bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth factor treatment prior to low-dose total body irradiation increases donor cell engraftment after bone marrow transplantation in mice
Authors: Noach Estelle J. K. , Ausema Albertina , Dillingh Jan H. , Dontje Bert , Weersing Ellen , Akkerman Imre , Vellenga Edo , de Haan Gerald
Published: October 2002

Low-toxicity conditioning regimens prior to bone marrow transplantation (BMT) are widely explored. We developed a new protocol using hematopoietic growth factors prior to low-dose total body irradiation (TBI) in recipients of autologous transplants to establish high levels of long-term donor cell engraftment. We hypothesized that treatment of recipient mice with growth factors would selectively deplete stem cells, resulting in successful long-term donor cell engraftment after transplantation. Recipient mice were treated for 1 or 7 days with growth factors (stem cell factor [SCF] plus interleukin 11 [IL-11], SCF plus Flt-3 ligand [FL], or granulocyte colony-stimulating factor [G-CSF]) prior to low-dose TBI (4 Gy). Donor cell chimerism was measured after transplantation of congenic bone marrow cells. High levels of donor cell engraftment were observed in recipients pretreated for 7 days with SCF plus IL-11 or SCF plus FL. Although 1-day pretreatments with these cytokines initially resulted in reduced donor cell engraftment, a continuous increase in time was observed, finally resulting in highly significantly increased levels of donor cell contribution. In contrast, G-CSF treatment showed no beneficial effects on long-term engraftment. In vitro stem cell assays demonstrated the effect of cytokine treatment on stem cell numbers. Donor cell engraftment and number of remaining recipient stem cells after TBI were strongly inversely correlated, except for groups treated for 1 day with SCF plus IL-11 or SCF plus FL. We conclude that long-term donor cell engraftment can be strongly augmented by treatment of recipient mice prior to low-dose TBI with hematopoietic growth factors that act on primitive cells.
Enhancement of G-CSF–induced stem cell mobilization by antibodies against the β2 integrins LFA-1 and Mac-1
Authors: Velders Gerjo A. , Pruijt Johannes F. M. , Verzaal Perry , van Os Ronald , van Kooyk Yvette , Figdor Carl G. , de Kruijf Evert-Jan F. M. , Willemze Roel , Fibbe Willem E.
Published: October 2002

The β2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34+ cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti–LFA-1 antibodies completely prevent the IL-8–induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-β2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the α chain of LFA-1 or against the α chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area–forming cells in vitro was significantly higher after mobilization with anti–LFA-1 antibodies followed by 5 μg G-CSF for 5 days than with G-CSF alone (119 ± 34 days vs 17 ± 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF–induced mobilization, suggesting that the enhancing effect required an interaction of the β2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1–deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1–mediated adhesion.
Glycoprotein V: the predominant target antigen in gold-induced autoimmune thrombocytopenia
Authors: Garner Stephen F. , Campbell Kate , Metcalfe Paul , Keidan Jane , Huiskes Elly , Dong Jing-Fei , López José A. , Ouwehand Willem H.
Published: October 2002

Autoimmune thrombocytopenia is generally caused by autoantibodies against glycoprotein (GP) IIb-IIIa or GPIb-IX and occasionally against GPIa-IIa or GPV. By investigating 38 rheumatoid arthritis (RA) patients on gold therapy, 10 with profound thrombocytopenia and 28 nonthrombocytopenic controls, we showed that in all 10 patients with thrombocytopenia, the platelet autoantibodies preferentially targeted GPV but the presence of gold was not required for their reactivity. Elevated levels of platelet-associated IgG (PAIgG) were observed in 8 of the 10 patients in whom the tests were performed. In 5 patients with sufficient autologous platelets, the GPV specificity of PAIgG was confirmed. Tests with GPV transfectants revealed that the antibodies reacted with GPV independent of GPIbα, GPIbβ, or GPIX. Autoantibodies recognizing GPV were not seen in the 28 nonthrombocytopenic control RA patients. Thus, GPV seems to be targeted in gold-induced autoimmune thrombocytopenia.
Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease
Authors: Schnittger Susanne , Schoch Claudia , Dugas Martin , Kern Wolfgang , Staib Peter , Wuchter Christian , Löffler Helmut , Sauerland Cristina Maria , Serve Hubert , Büchner Thomas , Haferlach Torsten , Hiddemann Wolfgang
Published: October 2002

