anti-EBV antibodies also normalized. No toxicity was observed. There were 4 patients who did not show any relapse of disease within 6 to 36 months follow-up; one patient had recurrence of fatigue and myalgia one year after CTL infusion. We suggest that adoptive immunotherapy with autologous EBV-CTLs may represent a safe and feasible alternative treatment for patients affected with mild/moderate CAEBV infection and that this approach should be evaluated in the more severe forms of the disease.
Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety
Authors: Noizat-Pirenne France , Lee Ketty , Pennec Pierre-Yves Le , Simon Philippe , Kazup Philippe , Bachir Dora , Rouzaud Anne-Marie , Roussel Michèle , Juszczak Geneviève , Ménanteau Cècile , Rouger Philippe , Kotb Rami , Cartron Jean-Pierre , Ansart-Pirenne Hélène
Published: November 2002

The molecular backgrounds of variants encountered in Afro-Caribbean black individuals and associated with the production of clinically significant antibodies against high-incidence antigens (anti-RH18, anti-RH34) and against Rhe epitopes were determined. We showed that RH:−18 phenotypes are produced by 3 distinct RHCEalleles: ceEK carrying 48G>C (exon 1), 712A>G, 787A>G, 800T>A (exon 5); ceBI carrying 48G>C (exon 1), 712A>G (exon 5), 818C>T (exon 6), 1132C>G (exon 8); and the already knownceAR allele carrying 48G>C (exon 1), 712A>G, 733C>G, 787A>G, 800T>A (exon 5), and 916A>G (exon 6). The RH:−34 phenotype is produced by the (C)ces haplotype described previously and composed of a hybrid D-CE(3-8)-D gene with 4 extra mutations next to a ces allele (733C>G; exon 5) with an extra mutation in exon 7 (1006G>T). Partial Rhe with risk of immunization against lacking epitopes can be produced by the new ces allele carrying an extra mutation in exon 3 (340C>T) and by the ceMO allele described previously. A population of sickle cell disease patients was screened to estimate the incidence of these rare alleles, with the conclusion that a procedure is required to detect the associated phenotypes in black donors to ensure transfusion safety for patients. We also described a new variant [ces(748)] and variants carrying different altered alleles in nonimmunized patients and for whom the risk of immunization is discussed.
Transplant-related mortality and long-term graft function are significantly influenced by cell dose in patients undergoing allogeneic marrow transplantation
Authors: Dominietto Alida , Lamparelli Teresa , Raiola Anna Maria , Van Lint Maria Teresa , Gualandi Francesca , Berisso Giovanni , Bregante Stefania , di Grazia Carmen , Soracco Monica , Pitto Anna , Frassoni Francesco , Bacigalupo Andrea
Published: November 2002

We have studied the impact of cell dose on short- and long-term graft function and outcome in 905 patients undergoing an unmanipulated allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling (n = 735), a one-antigen mismatched related donor (n = 35), or a matched unrelated donor (n = 135). Median number of nucleated cells infused was 3.4 × 108/kg (25th percentile 2.4 × 108/kg, 75th percentile 5 × 108/kg). Patients were stratified according to cells infused in 3 groups: ≤ 2.4 × 108/kg (n = 247; low dose); >2.4 × 108/kg and ≤ 5 × 108/kg (n = 452; intermediate dose); and >5 × 108/kg (n = 206; high dose). Patients receiving high cell dose had significantly higher platelet counts on days +20, +50, +100, +180, and +365 after BMT (P< .01) and higher white blood cell counts on days +50, +100, and +180 after BMT (P < .01) as compared with other patients. The actuarial 5-year transplant-related mortality (TRM) was 41% versus 36% versus 28% (P = .01); overall survival was 45% versus 51% versus 56% (P = .0008); and disease-free survival was 41% versus 42% versus 48%, respectively, (P = .04) in patients receiving low, intermediate, or high cell dose. The cell dose effect was more pronounced in patients older than 30 years of age, with advanced disease, with chronic myeloid leukemia, and with alternative donors. In multivariate Cox analysis on TRM, cell dose was a significant predictor (P = .002; relative risk 0.6) together with donor type (P = .0001), year of transplantation (P = .0001), conditioning regimen (P = .02), and recipient age (P = .02). In conclusion, transplantation of high marrow cell dose is associated with reduced transplant mortality and improved survival and results in improved graft function both short and long term.
Reovirus therapy of lymphoid malignancies
Authors: Alain Tommy , Hirasawa Kensuke , Pon Kelly J. , Nishikawa Sandra G. , Urbanski Stefan J. , Auer Yvonna , Luider Joanne , Martin Anita , Johnston Randal N. , Janowska-Wieczorek Anna , Lee Patrick W. K. , Kossakowska Anna E.
Published: November 2002

