bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation
Authors: Storek Jan , Dawson Monja A. , Storer Barry , Stevens-Ayers Terry , Maloney David G. , Marr Kieren A. , Witherspoon Robert P. , Bensinger William , Flowers Mary E. D. , Martin Paul , Storb Rainer , Appelbaum Frederick R. , Boeckh Michael
Published: July 2002

Allogeneic peripheral blood stem cell grafts contain about 10 times more T and B cells than marrow grafts. Because these cells may survive in transplant recipients for a long time, recipients of blood stem cells may be less immunocompromised than recipients of marrow. Immune reconstitution was studied in 115 patients randomly assigned to receive either allogeneic marrow or filgrastim-mobilized blood stem cell transplantation. Between day 30 and 365 after transplantation, counts of most lymphocyte subsets were higher in the blood stem cell recipients. The difference was most striking for CD4 T cells (about 4-fold higher counts for CD45RAhigh CD4 T cells and about 2-fold higher counts for CD45RAlow/−CD4 T cells;P < .05). On assessment using phytohemagglutinin and herpesvirus antigen-stimulated proliferation, T cells in the 2 groups of patients appeared equally functional. Median serum IgG levels were similar in the 2 groups. The rate of definite infections after engraftment was 1.7-fold higher in marrow recipients (P = .001). The rate of severe (inpatient treatment required) definite infections after engraftment was 2.4-fold higher in marrow recipients (P = .002). The difference in the rates of definite infections was greatest for fungal infections, intermediate for bacterial infections, and lowest for viral infections. Death associated with a fungal or bacterial infection occurred between day 30 and day 365 after transplantation in 9 marrow recipients and no blood stem cell recipients (P = .008). In conclusion, blood stem cell recipients have higher lymphocyte-subset counts and this appears to result in fewer infections.
Cardiac involvement in thalassemia intermedia: a multicenter study
Authors: Aessopos Athanasios , Farmakis Dimitrios , Karagiorga Markisia , Voskaridou Ersi , Loutradi Aphrodite , Hatziliami Antonia , Joussef Jacqueline , Rombos John , Loukopoulos Dimitris
Published: July 2002

Cardiac complications in 110 patients (mean age, 32.5 ± 11.4 years) with thalassemia intermedia (TI) were studied. Sixty-seven (60.9%) of them had not been transfused or were minimally transfused (group A). The rest had started transfusions after the age of 5 years (mean, 15.1 ± 10.1 years), initially on demand and later more frequently (group B). Overall mean hemoglobin and ferritin levels were 9.1 ± 1.1 g/dL and 1657 ± 1477 ng/mL, respectively. Seventy-six healthy controls were also studied. The investigation included thorough history taking, clinical examination, electrocardiography, chest radiograph, and full resting echocardiography. Of 110 patients, 6 (5.4%) had congestive heart failure (CHF), and 9 (8.1%) had a history of acute pericarditis. Echocardiography showed pericardial thickening, with or without effusion, in 34.5% of the patients. Valvular involvement included leaflet thickening (48.1%), endocardial calcification (20.9%), and left-sided valve regurgitation (aortic, 15.4%; mitral, 47.2%). All patients had normal left ventricular contractility (fractional shortening, 0.43 ± 0.05), and high cardiac output (CO; 9.34 ± 2.28 L/min). Pulmonary hypertension (PHT), defined as Doppler peak systolic tricuspid gradient greater than 30 mm Hg, developed in 65 patients (59.1%). PHT correlated positively with age and CO and did not differ significantly between groups. Cardiac catheterization in the 6 patients with CHF revealed severe PHT, increased pulmonary resistance (PVR), and normal capillary wedge pressure. It was concluded that in patients with TI, the heart is primarily affected by PHT, which is the leading cause of CHF. High CO resulting from chronic tissue hypoxia and increased PVR are the main contributing factors. Doppler tricuspid gradient measurement should be considered, in addition to other factors, when determining the value of transfusion therapy for patients with TI.
Signal transducer and activator of transcription 3 activation is required for Asp816 mutant c-Kit–mediated cytokine-independent survival and proliferation in human leukemia cells
Authors: Ning Zhi-Qiang , Li Jin , Arceci Robert J.
Published: July 2002