FLT3 length mutation (FLT3-LM) is a molecular marker potentially useful for the characterization of acute myeloid leukemia (AML). To evaluate the distribution of FLT3-LM within biologic subgroups, we screened 1003 patients with AML at diagnosis for this mutation. FLT3-LM was found in 234 (23.5%) of all patients and thus is the most frequent mutation in AML described so far. Of all positive patients, 165 (70.5%) revealed a normal karyotype. Of the 69 patients with chromosome aberrations, 24 (34.8%) had a t(15;17). The mutation was rare in AML with t(8;21), inv(16) 11q23 rearrangements, and complex karyotypes. FLT3-LM was not distributed equally within different French-American-British (FAB) subtypes and was correlated with a high peripheral blood count in FAB M1, M2, and M4 (P < .0001). In addition, the median age of patients with the mutation was lower (54.9 vs 57.6 years;P = .043), and, at a ratio of 1.36:1 (P = .023), the mutation was more frequent in females than in males. Within the AMLCG study, FLT3-LM was of intermediate prognostic significance. The complete remission rate of 70.3% in patients with FLT3-LM was similar to that (70.4%) in patients without FLT3-LM. Overall survival was not different between patients with or without FLT3-LM. In contrast, patients with FLT3-LM had a significantly shorter event-free survival (7.4 vs 12.6 months;P = .0072) because of a higher relapse rate. Besides the importance of FLT3-LM for biologic and clinical characterization of AML, we show its value as a marker for disease monitoring based on 120 follow-up samples of 34 patients.
Transcriptional regulation of myeloid differentiation primary response (MyD) genes during myeloid differentiation is mediated by nuclear factor Y
Authors: Tjin Tham Sjin Robert M. , Krishnaraju Kandasamy , Hoffman Barbara , Liebermann Dan A.
Published: October 2002

To understand the molecular mechanism by which interleukin-6 (IL-6) regulates myeloid differentiation primary response (MyD) genes at the onset of M1 myeloid differentiation, we used JunB as a representative MyD gene to isolate and characterize IL-6 responsive elements. An IL-6 responsive element was localized between −65 and −52 of the JunB promoter (−65/−52 IL-6RE). By using antibody and oligonucleotide competition assays in electrophoretic mobility shift assay experiments, we have shown that the heterotrimeric transcription nuclear factor Y (NF-Y) complex binds to this element. A dominant-negative form of NF-YA, ectopically expressed in M1 cells, blocked NF-Y binding to the −65/−52 IL-6RE and reduced induction of JunB by IL-6. Furthermore, inhibition of NF-Y binding also reduced MyD gene induction by IL-6 and dampened the IL-6–induced M1 differentiation program. These findings are consistent with the observation that most MyD genes contain intact NF-Y binding motifs in their promoter regions. In contrast to M1 cells, during myeloid differentiation of bone marrow (BM), there was induction of NF-Y binding to the −65/−52 IL-6RE. This induced binding can be attributed to the observed induction of NF-YA protein expression and may reflect the molecular mechanism that couples proliferation to terminal differentiation of normal myeloblasts. Similar to M1 cells, blocking NF-Y binding in BM resulted in a reduction in mature macrophages. It can be concluded that NF-Y plays a role in the transcriptional regulation of MyD genes and is required for optimum myeloid differentiation.
Granulocyte inducer C/EBPα inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions
Authors: Reddy Venkateshwar A. , Iwama Atsushi , Iotzova Guergana , Schulz Mathias , Elsasser Annika , Vangala Rajani K. , Tenen Daniel G. , Hiddemann Wolfgang , Behre Gerhard
Published: October 2002

Several transcription factors have been implicated as playing a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of myeloid and lymphoid lineages, whereas the transcription factor CCAAT–enhancer binding protein family member C/EBPα is essential for granulocyte development. We present here the first evidence that C/EBPα blocks the function of PU.1. PU.1 and C/EBPα interact physically and colocalize in myeloid cells. As a consequence of this interaction, C/EBPα can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA-binding sites. We further demonstrate that the leucine zipper in the DNA-binding domain of C/EBPα interacts with the β3/β4 region in the DNA-binding domain of PU.1 and as a result displaces the PU.1 coactivator c-Jun. Finally, C/EBPα blocks PU.1-induced dendritic cell development from CD34+ human cord blood cells. The functional blocking of PU.1 by C/EBPα could be the mechanism by which C/EBPα inhibits cell fates specified by PU.1 and directs cell development to the granulocyte lineage.
Activated protein C cleaves factor Va more efficiently on endothelium than on platelet surfaces
Authors: Oliver Julie A. , Monroe Dougald M. , Church Frank C. , Roberts Harold R. , Hoffman Maureane
Published: October 2002