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.
Absent phenotypic expression of X-linked sideroblastic anemia in one of 2 brothers with a novel ALAS2 mutation
Authors: Cazzola Mario , May Alison , Bergamaschi Gaetano , Cerani Paola , Ferrillo Sara , Bishop David F.
Published: November 2002

X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinic acid synthase (ALAS2)gene. Hemizygous males have microcytic anemia and iron overload. A 38-year-old male presented with this phenotype (hemoglobin [Hb] 7.6 g/dL, mean corpuscular volume [MCV] 64 fL, serum ferritin 859 μg/L), and molecular analysis of ALAS2 showed a mutation 1731G>A predicting an Arg560His amino acid change. A 36-year-old brother was hemizygous for this mutation and expressed the mutated ALAS2 mRNA in his reticulocytes, but showed almost no phenotypic expression. All 5 heterozygous females from this family, including the 3 daughters of the nonanemic hemizygous male, showed marginally increased red-cell distribution width (RDW). Although variable penetrance for XLSA in males has been previously described, this is the first report showing that phenotypic expression can be absent in hemizygous males. This observation is relevant to genetic counseling, emphasizing the importance of gene-based diagnosis.
Human T cells resistant to complement lysis by bivalent antibody can be efficiently lysed by dimers of monovalent antibody
Authors: Nielsen Soren U. , Routledge Edward G.
Published: November 2002

We have previously shown that bivalent human γ1 CD3 monoclonal antibody (mAb) is ineffective at mediating lysis of human T cells with human complement. In this paper we have used genetic engineering and sulfur chemistry to prepare 2 types of human γ1 CD3 mAb dimer, with the aim of improving complement lysis activity. The IgG molecules forming the dimers were linked together at their C-termini by stable bismaleimide thioether bridges. The first dimer was composed of 2 bivalent mAb molecules. This dimer proved incapable of lysing human T-cell blasts with human, rabbit, or guinea pig complement. The second dimer consisted of 2 molecules of a monovalent derivative (possessing a single Fab domain) of the bivalent mAb. This dimer was highly lytic with human complement, with a lytic titer 64-fold greater than that of the nondimerized monovalent mAb. The maximum level of lysis of human T-cell blasts achieved with this monovalent mAb dimer was equal to that obtained with the therapeutic antilymphocyte mAb alemtuzumab, but its lytic titer was 4-fold greater. The monovalent mAb dimer was also found to be lytic in the presence of rabbit and guinea pig complement. Dimerization of monovalent antibodies may provide a general strategy for improving the cytolytic activity of other mAbs that are normally unable to induce lysis with complement. The monovalent CD3 mAb dimer may have potential for development as an agent for immunotherapy of T-cell leukemia.
Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria–type cells in bone marrow failure syndrome
Authors: Wang Hongbo , Chuhjo Tatsuya , Yasue Shizuka , Omine Mitsuhiro , Nakao Shinji
Published: November 2002

A minor population of blood cells deficient of glycosylphosphatidylinositol (GPI)–anchored membrane proteins is often detected in patients with aplastic anemia (AA), though the clinical significance of such paroxysmal nocturnal hemoglobinuria (PNH)–type cells remains unclear. To clarify this issue, we studied 164 patients with myelodysplastic syndrome (MDS) for the presence of CD55−CD59− granulocytes and red blood cells using sensitive flow cytometry. Among the different subgroups of MDS, a significant increase (ie, at least 0.003%) of PNH-type cells was detected in 21 of 119 patients with refractory anemia (RA); this frequency (17.6%) of RA patients with increased PNH-type cells (PNH+ patients) was much lower than what we previously reported (52.0%) for AA patients. PNH+ RA patients had distinct clinical features compared with RA patients without increased PNH-type cells (PNH− patients), such as less pronounced morphologic abnormality of blood cells, more severe thrombocytopenia, lower rates of karyotypic abnormality (4.8% vs 32.8%) and of progression to acute leukemia (0% vs 6.2%), higher probability of response to cyclosporine therapy (77.8% vs 0%), and higher incidence of HLA-DR15 (90.5% vs 18.5%). These data indicate that the presence of a minor population of PNH-type cells suggests a benign type of bone marrow failure, probably caused by an immunologic mechanism. To choose an appropriate therapy, peripheral blood should be tested using sensitive flow cytometry for the presence of PNH-type cells in all patients with bone marrow failure before treatment.
The first 3 days of B-cell development in the mouse embryo
Authors: de Andrés Belén , Gonzalo Pilar , Minguet Susana , Martı́nez-Marı́n José A. , Soro Pilar G. , Marcos Miguel Angel R. , Gaspar Marı́a Luisa
Published: November 2002