Activating mutations of c-kit at codon 816 (Asp816) have been implicated in a variety of malignancies, including acute myeloid leukemia (AML). The mutant c-Kit receptor confers cytokine-independent survival of leukemia cells and induces tumorigenicity. Changes in the signal transduction pathways responsible for Asp816 mutant c-Kit–mediated biologic effects are largely undefined. The results of this study show that Asp816 mutant c-Kit induces constitutive activation of signal transducer and activator of transcription 3 (STAT3) and STAT1, and up-regulates STAT3 downstream targets, Bcl-xL and c-myc. The phosphatidylinositol-3-kinase (PI-3K)/Akt pathway, but not the Ras-mediated mitogen-activated protein (MAP) kinase pathway, is also constitutively activated by Asp816 mutant c-Kit. Suppression of STAT3 activation by a dominant negative molecule in MO7e leukemia cells transduced with mutant c-kit inhibits stem cell factor (SCF)-independent survival and proliferation, accompanied by the down-regulation of Bcl-xL and c-myc. However, activated STAT3 does not appear to be the sole mediator that is responsible for the phenotypic changes induced by Asp816 mutant c-Kit, because expression of constitutively activated STAT3 in MO7e cells does not completely reconstitute cytokine independence. Activation of other signaling components by mutant c-Kit, such as those in the PI-3K/Akt pathway, is demonstrated and may also be needed for the mutant c-Kit–mediated biologic effects. The investigation of altered signal transduction pathways and the resulting functional consequences mediated by Asp816 mutant c-Kit should provide important information for the characterization of subsets of leukemia and potential molecular pathways for therapeutic targeting.
The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity
Authors: Lieby Patricia , Soley Anne , Levallois Honey , Hugel Benedicte , Freyssinet Jean-Marie , Cerutti Martine , Pasquali Jean-Louis , Martin Thierry
Published: July 2002

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins.
Allogeneic chimerism with low-dose irradiation, antigen presensitization, and costimulator blockade in H-2 mismatched mice
Authors: Quesenberry Peter J. , Zhong Suju , Wang Han , Stewart Marc
Published: July 2002

We have previously shown that the keys to high-level nontoxic chimerism in syngeneic models are stem cell toxic, nonmyelotoxic host treatment as provided by 100-cGy whole-body irradiation and relatively high levels of marrow stem cells. This approach was unsuccessful in H-2 mismatched B6.SJL to BALB/c marrow transplants, but with tolerization, stable multilineage chimerism was obtained. Ten million B6.SJL spleen cells were infused intravenously into BALB/c hosts on day −10 and (MR-1) anti-CD40 ligand monoclonal antibody (mAb) injected intraperitoneally at varying levels on days −10, −7, −3, 0, and +3 and the BALB/c mice irradiated (100 cGy) and infused with 40 million B6.SJL/H-2 mismatched marrow cells on day 0. Stable multilineage chimerism at levels between 30% to 40% was achieved in the great majority of mice at 1.6 mg anti-CD40 ligand mAb per injection out to 64 weeks after transplantation, without graft-versus-host disease. The transplanted mice were also tolerant of donor B6.SJL, but not third-party CBA/J skin grafts at 8 to 9 and 39 to 43 weeks after marrow transplantation. These data provide a unique model for obtaining stable partial chimerism in H-2 mismatched mice, which can be applied to various clinical diseases of man such as sickle cell anemia, thalassemia, and autoimmune disorders.
Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation
Authors: Nishibe Toshiya , Parry Graham , Ishida Atsushi , Aziz Salim , Murray Jacqueline , Patel Yatin , Rahman Salman , Strand Kurt , Saito Keiko , Saito Yuji , Hammond William P. , Savidge Geoffrey F. , Mackman Nigel , Wijelath Errol S.
Published: July 2002