The protein C/protein S system is known to regulate thrombin generation in vivo by cleaving factors Va and VIIIa. We have examined the activity of activated protein C in several tissue factor–initiated models of coagulation. We used 4 models: monocytes as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as both the tissue factor source and the thrombin-generating surface; and relipidated tissue factor with lipid vesicles providing the surface for thrombin generation. With the lipid surface, activated protein C dose-dependently reduced thrombin generation. Similarly, when endothelial cells provided the only surface for thrombin generation, activated protein C dose-dependently decreased thrombin generation significantly. By contrast, whenever platelets were present, activated protein C only minimally affected the amount of thrombin generated. When endothelial cells were the tissue factor source with platelets providing the surface for thrombin generation, activated protein C did increase the time until the burst of thrombin generation but had minimal effects on the total amount of thrombin generated. Activated protein C had essentially no effect on thrombin generation when monocytes were the tissue factor source with platelets providing the surface for thrombin generation. From the studies reported here, we conclude that in vivo, despite the important role of the protein C system in regulating thrombosis, activated protein C does not serve as a primary regulator of platelet-dependent thrombin generation.
Absence of biologically important Kaposi sarcoma–associated herpesvirus gene products and virus-specific cellular immune responses in multiple myeloma
Authors: Brander Christian , Raje Noopur , O'Connor Paula G. , Davies Faith , Davis Jennifer , Chauhan Darminder , Hideshima Teru , Martin Jeff , Osmond Dennis , Kedes Dean H. , Walker Bruce D. , Scadden David T. , Anderson Kenneth C.
Published: October 2002

Kaposi sarcoma–associated herpesvirus (KSHV) has been associated with several diseases, but the association between KSHV and multiple myeloma (MM) remains controversial. To address this issue, we studied patients with MM for the presence of viral RNA transcripts as well as KSHV-specific cellular immune responses. Highly sensitive reverse transcription–polymerase chain reaction assays for detection of viral transcripts of KSHV open reading frame (ORF) 26, ORF72, and ORF74 did not detect viral gene transcripts in long-term cultures of bone marrow stromal cells from 23 patients with MM. Moreover, sensitive assays for KSHV ORF65–specific and ORF73-specific cytotoxic T-lymphocyte (CTL) activity that readily and routinely detect CTLs specific for ORF65 and ORF73 in patients positive for human immunodeficiency virus and KSHV did not show any specific responses in 16 patients with MM, despite the presence of positive Epstein-Barr virus–specific CTLs in all cases. These data therefore do not show a biologically important association between ongoing KSHV infection and MM.
The expression pattern of c-mpl in megakaryocytes correlates with thrombotic risk in essential thrombocythemia
Authors: Teofili Luciana , Pierconti Francesco , Di Febo Annalaura , Maggiano Nicola , Vianelli Nicola , Ascani Stefano , Rossi Elena , Pileri Stefano , Leone Giuseppe , Larocca Luigi Maria , De Stefano Valerio
Published: October 2002

Using immunohistochemistry, we investigated the expression of c-mpl in bone marrow megakaryocytes of 88 patients with essential thrombocythemia (ET), 6 patients with secondary thrombocytosis (ST), and 20 patients with lymphoma (controls). Considering both the pattern of expression and the staining intensity, we identified a uniform and a heterogeneous pattern of c-mplexpression. The uniform pattern was found in all the controls, all the patients with ST, and 28 of the patients with ET, with a strong staining intensity observed in most megakaryocytes (> 80%). In contrast, c-mpl expression was heterogeneous in 60 patients with ET, 18 of whom (30%) presented with thrombosis at diagnosis, a significant difference from patients with a uniform c-mpl pattern (2 of 28; 7%; P = .026). In particular, the overrepresentation of thrombotic complications in patients with a heterogeneous c-mpl expression pattern was found mainly among patients with a significant percentage (10% to 40%) of weakly stained or c-mpl–negative megakaryocytes (heterogeneous-weak pattern; 13 of 30; 43%;P = .002). Accordingly, this pattern was associated with a 6.1-fold increased risk of thrombosis compared with that of patients with a uniform c-mpl pattern. In conclusion, the presence of a heterogeneous pattern of c-mpl distribution in bone marrow megakaryocytes could be a useful diagnostic criterion in the differential diagnosis of thrombocytosis. Furthermore, detection of a significant percentage of weakly stained or c-mpl–negative megakaryocytes can identify patients with a higher risk of thrombosis.
Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated
Authors: Billheimer Jeffrey T. , Dicker Ira B. , Wynn Richard , Bradley Jodi D. , Cromley Debra A. , Godonis Helen E. , Grimminger Lisa C. , He Bokang , Kieras Cathy J. , Pedicord Donna L. , Spitz Susan M. , Thomas Beth E. , Zolotarjova Nina I. , Gorko Mary A. , Hollis Gregory F. , Daly Robert N. , Stern Andrew M. , Seiffert Dietmar
Published: October 2002