B-lineage–committed cells are believed to arise in the liver of mouse embryos at 14 days after coitus (dpc). However, pre-B-specific gene transcripts and DJH gene rearrangements have been detected in earlier, midgestation embryos. We describe here a population of c-kit+AA4.1+CD19+Pax5+cells present in the aorta-gonad-mesonephros (AGM) area and in the livers of 11-dpc mouse embryos. In contrast to multipotent c-kit+AA4.1+CD19−hematopoietic stem cells (HSCs), these c-kit+AA4.1+CD19+ progenitors differentiated only to B-lineage cells in vitro. We propose that mouse embryonic B lymphopoiesis starts earlier than previously thought, at 10 to 11 dpc, both in liver and extra-liver hematopoietic sites. The B-cell differentiation program is not delayed with respect to the emerging lymphohematopoiesis events in the midgestation mouse embryo (8-9 dpc).
Hepatitic variant of graft-versus-host disease after donor lymphocyte infusion
Authors: Akpek Görgün , Boitnott John K. , Lee Linda A. , Hallick Jason P. , Torbenson Michael , Jacobsohn David A. , Arai Sally , Anders Viki , Vogelsang Georgia B.
Published: November 2002

Graft-versus-host disease (GVHD) of the liver is characterized by bile duct damage and portal lymphocytic infiltrate. We report acute hepatitislike presentation of GVHD after donor lymphocyte infusion (DLI). Between April 1998 and September 2001, 73 patients received 94 DLI treatments. Liver GVHD developed after DLI in 22 (30%) patients whose median age was 43 years (range, 21 to 61 years). Onset of liver dysfunction was at 35 days (range, 11 to 406 days) after DLI. Fifteen patients underwent liver biopsy, and the diagnosis of liver GVHD was confirmed in 13 (87%) patients. After viral hepatitis and recent drug exposure were excluded, 11 (50%) patients were given a diagnosis of a hepatitic variant of GVHD based on histologic evidence of lobular hepatitis (n = 5), elevation of maximum serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level more than 10 times the upper normal limit (n = 9), or both. There was a significant difference in maximum ALT (P = .002) and AST (P = .01) level between the hepatitic-variant and classical GVHD groups. GVHD progressed in 14 (64%) patients, and 10 patients died after a median follow-up of 221 days (range, 31-1284 days). These observations suggest that GVHD that occurs after DLI may have distinct clinical features. Hepatitic-variant GVHD should be considered in the differential diagnosis in DLI recipients with unexplained hepatitis.
Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations
Authors: Zheng Rui , Friedman Alan D. , Small Donald
Published: November 2002

Internal tandem duplication (ITD) mutations of the juxtamembrane domain–coding sequence of the FLT3 gene are found in up to 34% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITDs result in constitutive activation of the tyrosine kinase domain and transform growth factor–dependent cell lines. FLT3 activation leads to antiapoptotic and proliferative signals, but little is known about the impact of FLT3/ITDs on differentiation. This study was designed to investigate the effect of FLT3/ITD expression on the differentiation of the 32Dcl3 (32D) myeloblastic cell line to neutrophils in response to granulocyte colony-stimulating factor (G-CSF). Expression of FLT3/ITD completely blocked morphologic differentiation and induction of myeloperoxidase (MPO), lysozyme, and CCAAT/enhancer-binding protein ε (C/EBPε) in response to G-CSF. Wild-type FLT3 and vector-transfected 32D cells were able to differentiate, although the maturation of FLT3-transfected cells was delayed by FLT3 ligand (FL) stimulation. CEP-701, a potent FLT3 tyrosine kinase inhibitor, overcame the morphologic block in differentiation caused by FLT3/ITD expression and allowed G-CSF induction of myeloid maturation markers. These findings suggest that blocking differentiation may be one of the mechanisms by which FLT3/ITDs contribute to leukemogenesis. CEP-701 and other FLT3 inhibitors may be useful for overcoming the block to differentiation (as well as the block to apoptosis) in the leukemic cells of patients with AML.
Aging and obesity augment the stress-induced expression of tissue factor gene in the mouse
Authors: Yamamoto Koji , Shimokawa Takayoshi , Yi Hong , Isobe Ken-ichi , Kojima Tetsuhito , Loskutoff David J. , Saito Hidehiko
Published: November 2002