Tissue factor (TF), a transmembrane glycoprotein, initiates the extrinsic coagulation cascade. TF is known to play a major role in mediating thrombosis and thrombotic episodes associated with the progression of atherosclerosis. Macrophages at inflammatory sites, such as atherosclerotic lesions, release numerous cytokines that are capable of modulating TF expression. This study examined the role of oncostatin M (OSM), a macrophage/ T-lymphocyte–restricted cytokine, in the expression of TF in vascular smooth muscle cells (SMCs). It is reported here that OSM stimulated a biphasic and sustained pattern of TF messenger RNA (mRNA). The effect of OSM on TF mRNA expression was regulated at the transcriptional level as determined by nuclear run-offs and transient transfection of a TF promoter-reporter gene construct. OSM-induced TF expression was regulated primarily by the transcription factor NF-κB. Activation of NF-κB by OSM did not require IκB-α degradation. Inhibition of MEK activity by U0126 prevented OSM-induced TF expression by suppressing NF-κB DNA binding activity as determined by gel-shift analysis. Further, inhibition of Erk-1/2 protein by antisense treatment resulted in suppression of TF mRNA expression, indicating a role for Erk-1/2 in modulating NF-κB DNA binding activity. These studies suggest that the induced expression of TF by OSM is primarily through the activation of NF-κB and that activation of NF-κB is regulated in part by the MEK/Erk-1/2 signal transduction pathway. This study indicates that OSM may play a key role in promoting TF expression in SMCs within atherosclerotic lesions.
Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes
Authors: Buechler Christa , Ullrich Heidrun , Ritter Mirko , Porsch-Oezcueruemez Mustafa , Lackner Karl J. , Barlage Stefan , Friedrich Sven Olaf , Kostner Gert M. , Schmitz Gerd
Published: July 2002

Elevated plasma lipoprotein (a) (Lp[a]) and cardiac events show a modest but significant association in various clinical studies. However, the influence of high Lp(a) on the gene expression in blood monocytes as a major cell involved in atherogenesis is poorly described. To identify genes influenced by elevated serum Lp(a), the gene expression was analyzed on a complementary DNA microarray comparing monocytes from a patient with isolated Lp(a) hyperlipidemia and coronary heart disease with monocytes from a healthy blood donor with low Lp(a). By using this approach, numerous genes were found differentially expressed in patient-versus-control monocytes. Verification of these candidates by Northern blot analysis or semiquantitative polymerase chain reaction in monocytes from additional patients with Lp(a) hyperlipidemia and healthy blood donors with elevated Lp(a) confirmed a significant induction of plasminogen activator inhibitor type 2 (PAI-2) messenger RNA (mRNA) in monocytes from male, but not from female, individuals with high Lp(a), indicating that this observation is gender specific. This led also to increased intracellular and secreted PAI-2 protein in monocytes from male probands with Lp(a) hyperlipidemia. Plasminogen activator inhibitor type 1 (PAI-1) mRNA was found suppressed only in the patients′ monocytes and not in healthy probands with high Lp(a) levels. Purified Lp(a) induced PAI-2 mRNA and protein and reduced PAI-1 expression in monocytes isolated from various controls. The finding that PAI-2 is elevated in monocytes from male patients with isolated Lp(a) hyperlipidemia and male healthy probands with high Lp(a) and that purified Lp(a) up-regulates PAI-2 in control monocytes in vitro indicate a direct, but gender-specific, effect of Lp(a) for the induction of PAI-2 expression.
Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta
Authors: Horwitz Edwin M. , Prockop Darwin J. , Gordon Patricia L. , Koo Winston W. K. , Fitzpatrick Lorraine A. , Neel Michael D. , McCarville M. Elizabeth , Orchard Paul J. , Pyeritz Reed E. , Brenner Malcolm K.
Published: July 2002

Preclinical models have shown that transplantation of marrow mesenchymal cells has the potential to correct inherited disorders of bone, cartilage, and muscle. The report describes clinical responses of the first children to undergo allogeneic bone marrow transplantation (BMT) for severe osteogenesis imperfecta (OI), a genetic disorder characterized by defective type I collagen, osteopenia, bone fragility, severe bony deformities, and growth retardation. Five children with severe OI were enrolled in a study of BMT from human leukocyte antigen (HLA)–compatible sibling donors. Linear growth, bone mineralization, and fracture rate were taken as measures of treatment response. The 3 children with documented donor osteoblast engraftment had a median 7.5-cm increase in body length (range, 6.5-8.0 cm) 6 months after transplantation compared with 1.25 cm (range, 1.0-1.5 cm) for age-matched control patients. These patients gained 21.0 to 65.3 g total body bone mineral content by 3 months after treatment or 45% to 77% of their baseline values. With extended follow-up, the patients' growth rates either slowed or reached a plateau phase. Bone mineral content continued to increase at a rate similar to that for weight-matched healthy children, even as growth rates declined. These results suggest that BMT from HLA-compatible donors may benefit children with severe OI. Further studies are needed to determine the full potential of this strategy.
Thymic emigrants isolated by a new method possess unique phenotypic and functional properties
Authors: Lee Chong-kil , Kim Kyungjae , Welniak Lisbeth A. , Murphy William J. , Muegge Kathrin , Durum Scott K.
Published: July 2002