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.
Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma
Authors: Wiesener Michael S. , Seyfarth Melchior , Warnecke Christina , Jürgensen Jan Steffen , Rosenberger Christian , Morgan Neil V. , Maher Eamonn R. , Frei Ulrich , Eckardt Kai-Uwe
Published: October 2002

The von Hippel-Lindau (VHL) tumor suppressor gene targets hypoxia-inducible transcription factors (HIFs) for proteasomal degradation. Erythrocytosis due to inappropriate production of erythropoietin (EPO), one of the HIF target genes, is a classic albeit rare finding in patients with renal cancer. We report the clinical to molecular analysis in a patient in whom a thrombotic myocardial infarction was the first manifestation of a clear cell renal carcinoma associated with an elevated serum EPO level (109 U/L) and erythrocytosis (hemoglobin 200 g/L [20 g/dL]). The tumor strongly expressed EPO messenger RNA and the 2 regulatory subunits HIF-1α and HIF-2α. Sequence analysis of tumor tissue identified a point mutation of the VHL gene (nucleotide 701 T>C) with a predicted amino acid exchange (Leu163Pro). This structural change, although located at distance to the HIF-binding region, was found to inhibit binding of HIF-1α to VHL, thus leading to accumulation of HIF, which drives EPO production.
Fibronectin- and protein kinase C–mediated activation of ERK/MAPK are essential for proplateletlike formation
Authors: Jiang Fang , Jia Yuzhi , Cohen Isaac
Published: October 2002

The megakaryoblastic CHRF-288 cell line was used to investigate signal transduction pathways responsible for proplateletlike formation (PPF). The role of fibronectin (FN) and protein kinase C (PKC) activation in PPF were examined. In the presence of serum and phorbol 12-myristate 13-acetate (PMA), a PKC activator, cells exhibited full megakaryocytic differentiation, manifested by adhesion, shape change, increased cell size, polyploidy, PPF, and expression of CD41+, CD61+, and CD62P+. The same morphologic and phenotypic features were observed in serum-free cultures in the presence of FN/PMA. Only partial differentiation occurred when other integrin ligands were substituted for FN. FN alone induced minimal cell adhesion and spreading, while PMA alone induced only polyploidy without adhesion. Signal transduction changes involved the activation of the extracellular signal–regulated protein kinase 1 (ERK1)/ERK2 as well as c-Jun amino-terminal kinase 1 (JNK1)/stress-activated protein kinase (SAPK). Phosphoinositide-3 kinase and p38 were not stimulated under these conditions. Inhibitors were used to identify the causal relationship between signaling pathways and PPF. PD98059 and GF109203X, inhibitors of ERK1/ERK2 pathway and PKC, respectively, blocked PPF, while adhesion, spreading, and polyploidy were normal. These studies show that activation of ERK1/ERK2 mitogen-activated protein kinase pathway plays a critical role in PPF. The elucidation of the signal transduction pathway on megakaryocyte development and PPF is of crucial importance for understanding this unique biological process.
Fibrinogen Hillsborough: a novel γGly309Asp dysfibrinogen with impaired clotting
Authors: Mullin Jennifer L. , Brennan Stephen O. , Ganly Peter S. , George Peter M.
Published: October 2002

We present a novel γ-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed a γ-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal γ chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)→GAC (Asp) at codon 309 of the γ chain gene. This Gly→ Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue γ309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. γGly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting γD domains of condensing fibrin monomers during polymerization.
Platelet factor 4 binds to low-density lipoprotein receptors and disrupts the endocytic itinerary, resulting in retention of low-density lipoprotein on the cell surface
Authors: Sachais Bruce S. , Kuo Alice , Nassar Taher , Morgan Jeanelle , Kariko Katalin , Williams Kevin Jon , Feldman Michael , Aviram Michael , Shah Neelima , Jarett Leonard , Poncz Mortimer , Cines Douglas B. , Higazi Abd Al-Roof
Published: October 2002