Hypercoagulability and thrombotic tendency are frequently induced by a variety of stressors. Clinically, aged subjects and obese patients are more susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We investigated the expression of a procoagulant gene, tissue factor (TF), in a mouse model of restraint stress. Twenty hours of restraint stress to mice caused a substantial induction of TF mRNA in several tissues. Importantly, the magnitude of induction of TF mRNA by restraint stress was larger in aged mice compared with young mice. In situ hybridization analysis of the stressed aged mice revealed that strong signals for TF mRNA were localized to renal epithelial cells, smooth muscle cells, adventitial cells, and adipocytes but not to vascular endothelial cells. These observations suggest that restraint stress induces the TF expression in a tissue-specific and cell type–specific manner. Genetically obese mice were also hyperresponsive to restraint stress in the induction of TF gene, especially in their livers and adipose tissues. Stress-induced microthrombi formation was pronounced in renal glomeruli and within the vasculature in adipose tissues of aged mice. Tumor necrosis factor-α (TNF-α) antigen in plasma was elevated by stress in aged mice and obese mice, and pretreatment of mice with anti–TNF-α antibody partially attenuated the stress-mediated induction of TF gene in adipose tissues in these mice. These results suggest that the induction of TF gene may increase the risk of stress-associated thrombosis in older and obese subjects and that TNF-α may be involved.
Differential regulation of the von Willebrand factor and Flt-1 promoters in the endothelium of hypoxanthine phosphoribosyltransferase–targeted mice
Authors: Minami Takashi , Donovan Diana J. , Tsai Jo C. , Rosenberg Robert D. , Aird William C.
Published: November 2002

An important limitation of standard transgenic assays is that multiple copies of the transgene are inserted randomly into the mouse genome, resulting in line-to-line variation in expression. One way to control for these variables is to target a single copy of the transgene to a defined locus of the mouse genome by homologous recombination. In the present study, we have used such an approach to target the promoters of 2 different genes, namely von Willebrand factor (VWF) and Flt-1, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus. Consistent with previous findings in standard transgenic animals, we report that the VWF promoter contains information for expression in a subset of endothelial cells in the heart, skeletal muscle, and brain. In contrast, the Flt-1 promoter directs expression in all vascular beds except for the liver. The Flt-1 transgene was active in the endothelium of tumor xenografts, whereas the VWF promoter was not. Under in vitro conditions, conditioned medium from tumor cells resulted in a significant up-regulation of Flt-1 mRNA and promoter activity, but no change in VWF levels. Taken together, these results suggest that (1) Hprt locus targeting is a valuable tool for studying vascular bed–specific gene regulation, (2) the VWF and Flt-1 promoters are regulated by distinct transcriptional mechanisms in the intact endothelium, and (3) tumor angiogenesis results in the differential activation of endothelial cell–specific promoters.
Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation
Authors: Rocha Vanderson , Franco Rendrik F. , Porcher Raphaël , Bittencourt Henrique , Silva Wilson A. , Latouche Aurelien , Devergie Agnès , Espérou Hélène , Ribaud Patricia , Socié Gérard , Zago Marco Antonio , Gluckman Eliane
Published: November 2002

We made the hypothesis that donor and recipient gene polymorphisms that drive the host response to microorganisms could be associated with infections after bone marrow transplantation (BMT). HLA-identical BMT was performed for patients with acute (n = 39) or chronic leukemia (n = 68). Genotyping was performed in 107 D/R DNA pairs for gene polymorphisms of cytokines (tumor necrosis factor-α [TNF-α] and TNF-β, interleukin-1 receptor antagonist [IL-1Ra], IL-6, and IL-10), adhesion molecules (CD31 and CD54), Fcγreceptors (FcγRIIa, IIIa, IIIb), mannose-binding lectin (MBL), and myeloperoxidase (MPO). First infection (overall) and first episodes of bacterial, viral, or invasive fungal infection were studied retrospectively for 180 days after BMT. Univariate and multivariate analyses, using death as a competing event, were performed to study risk factors. In multivariate analysis, first overall infections were increased in patients with the FcγRIIa R-131 genotype (hazard ratio [HR] = 1.92; P = .04), and severe bacterial infections were increased when the MPO donor genotype was AG or AA (HR = 2.16; P = .03). Viral and invasive fungal infections were not influenced by any genetic factor studied. Interestingly, we also found that (1) time to neutrophil recovery was shorter when donors were FcγRIIIb HNA-1a/HNA-1b (HR = 1.77; P = .002); (2) donor IL-1Ra (absence of IL-1RN*2) increased the risk for acute graft-versus-host disease (GVHD) (II-IV) (HR = 2.17; P = .017); and (3) recipient IL-10 (GG) and IL-1Ra genotypes increased the risk for chronic GVHD (P = .03 and P = .03, respectively). Finally, 180-day transplantation-related mortality rates were increased when donors were FcγRIIIb HNA-1a/HNA-1a or HNA-1b/HNA-1b (HR = 2.57; P = .05) and donor MPO genotype was AA (HR = 5.14; P = .004). In conclusion, donor and recipient gene polymorphisms are informative genetic risk factors for selecting donor/recipient pairs and could help in the understanding of mechanisms involved in host defenses of BM transplant recipients.
Comparative analysis of murine marrow–derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses
Authors: Weigel Brenda J. , Nath Narender , Taylor Patricia A. , Panoskaltsis-Mortari Angela , Chen Wei , Krieg Arthur M. , Brasel Kenneth , Blazar Bruce R.
Published: November 2002