T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-αβ) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, orClostridium difficile toxin B, implicating an active motility process. Most of the surface markers on αβ-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, α4-integrin, α5-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.
Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte-macrophage colony-stimulating factor or interleukin-3
Authors: Tomasson Michael H. , Williams Ifor R. , Li Shaoguang , Kutok Jeffrey , Cain Danielle , Gillessen Silke , Dranoff Glenn , Van Etten Richard A. , Gilliland D. Gary
Published: July 2002

Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFβR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFβR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFβR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFβR-expressing cells. In contrast, recipients of mixed GM-CSF–transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested.
Nucleotide sequence, transcription map, and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia
Authors: Migliazza Anna , Bosch Francesc , Komatsu Hirokazu , Cayanis Eftihia , Martinotti Stefano , Toniato Elena , Guccione Ernesto , Qu Xiaoyan , Chien Minchen , Murty V. V. V. , Gaidano Gianluca , Inghirami Giorgio , Zhang Peisen , Fischer Stuart , Kalachikov Sergey M. , Russo James , Edelman Isidore , Efstratiadis Argiris , Dalla-Favera Riccardo
Published: July 2002

Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses. The MDR contains 2 pseudogenes and 3 transcribed genes: CAR, encoding a putative RING-finger containing protein; 1B4/Leu2, generating noncoding transcripts; and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons). These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion. The nondeleted allele of theCAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status. These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL. However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis.
The C-class chemokine lymphotactin costimulates the apoptosis of human CD4+ T cells
Authors: Cerdan Chantal , Devilard Elisabeth , Xerri Luc , Olive Daniel
Published: July 2002

Clonal expansion of activated T cells is controlled by homeostatic mechanisms leading to cell death of a large proportion of the cells. The CD3/TcR pathway induces cell death, mostly when triggered in the absence of costimulatory signal. The unique T cell–specific chemokine of the C class, lymphotactin (Lptn), has recently been shown to inhibit the production of Th1-type lymphokines in human CD4+ T cells. The present study shows the ability of Lptn to costimulate the death of CD4+ T lymphocytes triggered through CD3/TCR. The Lptn-mediated increased cell death exhibited characteristic features of apoptosis, as mainly determined by DNA fragmentation and exposure of an apoptotic-specific mitochondrial antigen. This apoptosis was dependent on Fas/FasL signaling, was not rescued by addition of interleukin 2, and proceeded with a predominant processing of both initiator procaspase-9 and effector procaspase-7. These caspase activities were further evidenced by specific cleavage of poly(ADP-ribose) polymerase (PARP) and CD3/TCR ζ-chain, but not DNA fragmentation factor (DFF45). This study demonstrates that the functional repertoire of Lptn in the regulation of human CD4+ T-lymphocyte activation includes the ability to costimulate apoptosis.
Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets
Authors: Rosado Juan A. , Meijer Else M. Y. , Hamulyak Karly , Novakova Irena , Heemskerk Johan W. M. , Sage Stewart O.
Published: July 2002

Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.
Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34+CD38−hematopoietic cells
Authors: Seiffert Martina , Brossart Peter , Cant Charles , Cella Marina , Colonna Marco , Brugger Wolfram , Kanz Lothar , Ullrich Axel , Bühring Hans-Jörg
Published: July 2002