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R–dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 μg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.
Constitutive nuclear factor–κB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins
Authors: Bureau Fabrice , Vanderplasschen Alain , Jaspar Fabrice , Minner Frédéric , Pastoret Paul-Pierre , Merville Marie-Paule , Bours Vincent , Lekeux Pierre
Published: October 2002

Constitutive nuclear factor kappaB (NF-κB) activity protects quiescent mature immune cells from spontaneous apoptosis. Here, we examined whether NF-κB exerts its antiapoptotic function in these cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-κB were used to achieve total NF-κB inactivation in quiescent human blood lymphocytes, granulocytes, and monocytes. NF-κB inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte apoptosis. T- and B-cell apoptosis was slow and associated with a gradual down-regulation of the prosurvival Bcl-2 family proteins Bcl-xL and Bcl-2, respectively. By contrast, granulocyte apoptosis was fast and accompanied by a rapid cellular accumulation of Bcl-xS, the proapoptotic Bcl-x isoform that is generated from alternative splicing of the bcl-x pre-mRNA. Finally, antisense bcl-xL and bcl-2knockdown in T and B cells, respectively, and induction of Bcl-xS expression in granulocytes through antisense oligonucleotide-mediated redirection of bcl-x pre-mRNA splicing were sufficient to induce significant apoptosis in these cells. Taken together, these results reveal that basal NF-κB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes through regulation of distinct members of the Bcl-2 family. This study sheds light on the constitutive mechanisms by which NF-κB maintains defense integrity.
Antitumorigenic effects of HIV protease inhibitor ritonavir: inhibition of Kaposi sarcoma
Authors: Pati Shibani , Pelser Colleen B. , Dufraine Joseph , Bryant Joseph L. , Reitz Marvin S. , Weichold Frank F.
Published: October 2002

Treatment of patients with human immunodeficiency virus (HIV) protease inhibitors such as ritonavir can result in increases in CD4+ T-cell counts that are independent of a reduction in HIV-1 viral load. This lack of correlation between the 2 has led to the identification of additional effects of ritonavir that potentially alter HIV disease pathogenesis. Our previous studies indicated that ritonavir directly affects immune cell activation, proliferation, and susceptibility to apoptosis. We show here that ritonavir inhibited the activation and proliferation of primary endothelial cells and decreased the production of tumor necrosis factor α (TNF-α) interleukin 6 (IL-6), IL-8, and vascular endothelial growth factor, factors that all contribute to tumor neovascularization and to the development of Kaposi sarcoma (KS) lesions. Ritonavir also suppressed the expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin, which correlated with a functional decrease in leukocyte adhesion. Transcriptional activation of nuclear factor-κB, as induced by the KS-promoting factor TNF-α, the HIV-1 Tat protein, or the human herpesvirus 8 protein ORF74, was inhibited by ritonavir. KS-derived cell lines underwent apoptosis in vitro after treatment with ritonavir at concentrations that are obtained in clinical therapy (3-15 μM). In a KS mouse xenotransplantation model, ritonavir inhibited tumor formation and progression by KS-derived cells. Taken together, these data suggest that ritonavir has antineoplastic effects that are independent from its ability to inhibit the HIV protease.
Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization
Authors: Becker Erika M. , Greer Judith M. , Ponka Prem , Richardson Des R.
Published: October 2002

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (β-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and β-globinmRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis.
Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli
Authors: Di Nicola Massimo , Carlo-Stella Carmelo , Magni Michele , Milanesi Marco , Longoni Paolo D. , Matteucci Paola , Grisanti Salvatore , Gianni Alessandro M.
Published: October 2002