Bone marrow (BM)–derived dendritic cells (DCs) cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) have been used to generate antitumor immune responses. The cytokine Flt3 ligand (Flt3L) also has been shown to generate BM DCs. We sought to determine if DCs generated by using Flt3L then matured with lipopolysaccharide (LPS) could lead to DCs with in vivo anti–acute myelogenous leukemia (anti-AML) activity. LPS and tumor necrosis factor α (TNF-α) are effective agents for maturing DCs; however, they have potential in vivo toxicities. Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpGs) are considered relatively nontoxic, potent activators of DC function and maturation in vitro and in vivo. We investigated whether CpGs would be comparable to TNF-α or LPS for the maturation of GM-CSF/IL-4–generated DCs. DCs cultured with GM-CSF/IL-4 and matured with TNF-α, LPS, or CpG produced a greater allogeneic T-cell response compared with Flt3L/LPS-generated DCs. All 4 distinct DC types were pulsed with AML-lysate and administered before tumor challenge produced an increase in the total number of splenic anti-AML–specific cytotoxic T-lymphocyte precursors and led to significantly (P ≤ .0001) improved survival compared with nonvaccinated controls. GM-CSF/IL-4/LPS was superior to Flt3L/LPS for generating anti-AML effects in vivo. Whereas TNF-α was comparable to LPS in conferring on GM-CSF/IL-4 DCs anti-AML effects in vivo, CpGs were superior to LPS. These data have important clinical implications and are the first to show that Flt3L-generated DCs can provide antitumor protection and that nontoxic agents such as CpGs and Flt3L may be useful in the clinical development of DC vaccines.
GM-CSF, via PU.1, regulates alveolar macrophage FcγR-mediated phagocytosis and the IL-18/IFN-γ–mediated molecular connection between innate and adaptive immunity in the lung
Authors: Berclaz Pierre-Yves , Shibata Yoko , Whitsett Jeffrey A. , Trapnell Bruce C.
Published: November 2002

Severely impaired pulmonary microbial clearance was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)–deficient mice. To determine mechanisms by which GM-CSF mediates lung host defense, FcγR-mediated phagocytosis (opsonophagocytosis) by alveolar macrophages (AMs) was assessed in GM-CSF–sufficient (GM+/+) and –deficient (GM−/−) mice and in GM−/− mice expressing GM-CSF only in the lungs from a surfactant protein C (SPC) promoter (SPC-GM+/+/GM−/−). Opsonophagocytosis by GM−/− AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Defective opsonophagocytosis by GM−/− AMs was associated with decreased FcγR expression. Because interferon-γ (IFN-γ) augments macrophage FcγR levels, the role of GM-CSF/PU.1 in the regulation of AM FcγR expression by IFN-γ was assessed during adenoviral lung infection. Adenoviral infection stimulated IFN-γ production and augmented FcγR levels on AMs in GM-CSF–expressing but not GM−/− mice. However, IFN-γ exposure ex vivo stimulated FcγR expression on GM−/− AMs. Because interleukin-18 (IL-18) and IL-12 stimulate IFN-γ production during adenoviral infection, their role in GM-CSF/PU.1 regulation of IFN-γ–augmented FcγR expression on AMs was assessed. Adenoviral infection stimulated IL-18 and IL-12 production in GM-CSF–expressing mice, but both were markedly reduced or absent in GM−/−mice. IL-18 expression by GM−/− AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Pulmonary administration of IL-18 in GM−/− mice stimulated IFN-γ production and restored FcγR expression on AMs. These results show that GM-CSF, via PU.1, regulates constitutive AM FcγR expression and opsonophagocytosis and is required for the IFN-γ–dependent regulation of AM FcγR expression, enabling AMs to release IL-18/IL-12 during lung infection.
CD8αα memory effector T cells descend directly from clonally expanded CD8α+βhigh TCRαβ T cells in vivo
Authors: Konno Akihiro , Okada Kanae , Mizuno Kazunori , Nishida Mika , Nagaoki Shuya , Toma Tomoko , Uehara Takahiro , Ohta Kazuhide , Kasahara Yoshihito , Seki Hidetoshi , Yachie Akihiro , Koizumi Shoichi
Published: November 2002