Signal-regulatory proteins (SIRPs) represent a new family of inhibitory/activating receptor pairs. They consist of 3 highly homologous immunoglobulin (Ig)–like domains in their extracellular regions, but differ in their cytoplasmic regions by the presence (SIRPα) or absence (SIRPβ) of immunoreceptor tyrosine-based inhibitory motifs (ITIMs). To analyze the differential expression on hematopoietic cells, function and ligand binding capacity of SIRPα and SIRPβ molecules, soluble fusion proteins consisting of the extracellular domains of SIRPα1, SIRPα2, and SIRPβ1, as well as SIRPα/β-specific and SIRPβ-specific monoclonal antibodies (MoAbs) were generated. In contrast to SIRPα1 and SIRPα2, no adhesion of SIRPβ1 to CD47 could be detected by cell attachment assays and flow cytometry. Using deletion constructs of SIRPα1, the epitope responsible for SIRPα1 binding to CD47 could be confined to the N-terminal Ig-like loop. Flow cytometry analysis with SIRPα/β- and SIRPβ-specific MoAbs revealed that SIRPα but not SIRPβ is expressed on CD34+CD38− hematopoietic cells. In addition, a strong SIRPα expression was also observed on primary myeloid dendritic cells (DCs) from peripheral blood as well as on in vitro generated DCs. Analysis of the T-cell stimulatory capacity of in vitro generated DCs in the presence of soluble SIRPα1 fusion proteins as well as SIRPα/β-specific and CD47-specific MoAbs revealed a significant reduction of T-cell proliferation in mixed lymphocyte reaction and inhibition of induction of primary T-cell responses under these conditions. In contrast, soluble SIRPα or SIRPβ-specific antibodies had no effect. The data suggest that the interaction of SIRPα with CD47 plays an important role during T-cell activation and induction of antigen-specific cytotoxic T-lymphocyte responses by DCs.
Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1
Authors: Moulds Joann M. , Zimmerman Peter A. , Doumbo Ogobara K. , Kassambara Lalla , Sagara Issaka , Diallo Dapa A. , Atkinson John P. , Krych-Goldberg Malgorzata , Hauhart Richard E. , Hourcade Dennis E. , McNamara David T. , Birmingham Daniel J. , Rowe J. Alexandra , Moulds John J. , Miller Louis H.
Published: July 2002

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum–infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sla), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparummalaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Slato long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sla with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sla and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.
Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region–derived stromal cells
Authors: Matsuoka Sahoko , Tsuji Kohichiro , Hisakawa Hiroaki , Xu Ming-jiang , Ebihara Yasuhiro , Ishii Takefumi , Sugiyama Daisuke , Manabe Atsushi , Tanaka Ryuhei , Ikeda Yasuo , Asano Shigetaka , Nakahata Tatsutoshi
Published: July 2002

There is controversy as to whether murine definitive hematopoiesis originates from yolk sac (YS) or the intraembryonic region. This study reports the generation of definitive hematopoietic stem cells (HSCs) from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (dpc). YS and P-Sp cells at 8.5 dpc generated no definitive hematopoiesis-derived colony-forming cells in cocultures with AGM-S3 cells, but spleen colony-forming cells and HSCs capable of reconstituting definitive hematopoiesis in adult mice simultaneously appeared on day 4 of coculture. Precursors for definitive HSCs were present in YS and P-Sp at 8.0 dpc, a time when YS and embryo were not connected by blood vessels. It is proposed that precursors with the potential to generate definitive HSCs appear independently in YS and intraembryonic P-Sp and that the P-Sp or AGM region affords the microenvironment that facilitates generation of definitive hematopoiesis from precursors.
Rescue of the colony-stimulating factor 1 (CSF-1)–nullizygous mouse (Csf1op/Csf1op) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis
Authors: Ryan Gregory R. , Dai Xu-Ming , Dominguez Melissa G. , Tong Wei , Chuan Fenchi , Chisholm Orin , Russell Robert G. , Pollard Jeffrey W. , Stanley E. Richard
Published: July 2002

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1–nullCsf1op/Csf1opmice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects ofCsf1op/Csf1opmice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed β-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.
Interferon-γ and interleukin-6 gene polymorphisms associate with graft-versus-host disease in HLA-matched sibling bone marrow transplantation
Authors: Cavet James , Dickinson Anne M. , Norden Jean , Taylor Penelope R. A. , Jackson Graham H. , Middleton Peter G.
Published: July 2002