CD2+ T lymphocytes obtained from either the donor of bone marrow stromal cells (BMSCs) or a third party were cultured in mixed lymphocyte reactions (MLRs) with either allogeneic dendritic cells (DCs) or peripheral blood lymphocytes (PBLs). When autologous or allogeneic BMSCs were added back to T cells stimulated by DCs or PBLs, a significant and dose-dependent reduction of T-cell proliferation, ranging from 60% ± 5% to 98% ± 1%, was evident. Similarly, addition of BMSCs to T cells stimulated by polyclonal activators resulted in a 65% ± 5% (P = .0001) suppression of proliferation. BMSC- induced T-cell suppression was still evident when BMSCs were added in culture as late as 5 days after starting of MLRs. BMSC-inhibited T lymphocytes were not apoptotic and efficiently proliferated on restimulation. BMSCs significantly suppressed both CD4+ and CD8+ T cells (65% ± 5%, [P = .0005] and 75% ± 15% [P = .0005], respectively). Transwell experiments, in which cell-cell contact between BMSCs and effector cells was prevented, resulted in a significant inhibition of T-lymphocyte proliferation, suggesting that soluble factors were involved in this phenomenon. By using neutralizing monoclonal antibodies, transforming growth factor β1 and hepatocyte growth factor were identified as the mediators of BMSC effects. In conclusion, our data demonstrate that (1) autologous or allogeneic BMSCs strongly suppress T-lymphocyte proliferation, (2) this phenomenon that is triggered by both cellular as well as nonspecific mitogenic stimuli has no immunologic restriction, and (3) T-cell inhibition is not due to induction of apoptosis and is likely due to the production of soluble factors.
Herpes simplex virus type 2–specific CD8 cytotoxic T lymphocyte cross-reactivity against prevalent HLA class I alleles
Authors: Koelle David M. , Chen Hongbo B. , McClurkan Christopher M. , Petersdorf Effie W.
Published: October 2002

Clonally expressed T-cell receptor αβ heterodimers are able to bind many different major histocompatibility complex/peptide combinations. This promiscuity is thought to be required for adequate surveillance against microbial and malignancy-associated antigens. After transplantation, T cells may react with nonself structures, contributing to graft-versus-host disease, in the case of hematopoietic stem cell transplantation, or graft failure, when the host immune system is preserved. We describe 2 distinct HLA A*0201–restricted, cytotoxic CD8 T-cell responses to the prevalent chronic pathogen, herpes simplex virus type 2, that cross-react with cells bearing specific alleles of the common HLA B44 family. Transfection of human or primate renal epithelial cells with HLA class I complementary DNA confirmed these results. Given the prevalence of this viral infection and the HLA alleles involved, it is possible that this cross-reactivity may be involved in clinically significant events.
Aotus New World monkeys: model for studying malaria-induced anemia
Authors: Egan Andrea F. , Fabucci Maria Elena , Saul Allan , Kaslow David C. , Miller Louis H.
Published: October 2002

Falciparum malaria is a major cause of disease and death in African children and pregnant women, primarily due to severe anemia. We studied anemia in vaccinated Aotus monkeys during a second infection where the animals were considered to be semi-immune. Most animals had extremely low or undetectable levels of parasitemia; in some, anemia did not develop and reticulocytemia remained unchanged; in others, moderate to severe anemia developed with inappropriately low reticulocytemia indicating bone marrow dysfunction. Bone marrow rapidly responded after parasite clearance. The rapid drop in hematocrit despite extremely low to undetectable parasitemia indicated massive removal of uninfected red blood cells from the circulation that, in the presence of bone marrow dysfunction, led to severe anemia—the problem that occurs in African children. We demonstrate that Aotusmonkeys are a nonhuman primate model to gain insight into the pathogenesis of severe anemia in African children.
CD44 variant–specific antibodies trigger hemopoiesis by selective release of cytokines from bone marrow macrophages
Authors: Khaldoyanidi Sophia , Karakhanova Svetlana , Sleeman Jonathan , Herrlich Peter , Ponta Helmut
Published: October 2002

Hemopoiesis is regulated by the complex interplay between the bone marrow microenvironment and hemopoietic stem cells and progenitors. The local production of cytokines plays a critical role in this process. Using long-term bone marrow cultures, we show here that monoclonal antibodies directed against the CD44 v4 and CD44 v6 epitopes stimulate myelopoiesis (CD44 v4 and CD44 v6) and lymphopoiesis (CD44 v6). In the bone marrow cell population, CD44 v4 and CD44 v6 epitopes are found virtually exclusively on double-positive bone marrow macrophages. The anti-CD44 v4 and v6 antibodies act on bone marrow macrophages to stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF) production (v4 and v6) and interleukin-6 (IL-6) production (v6). This profile of cytokine production explains the differential stimulation of hemopoiesis by the 2 antibodies. We suggest that the antibodies mimic ligand(s) that stimulate GM-CSF or IL-6 production by bone marrow–derived macrophages by binding to CD44 family members that bear CD44 v4 and CD44 v6 epitopes on these cells.
Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth
Authors: Iversen Per Ole , Emanuel Peter D. , Sioud Mouldy
Published: October 2002