Whereas most peripheral CD8+ αβ T cells highly express CD8αβ heterodimer in healthy individuals, there is an increase of CD8α+βlow or CD8αα αβ T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8α+βhigh cells belong to different ontogenic lineages or whether a fraction of CD8α+βhigh cells have down-regulated CD8β chain. Here we assessed clonality of CD8αα and CD8α+βlow αβ T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8α+βlow cells are exclusively composed of effector memory cells. CD8αα cells comprise effector memory cells and terminally differentiated CD45RO−CCR7−memory cells. T-cell receptor (TCR) Vβ complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8α+βhigh cells and oligoclonality of CD8α+βlow and CD8αα cells. Importantly, some expanded clones within CD8αα cells were also identified within CD8α+βhigh and CD8α+βlow subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8β expression. These results indicated that some specific CD8α+βhigh αβ T cells expand clonally, differentiate, and simultaneously down-regulate CD8β chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8αα αβ T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.
Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality
Authors: Kröger Nicolaus , Sayer Herbert Gottfried , Schwerdtfeger Rainer , Kiehl Michael , Nagler Arnon , Renges Helmut , Zabelina Tatjana , Fehse Boris , Ayuk Francis , Wittkowsky Georg , Schmitz Norbert , Zander Axel Rolf
Published: November 2002

We investigated the feasibility of unrelated stem cell transplantation in 21 patients with advanced stage II/III multiple myeloma after a reduced-intensity conditioning regimen consisting of fludarabine (150 mg/m2), melphalan (100-140 mg/m2), and antithymocyte globulin (ATG; 10 mg/kg on 3 days). The median patient age was 50 years (range, 32-61 years). All patients had received at least one prior autologous transplantation, in 9 cases as part of an autologous-allogeneic tandem protocol. No graft failure was observed. At day 40 complete donor chimerism was detected in all patients. Grade II to IV acute graft-versus-host disease (GVHD) was seen in 8 patients (38%), and severe grade III/IV GVHD was observed in 4 patients (19%). Six patients (37%) developed chronic GVHD, but only 2 patients (12%) experienced extensive chronic GVHD. The estimated probability of nonrelapse mortality at day 100 was 10% and at 1 year was 26%. After allografting, 40% of the patients achieved a complete remission, and 50% achieved a partial remission, resulting in an overall response rate of 90%. After a median follow-up of 13 months, the 2-year estimated overall and progression-free survival rates are 74% (95% CI, 54%-94%) and 53% (95% CI, 29%-87%), respectively. A shorter progression-free survival was seen in patients who already experienced relapse to prior autograft (26% versus 86%, P = .04). Dose-reduced conditioning with pretransplantation ATG followed by unrelated stem cell transplantation provides durable engraftment and donor chimerism, reduces substantially the risk of transplant-related organ toxicity, and induces high remission rates.
Oncogenic interaction between BCR-ABL andNUP98-HOXA9 demonstrated by the use of an in vitro purging culture system
Authors: Mayotte Nadine , Roy Denis-Claude , Yao Jing , Kroon Evert , Sauvageau Guy
Published: November 2002

