bluuebunny/crossref_metadata_2025 huggingface dataset:
Male microchimerism in healthy women and women with scleroderma: cells or circulating DNA? A quantitative answer
Authors: Lambert Nathalie C. , Lo Y. M. Dennis , Erickson Timothy D. , Tylee Tracy S. , Guthrie Katherine A. , Furst Daniel E. , Nelson J. Lee
Published: September 2002

Male DNA, of presumed fetal origin, can be detected in the maternal circulation decades after delivery and is referred to as fetal microchimerism (FM). We previously found quantitatively greater FM in the circulation of women with the autoimmune disease scleroderma (SSc) than of healthy women. However, it is unknown whether this difference is due to intact circulating cells or free DNA released from breakdown in disease-affected tissues. To distinguish the origin of FM, we developed a real-time quantitative polymerase chain reaction (PCR) assay for the Y-chromosome–specific sequenceDYS14, and tested 114 women in peripheral blood mononuclear cells (PBMCs) and/or plasma. Fifty-seven controls and 57 SSc patients were studied, 48 and 43 of whom, respectively, had given birth to at least one son. Circulating FM was quantitatively greater in PBMCs from SSc patients (n = 39; range, 0.0-12.5 male genome–equivalent cells per million maternal cells), compared with healthy women (n = 39; range, 0.0-4.4; P = .03). In contrast, there was no difference between patients (n = 25) and controls (n = 22) in plasma, and no evidence of free DNA. FM was enriched among T lymphocytes compared with PBMCs (P = .01) in controls (n = 14), but not in SSc patients (n = 14); the latter finding was most likely due to immunosuppressive medications. In conclusion, this real-time quantitative assay showed that quantitative differences in the circulation of women with SSc are due to cells and not to free DNA. As FM was not uncommon in healthy women, including among T cells, and because graft-versus-host disease has similarities to SSc, these results also suggest that FM merits investigation in pheresis products used for stem cell transplantation.
Thrombospondin and fibrinogen bind serotonin-derivatized proteins on COAT-platelets
Authors: Szasz Robert , Dale George L.
Published: September 2002

Activation of platelets with 2 agonists, collagen and thrombin, reveals a subpopulation of cells referred to as COAT-platelets (collagen and thrombin activated). These cells are enriched in several membrane-bound, procoagulant proteins, including fibrinogen, thrombospondin, factor V, von Willebrand factor, and fibronectin. α-Granule proteins bound to COAT-platelets are derivatized with serotonin by a transglutaminase-mediated process, and the interaction of conjugated serotonins with unidentified serotonin binding sites on the platelet surface enhances retention of these proteins. We now demonstrate that both thrombospondin and fibrinogen provide the requisite serotonin binding sites. Thrombospondin and fibrinogen were identified using photoreactive cross-linking to an albumin-(serotonin)6conjugate during COAT-platelet production. We subsequently verified that biotin-albumin-(serotonin)6 binds in vitro to thrombospondin, fibrinogen, and fibrinogen fragment D in a saturable manner. These data support a model for COAT-platelets where serotonin-derivatized procoagulant proteins interact with their respective receptors (eg, fibrinogen with glycoprotein IIb/IIIa or factor V with phosphatidylserine) as well as serotonin binding sites on fibrinogen and thrombospondin, resulting in a stable, multivalent complex on the cell surface.
Wiskott-Aldrich syndrome in a female
Authors: Lutskiy Maxim I. , Sasahara Yoji , Kenney Dianne M. , Rosen Fred S. , Remold-O'Donnell Eileen
Published: September 2002

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema, and various degrees of immune deficiency. Carriers of mutated WASP have nonrandom X chromosome inactivation in their blood cells and are disease-free. We report data on a 14-month-old girl with a history of WAS in her family who presented with thrombocytopenia, small platelets, and immunologic dysfunction. Sequencing of the WASP gene showed that the patient was heterozygous for the splice site mutation previously found in one of her relatives with WAS. Sequencing of all WASP exons revealed no other mutation. Levels of WASP in blood mononuclear cells were 60% of normal. Flow cytometry after intracellular staining of peripheral blood mononuclear cells with WASP monoclonal antibody revealed both WASPbright and WASPdimpopulations. X chromosome inactivation in the patient's blood cells was found to be random, demonstrating that both maternal and paternal active X chromosomes are present. These findings indicate that the female patient has a defect in the mechanisms that lead in disease-free WAS carriers to preferential survival/proliferation of cells bearing the active wild-type X chromosome. Whereas the patient's lymphocytes are skewed toward WASPbright cells, about 65% of her monocytes and the majority of her B cells (CD19+) are WASPdim. Her naive T cells (CD3+CD45RA+) include WASPbrightand WASPdim populations, but her memory T cells (CD3+CD45RA−) are all WASPbright. After activation in vitro of T cells, all cells exhibited CD3+CD45RA− phenotype and most were WASPbright with active paternal (wild-type) X chromosome, suggesting selection against the mutated WASP allele during terminal T-cell maturation/differentiation.
Developmental dissociation of T cells from B, NK, and myeloid cells revealed by MHC class II–specific chimeric immune receptors bearing TCR-ζ or FcR-γ chain signaling domains
Authors: Lin Wei Yu , Roberts Margo R.
Published: September 2002