Proinflammatory cytokines including interferon-γ (IFNγ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) are implicated in the pathogenesis of acute graft-versus-host disease (aGVHD). Cytokine gene polymorphism is associated with functional differences in cytokine regulation and altered clinical performance in a variety of diseases. Polymorphism in the IFNγIntron1 microsatellite (CA)n repeat has been linked with in vitro IFNγ production and renal transplant rejection. The IL-6−174(G/C) single nucleotide polymorphism has been linked to in vitro and in vivo IL-6 production, juvenile chronic arthritis, and renal transplant rejection. This study examined the potential association of GVHD with IFNγ and IL-6 polymorphisms in 80 sibling bone marrow transplant (BMT) donor/recipient pairs. Patients homozygous for the IFNγIntron1 allele 3 had more severe (grade III-IV) aGVHD. Patients possessing the IL-6−174G allele had a trend toward higher grades of aGVHD, and those homozygous for the IL-6−174G allele were more likely to develop chronic GVHD (cGVHD). The associations of previously identified aGVHD severity-associated cytokine gene polymorphisms (TNFd and IL-10−1064) with severe aGVHD were reconfirmed. Logistic regression analysis confirmed the association of severe aGVHD with recipient genotype at IFNγIntron1 (odds ratio [OR] 3.92;P = .02), IL-10−1064 (OR 4.61;P = .026) and TNFd (OR 3.29; P = .039), and that of cGVHD with recipient IL-6−174 genotype (OR 4.25;P = .007), in addition to age, gender mismatch, and underlying disease. Assessment of cytokine genotype may potentially allow more accurate prediction of GVHD and appropriate adjustment of GVHD prophylaxis, as well as indicating novel areas for future studies of GVHD pathogenesis.
The deubiquitinating enzyme DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal
Authors: Migone Thi-Sau , Humbert Martine , Rascle Anne , Sanden David , D'Andrea Alan , Johnston James A.
Published: July 2002

Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 but as yet its function has not been determined. The results of this study show that DUB-2 is expressed in human T-cell lymphotropic virus-I(HTLV-1)–transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB-2 markedly prolonged IL-2–induced STAT5 phosphorylation. Although DUB-2 did not enhance IL-2–mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2–induced genescis and c-myc. Moreover, DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Taken together these data suggest that DUB-2 can enhance signaling through the JAK/STAT pathway, prolong lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells.
Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ
Authors: Fujii Shin-ichiro , Shimizu Kanako , Shimizu Takashi , Lotze Michael T.
Published: July 2002

Interleukin-10 (IL-10) is a multifunctional cytokine that can exert suppressive and stimulatory effects on T cells. It was investigated whether IL-10 could serve as an immunostimulant for specific CD8+ cytotoxic T cell (CTL) in vivo after vaccination and, if so, under what conditions. In tumor prevention models, administration of IL-10 before, or soon after, peptide-pulsed primary dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy. Analysis of spleen cells derived from these latter animals 3 weeks after IL-10 treatment revealed that the number of CD8+CD44hi CD122+ T cells had increased and that antigen-specific proliferation in vitro was enhanced. Although cytotoxicity assays did not support differences between the various treatment groups, 2 more sensitive assays measuring antigen-specific interferon-γ production at the single-cell level demonstrated increases in the number of antigen-specific responder T cells in animals in the vaccine/IL-10 treatment group. Thus, IL-10 may maintain the number of antitumor CD8+ T cells. In adoptive transfer studies, the ability of IL-10 to maintain CTL function could be enhanced by the depletion of CD4+ T cells. This suggests that IL-10 mediates contrasting effects on both CD4+ and CD8+ T cells that result in either immune dampening or immune potentiation in situ, respectively. Appreciation of this dichotomy in IL-10 immunobiology may allow for the design of more effective cancer vaccines designed to activate and maintain specific CD8+ T-cell effector function in situ.
A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype
Authors: Ruiz Catherine , Liu Chao-Yan , Sun Qi-Hong , Sigaud-Fiks Marianne , Fressinaud Edith , Muller Jean-Yves , Nurden Paquita , Nurden Alan T. , Newman Peter J. , Valentin Nathalie
Published: July 2002

This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of β3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa–transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.
An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element
Authors: Loyevsky Mark , LaVaute Timothy , Allerson Charles R. , Stearman Robert , Kassim Olakunle O. , Cooperman Sharon , Gordeuk Victor R. , Rouault Tracey A.
Published: July 2002