Chronic myelogenous leukemia (CML) is a clonal stem cell disease caused by the BCR-ABL oncoprotein and is characterized, in its early phase, by excessive accumulation of mature myeloid cells, which eventually leads to acute leukemia. The genetic events involved in CML's progression to acute leukemia remain largely unknown. Recent studies have detected the presence of theNUP98-HOXA9 fusion oncogene in acute leukemia derived from CML patients, which suggests that these 2 oncoproteins may interact and influence CML disease progression. Using in vitro purging of BCR-ABL–transduced mouse bone marrow cells, we can now report that recipients of bone marrow cells engineered to coexpressBCR-ABL with NUP98-HOXA9 develop acute leukemia within 7 to 10 days after transplantation. However, no disease is detected for more than 2 months in mice receiving bone marrow cells expressing either BCR-ABL orNUP98-HOXA9. We also provide evidence of high levels ofHOXA9 expressed in leukemic blasts from acute-phase CML patients and that it interacts significantly on a genetic level withBCR-ABL in our in vivo CML model. Together, these studies support a causative, as opposed to a consequential, role forNUP98-HOXA9 (and possibly HOXA9) in CML disease progression.
Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction
Authors: Spaggiari Grazia Maria , Contini Paola , Dondero Alessandra , Carosio Roberta , Puppo Francesco , Indiveri Francesco , Zocchi Maria Raffaella , Poggi Alessandro
Published: November 2002

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I–Cw4 or –Cw3 from untransfected or –Cw4 or –Cw3 alleles transfected HLA-A−, B−, C−, G−, E+, F+ 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR– or anti-KIR–specific monoclonal antibodies (mAbs). After sHLA-I–activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I–mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor–mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H+–adenosine triphosphatase (H+-ATPase) of granules, inhibited activating receptor–induced NK cell cytolysis, suggesting that activating receptor–mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-γ (IFN-γ) was detectable in culture supernatant of activating receptor+ NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor–mediated IFN-γ production. This suggests that activating receptor–induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-γ, an antiviral cytokine able to amplify immune responses.
Protein kinase C mediates mutant N-Ras–induced developmental abnormalities in normal human erythroid cells
Authors: Darley Richard L. , Pearn Lorna , Omidvar Nader , Sweeney Marion , Fisher Janet , Phillips Sarah , Hoy Terry , Burnett Alan K.
Published: November 2002

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]–independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34+ erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.
Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells
Authors: Horn Peter A. , Topp Max S. , Morris Julia C. , Riddell Stanley R. , Kiem Hans-Peter
Published: November 2002

Vector-containing medium harvested from murine packaging cell lines has been shown to contain factors that can negatively influence the transduction and maintenance of hematopoietic stem cells. Thus, we generated a human packaging cell line with a gibbon ape leukemia virus pseudotype (Phoenix-GALV), and we evaluated vectors produced by Phoenix-GALV for their ability to transduce hematopoietic progenitor/stem cells. In 3 baboons, we used a competitive repopulation assay to directly compare GALV-pseudotype retrovirus vectors produced by either Phoenix-GALV or by the NIH 3T3–derived packaging cell line, PG13. In 3 additional baboons we compared Phoenix-GALV–derived vectors to more recently developed lentiviral vectors. Gene transfer efficiency into hematopoietic repopulating cells was assessed by evaluating the number of genetically modified peripheral blood and marrow cells using flow cytometry and real-time polymerase chain reaction. Transduction efficiency of hematopoietic repopulating cells was significantly higher using the Phoenix-GALV–derived vector as compared with the PG13-derived vectors or lentiviral vectors, with stable transduction levels up to 25%. We followed 2 animals for more than one year. Flow cytometric analysis of hematopoietic subpopulations in these animals revealed transgene expression in CD13+ granulocytes, CD20+ B lymphocytes, CD3+ T lymphocytes, CD61+ platelets, as well as red blood cells, indicating multilineage engraftment of cells transduced by Phoenix-GALV–pseudotype vectors. In addition, transduction of human CD34+ cells was significantly more efficient than transduction of baboon CD34+ cells, suggesting that Phoenix-GALV–derived oncoretroviral vectors may be even more efficient in human stem cell gene therapy applications.
ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions
Authors: Dong Jing-fei , Moake Joel L. , Nolasco Leticia , Bernardo Aubrey , Arceneaux Wendy , Shrimpton Corie N. , Schade Alicia J. , McIntire Larry V. , Fujikawa Kazuo , López José A.
Published: November 2002

Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibα, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm2) and arterial (20 and 50 dyne/cm2) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.
Serum insulinlike growth factor is not elevated in patients with multiple myeloma but is still a prognostic factor
Authors: Standal Therese , Borset Magne , Lenhoff Stig , Wisloff Finn , Stordal Berit , Sundan Anders , Waage Anders , Seidel Carina
Published: November 2002