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood disorder with few therapeutic options. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor–α (TNF-α) promote JMML cell growth. A hyperactive function of the ras oncogene is a hallmark of JMML. We therefore targeted the protein kinase Raf-1 downstream of Ras using a DNA enzyme that degrades mRNA–Raf-1. Western blots of JMML cell lysates revealed phosphorylated Raf-1 protein, indicating constitutive activation. Addition of GM-CSF, but not TNF-α, increased phosphorylation of both Raf-1 and the mitogen-activated protein kinases (MAPKs) JNK-1 and ERK-1. Depletion of Raf-1 protein markedly impaired activation of MAPKs, induced substantial inhibition of JMML cell colony formation, and virtually abolished GM-CSF hypersensitivity in JMML cells. Exogenous TNF-α, but not GM-CSF, restored colony formation of JMML cells pretreated with the enzyme. We could not detect any effect of the enzyme on the proliferation of normal bone marrow cells, indicating its specificity and potential safety. When immunodeficient mice engrafted with JMML cells were treated continuously with the enzyme via a peritoneal osmotic mini-pump for 4 weeks, a profound reduction in the JMML cell numbers in the recipient murine bone marrows was found. We conclude that GM-CSF is a chief regulator of JMML growth and exerts its proleukemic effects primarily via the Ras/Raf-1 signaling cascade. TNF-α plays a permissive role, being dependent upon GM-CSF to induce JMML cell proliferation. The DNA enzyme efficiently catabolized mRNA–Raf-1 with subsequent inhibition of JMML cell growth, suggesting its potential as a mechanism-based therapy in this fatal leukemia.
Antidisialoganglioside/granulocyte macrophage–colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcγRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis
Authors: Metelitsa Leonid S. , Gillies Stephen D. , Super Michael , Shimada Hiroyuki , Reynolds C. Patrick , Seeger Robert C.
Published: October 2002

Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcγRII and FcγRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcγRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1–dependent PMN–target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined β2-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcγRII and Mac-1–dependent enhanced adhesion and degranulation.
Molecular basis of NB1 (HNA-2a, CD177) deficiency
Authors: Kissel Karin , Scheffler Steffi , Kerowgan Mohammed , Bux Jürgen
Published: October 2002

Alloimmunization to the neutrophil antigen NB1 (HNA-2a, CD177) can result in immune neutropenia and transfusion-related acute lung injury. Recently, we were able to elucidate the primary structure of NB1. To shed light also on the molecular basis of the NB1-negative phenotype, we studied the neutrophils of 2 women with NB1-specific alloantibodies for intracellular and extracellular NB1 expression, NB1-specific mRNA production, and the presence of the NB1 gene. No antibody binding to neutrophils was observed by immunofluorescence and immunoblot using a variety of human and monoclonal NB1-specific antibodies. By reverse transcription–polymerase chain reaction with NB1-specific primers we could not detect NB1 cDNAs without accessory sequences, which were found to be introns. The NB1 gene was present in the genome of both patients. Our data indicate that the NB1-negative phenotype is the result of different off-frame insertions on RNA level, resulting in NB1 deficiency on neutrophils.
Gemtuzumab ozogamicin with or without interleukin 11 in patients 65 years of age or older with untreated acute myeloid leukemia and high-risk myelodysplastic syndrome: comparison with idarubicin plus continuous-infusion, high-dose cytosine arabinoside
Authors: Estey Elihu H. , Thall Peter F. , Giles Francis J. , Wang Xue-Mei , Cortes Jorge E. , Beran Miloslav , Pierce Sherry A. , Thomas Deborah A. , Kantarjian Hagop M.
Published: October 2002