This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.
Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects
Authors: Dai Xu-Ming , Ryan Gregory R. , Hapel Andrew J. , Dominguez Melissa G. , Russell Robert G. , Kapp Sara , Sylvestre Vonetta , Stanley E. Richard
Published: July 2002

The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene was inactivated. The phenotype ofCsf1−/Csf1r− mice closely resembled the phenotype of CSF-1-nullizygous(Csf1op/Csf1op) mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in bothCsf1op/Csf1op andCsf1−/Csf1r− mice were significantly elevated, consistent with a negative regulatory role of CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic progenitor cells. The circulating CSF-1 concentration inCsf1r−/Csf1r− mice was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R–mediated endocytosis and intracellular destruction. Despite their overall similarity, several phenotypic characteristics of theCsf1r−/Csf1r− mice were more severe than those of theCsf1op/Csf1op mice. The results indicate that all of the effects of CSF-1 are mediated via the CSF-1R, but that subtle effects of the CSF-1R could result from its CSF-1–independent activation.
Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes
Authors: Werz Oliver , Bürkert Eva , Samuelsson Bengt , Rådmark Olof , Steinhilber Dieter
Published: July 2002

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. This study showed that various forms of cell stress, such as chemical stress (sodium arsenite), osmotic stress, or heat shock lead to substantial formation of 5-LO products in freshly isolated human polymorphonuclear leukocytes (PMNLs), when exogenous arachidonic acid (10 μM) was present. In parallel, cell stress led to activation of p38 MAPK (mitogen-activated protein kinase) and mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) kinases, which can phosphorylate 5-LO in vitro. Interestingly, arsenite also caused redistribution of 5-LO from the cytosol to the nuclear membrane. Only minor activation of extracellular signal-regulated kinases and c-jun NH2-terminal kinases was observed, implying that these MAPKs are less important for 5-LO product formation in stress-stimulated PMNLs. Stimulation of 5-LO product formation by Ca++-ionophore A23187 or thapsigargin depended on Ca++; almost no 5-LO product formation was observed in freshly isolated PMNLs when Ca++ was depleted by chelating agents. Also the response toN-formylmethionyl-leucyl-phenylalanine (fMLP) was clearly diminished, but some 5-LO product formation remained. In contrast, stress-induced product formation and translocation of 5-LO, as well as activation of p38 MAPK, occurred also after Ca++ depletion. Moreover, the p38 MAPK inhibitor SB203580 blocked stress-induced 5-LO product formation efficiently, whereas ionophore- or thapsigargin-induced formation of 5-LO products was less sensitive. These data show that cell stress can activate 5-LO in isolated PMNLs by a mechanism that does not involve Ca++ mobilization. This mechanism could function independently of Ca++-mediated 5-LO activation for stimulation of leukotriene biosynthesis under physiologic conditions as well as in inflammatory diseases.
Pathways for internalization and recycling of the chemokine receptor CCR5
Authors: Mueller Anja , Kelly Eamonn , Strange Philip G.
Published: July 2002

M-tropic human immunodeficiency virus (HIV-1) strains enter the cell after interaction with their receptors, CD4 and the G-protein–coupled chemokine receptor CCR5. The number of cell surface CCR5 molecules is thought to be important in determining the infection rate for HIV. Cell surface CCR5 is dependent on the rate of receptor internalization and recycling. Internalization of G-protein–coupled receptors after agonist activation is thought to occur either through clathrin-coated pits or through caveolae. In this study, the role of these different pathways was investigated in Chinese hamster ovary cells expressing CCR5 using specific inhibitors. Internalization of CCR5 after chemokine treatment was inhibited by sucrose, indicating a role for the clathrin-coated pit pathway. Activation of CCR5 leads to arrestin-2 movement in the cells, providing further evidence for the involvement of clathrin-coated pits. Nystatin and filipin also affected the rate of internalization of CCR5, indicating a role for caveolae. Using inhibitors of vesicle transport in the cell, it was found that the CCR5 recycling pathway is independent of the Golgi apparatus and late endosomes. Protein synthesis is not involved in receptor recovery. It seems likely that after internalization, CCR5 is directed to early endosomes and subsequently recycled to the cell surface.