Insulinlike growth factor 1 (IGF-1) has growth-promoting effects on myeloma cells in vitro as well as in vivo. In this study, we measured the concentration of IGF-1 and its major binding protein, IGF- binding protein 3 (IGFBP-3), in serum from 127 patients with multiple myeloma. Serum had been drawn at the time of diagnosis, before treatment with high-dose melphalan. IGFBP-3 in myeloma patients (1.6 ± 0.73 μg/mL; mean ± SD) was significantly decreased compared to healthy age- and sex-matched controls (2.2 ± 0.42 μg/mL). However, IGFBP-3 had no prognostic value in this study. The mean IGF-1 level did not differ between myeloma patients (17.8 ± 7.7 nM) and controls (17.3 ± 5.6 nM). Nevertheless, IGF-1 was a strong indicator of prognosis. After 80 months of follow-up, myeloma patients with low levels (< 13 nM) of serum IGF-1 had not reached median survival. In the patient group with IGF-1 levels above 13 nM, median survival was 62 months (P = .006). These findings support the hypothesis of a role for IGF-1 in myeloma disease progression.
Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells
Authors: Bradley Heath L. , Hawley Teresa S. , Bunting Kevin D.
Published: November 2002

Secreted growth factors are integral components of the bone marrow (BM) niche and can regulate survival, proliferation, and differentiation of committed hematopoietic stem cells (HSCs). However, downstream genes activated in HSCs by early-acting cytokines are not well characterized. To better define intracellular cytokine signaling in HSC function, we have analyzed mice lacking expression of both signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab−/−). These studies specifically avoided possible autoimmune and/or splenomegaly disease-mediated indirect effects on HSC function by using 2 independent approaches: (1) by crossing onto the C57Bl/6 RAG2−/− background, and (2) by generation of wild-type chimeric mice reconstituted with transplanted STAT5ab−/− BM cells. These experiments demonstrated that STAT5-deficient HSCs have cell autonomous defects in competitive long-term repopulating activity. Furthermore, in the chimeric mice, injected wild-type BM cells showed a progressive multilineage competitive repopulating advantage in vivo, demonstrating that steady-state hematopoiesis was also highly STAT5-dependent. Consistent with the in vivo repopulating deficiency, when Sca-1+c-kit+lin− (KLS) cells were isolated and stimulated with growth factors in vitro, up to a 13-fold reduced expansion of total nucleated cells was observed in response to cocktails containing interleukin 3 (IL-3), IL-6, stem cell factor (SCF), Flt3 ligand, and thrombopoietin. Notably, a 10-fold reduction in expansion was observed with IL-3 and SCF. However, STAT5 activation was not required for regeneration of the KLS pool in vivo following transplant or for secondary repopulating ability. These studies support a major role for STAT5 activation as a cellular determinant of cytokine-mediated HSC repopulating potential but not self-renewal capacity.
Transcriptional activation of endoglin and transforming growth factor-β signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury
Authors: Botella Luisa M. , Sánchez-Elsner Tilman , Sanz-Rodriguez Francisco , Kojima Soichi , Shimada Jun , Guerrero-Esteo Mercedes , Cooreman Michael P. , Ratziu Vlad , Langa Carmen , Vary Calvin P. H. , Ramı́rez Jose R. , Friedman Scott , Bernabéu Carmelo
Published: November 2002

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-β (TGF-β) signaling receptors to bind TGF-β family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-β, TGF-β signaling receptors, and TGF-β–stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglinpromoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated theendoglin promoter and those of collagen α1(I), urokinase-type plasminogen activator, TGF-β1, and TGF-β receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-β signaling complex in vascular repair.
Hodgkin and Reed-Sternberg cell–associated autoantigen CLIP-170/restin is a marker for dendritic cells and is involved in the trafficking of macropinosomes to the cytoskeleton, supporting a function-based concept of Hodgkin and Reed-Sternberg cells
Authors: Sahin Ugur , Neumann Frank , Türeci Özlem , Schmits Rudolf , Perez Franck , Pfreundschuh Michael
Published: November 2002

Little is known about the distribution in normal cells of CLIP-170, a linkage mediator between endocytic vesicles and microtubules, and restin, a splice variant encoded by the same gene and marker for Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin disease. Although only trace amounts of CLIP-170/restin are present in peripheral blood mononuclear cell subpopulations, monocyte-derived dendritic cells (DCs) and interleukin-4 (IL-4) + CD40L-activated B cells express high levels of CLIP-170/restin. CLIP-170/restin colocalizes preferentially with membranes of intermediate macropinocytic vesicles, suggesting a new function of CLIP-170/restin in the trafficking of macropinosomes to the cytoskeleton, which is a crucial step in antigen presentation. The strong expression of CLIP-170/restin in HRS cells, DCs, and activated B cells underscores their functional similarities supporting a function-based concept of HRS cells as professional antigen-presenting cells.