We investigated treatment with gemtuzumab ozogamicin (GO) in 51 patients aged 65 years or older with newly diagnosed acute myeloid leukemia (AML), refectory anemia (RA) with excess of blasts in transformation, or RA with excess blasts. GO was given in doses of 9 mg/m2 of body-surface area on days 1 and 8 or, therapeutically equivalently, on days 1 and 15, with or without interleukin 11 (IL-11; 15 μg/kg per day on days 3 to 28), with assignment to IL-11 treatment made randomly. Complete remission (CR) rates were 2 of 26 (8%) for GO without IL-11 and 9 of 25 (36%) for GO with IL-11. Regression analyses indicated that IL-11 was independently predictive of CR but not survival. We compared GO with or without IL-11 with idarubicin plus cytosine arabinoside (IA), as previously administered, in similar patients. The CR rate with IA was 15 of 31 (48%), and survival was superior with IA compared with GO with or without IL-11 (P = .03). Besides accounting for possible covariate effects on outcome, we also accounted for possible trial effects (TEs) arising because IA and GO with or without IL-11 were not arms of a randomized trial. Bayesian posterior probabilities that GO with or without IL-11 produced longer survival than IA, after accounting for covariates and TEs, were less than 0.01 in patients with abnormal cytogenetic findings (AC) and less than 0.15 in patients with normal cytogenetic findings (NC). Regarding CR, the analogous probabilities were less than 0.02 for GO without IL-11 (all cytogenetic groups), and for GO with IL-11, less than 0.25 for AC groups and about 0.50 for NC groups. TEs 2 to 5 times the magnitude of those previously observed would be needed to conclude that survival with GO with or without IL-11 is likely longer than with IA. Thus, there is little evidence to suggest that GO with or without IL-11 should be used instead of IA in older patients with newly diagnosed AML or myelodysplastic syndrome.
The cysteine knot of platelet glycoprotein Ibβ (GPIbβ) is critical for the interaction of GPIbβ with GPIX
Authors: Kenny Dermot , Morateck Patricia A. , Montgomery Robert R.
Published: October 2002

The glycoprotein Ib (GPIb) complex is composed of GPIbα covalently attached to GPIbβ and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIbβ or GPIX result in an absence of platelet GPIbα. This occurs through the interaction of GPIX with GPIbβ. The precise sites of interaction of GPIbβ with GPIX are not known. To characterize the interaction of GPIbβ and GPIX, we developed an anti-GPIbβ monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIbβ. N-terminal truncations of GPIbβ were expressed in mammalian cells. N-terminal truncations of GPIbβ, missing the first 14, 26, or 31 amino acids, were surface-expressed but did not enable coexpressed GPIX to be surface expressed, suggesting that the site of interaction with GPIX was modified by these deletions. GPIbβ and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIbβ with GPIX. Replacing the N-terminal disulfide loops of GPIbβ (amino acids 1-14) with the corresponding disulfide loops of GPIX (amino acids 1-22) resulted in surface expression of coexpressed wildtype GPIX. However, when the N terminus of GPIbβ was replaced to residue 32 with the N terminus of GPIX (amino acids 1-36), GPIX did not surface express with this chimera. These results suggest that the cysteine knot region of GPIbβ in the N terminus is critical for the conformation of GPIbβ that interacts with GPIX and further suggests that a critical interaction of GPIbβ with GPIX involve residues 15 through 32 of GPIbβ.
Identification of integrin αMβ2 as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions
Authors: Schober Joseph M. , Chen Ningyu , Grzeszkiewicz Tatiana M. , Jovanovic Igor , Emeson Eugene E. , Ugarova Tatiana P. , Ye Richard D. , Lau Lester F. , Lam Stephen C.-T.
Published: October 2002

Cysteine-rich 61 (Cyr61, CCN1) and connective tissue growth factor (CTGF, CCN2) are growth factor–inducible immediate-early gene products found in blood vessel walls and healing cutaneous wounds. We previously reported that the adhesion of endothelial cells, platelets, and fibroblasts to these extracellular matrix–associated proteins is mediated through integrin receptors. In this study, we demonstrated that both Cyr61 and CTGF are expressed in advanced atherosclerotic lesions of apolipoprotein E–deficient mice. Because monocyte adhesion and transmigration are important for atherosclerosis, wound healing, and inflammation, we examined the interaction of THP-1 monocytic cells and isolated peripheral blood monocytes with Cyr61 and CTGF. THP-1 cells and monocytes adhered to Cyr61- or CTGF-coated wells in an activation-dependent manner and this process was mediated primarily through integrin αMβ2. Additionally, expression of αMβ2 on human embryonic kidney 293 cells resulted in enhanced cell adhesion to Cyr61. Consistent with these data, a GST-fusion protein containing the I domain of the integrin αM subunit bound specifically to immobilized Cyr61 or CTGF. We have also investigated the requirement of cell surface heparan sulfate proteoglycans (HSPGs) as coreceptors for monocyte adhesion to Cyr61. Pretreatment of monocytes with heparin or heparinase I resulted in partial inhibition of cell adhesion to Cyr61. However, monocytes, but not fibroblasts, were capable of adhering to a Cyr61 mutant deficient in heparin binding activity. Collectively, these results show that activated monocytes adhere to Cyr61 and CTGF through integrin αMβ2 and cell surface HSPGs. However, unlike fibroblast adhesion to Cyr61, cell surface HSPGs are not absolutely required for this adhesion process.