The T-cell receptor ζ (TCR-ζ) and FcR-γ chains play a critical role in mediating signal transduction. We have previously described HIV glycoprotein 120 (gp120)–specific chimeric immune receptors (CIRs) in which the extracellular domain of CD4 is linked to the signaling domain of ζ (CD4ζ) or γ (CD4γ). Such CIRs are efficiently expressed following retroviral transduction of mature T cells and specifically redirect effector functions toward HIV-infected targets. In this report, we examine development of CD4ζ- or CD4γ-expressing T cells from retrovirally transduced hematopoietic stem cells following bone marrow transplantation. Although CD4ζ/γ-expressing myeloid, NK, and B cells were efficiently reconstituted, parallel development of CD4ζ/γ-expressing T cells was blocked prior to the CD25+CD44+prothymocyte stage. In contrast, T cells expressing a signaling-defective CIR were efficiently generated. When major histocompatibility complex (MHC) class II–deficient mice were used as transplant recipients, development of CD4ζ/γ-expressing T cells was restored. We conclude that CD4ζ/γ signaling generated following engagement of MHC class II selectively arrests T-lineage development.
Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibα mechanoreceptor
Authors: Mazzucato Mario , Pradella Paola , Cozzi Maria Rita , De Marco Luigi , Ruggeri Zaverio M.
Published: September 2002

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca++ concentration ([Ca++]i) that are associated with sequential stages of integrin αIIbβ3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibα (GPIbα) and the VWF A1 domain led to Ca++ release from intracellular stores (type α/β peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca++]ioscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbα. Once adhesion was established through the integrin αIIbβ3, new [Ca++]i oscillations (type γ) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbα-VWF bonds leads to αIIbβ3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied αIIbβ3, with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbα and αIIbβ3 and lead to platelet adhesion and aggregation on immobilized VWF.
SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase
Authors: Yee Kevin W. H. , O'Farrell Anne Marie , Smolich Beverly D. , Cherrington Julie M. , McMahon Gerald , Wait Cecily L. , McGreevey Laura S. , Griffith Diana J. , Heinrich Michael C.
Published: September 2002

Internal tandem duplication (ITD) in the juxtamembrane portion of Fms-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase (RTK), is the most common molecular defect associated with acute myeloid leukemia (AML). The high prevalence of this activating mutation makes it a potential target for molecularly based therapy. Indolinone tyrosine kinase inhibitors have known activity against KIT, another member of the type III RTK family. Given the conserved homology between members of this family, we postulated that the activity of some KIT inhibitors would extend to FLT3. We used various leukemic cell lines (BaF3, MV 4-11, RS 4;11) to test the activity of indolinone compounds against the FLT3 kinase activity of both wild-type (WT) and ITD isoforms. Both SU5416 and SU5614 were capable of inhibiting autophosphorylation of ITD and WT FLT3 (SU5416 concentration that inhibits 50% [IC50], 100 nM; and SU5614 IC50 10 nM). FLT3-dependent activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) was also inhibited by treatment in the same concentration ranges. FLT3 inhibition by SU5416 and SU5614 resulted in reduced proliferation (IC50, 250 nM and 100 nM, respectively) and induction of apoptosis of FLT3 ITD-positive leukemic cell lines. Treatment of these cells with an alternative growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF]) restored MAPK signaling and cellular proliferation, demonstrating specificity of the observed inhibitory effects. We conclude that SU5416 and SU5614 are potent inhibitors of FLT3. Our finding that inhibition of FLT3 induces apoptosis of leukemic cells supports the feasibility of targeting FLT3 as a novel treatment strategy for AML.
Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival
Authors: Novak Anne J. , Bram Richard J. , Kay Neil E. , Jelinek Diane F.
Published: September 2002

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.
Human CD34+CXCR4− sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation
Authors: Kollet Orit , Petit Isabelle , Kahn Joy , Samira Sarit , Dar Ayelet , Peled Amnon , Deutsch Varda , Gunetti Monica , Piacibello Wanda , Nagler Arnon , Lapidot Tsvee
Published: September 2002

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34+stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34+CD38−CXCR4− and CXCR4+ cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34+CXCR4+ (R4+) and CD34+CXCR4− (R4−) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1–migrated CD34+ cells. Coinjection of purified cells with 10 μg anti-CXCR4 mAb significantly reduced engraftment of all CD34+ subsets, and 50 μg completely abrogated engraftment by R4− and CD34+ cells. Importantly, R4− cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4− cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34+ cells but yet CXCR4 dependent. In conclusion, R4− cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1–dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34+ stem and progenitor cells, regulating their motility and repopulation capacities.
Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel
Authors: Hassock Sheila R. , Zhu Michael X. , Trost Claudia , Flockerzi Veit , Authi Kalwant S.
Published: September 2002

Store-operated Ca++ entry (SOCE) is thought to comprise the major pathway for Ca++ entry in platelets. Recently, a number of transient receptor potential (TRP) proteins, which have been divided into 3 groups (TRPC, TRPM, and TRPV), have been suggested as SOCE channels. We report the expression and function of TRPC proteins in human platelets. TRPC6 is found at high levels and TRPC1 at low levels. Using purified plasma (PM) and intracellular membranes (IM), TRPC6 is found in the PM, but TRPC1 is localized to the IM. Using Fura-2–loaded platelets, we report that, in line with TRPC6 expression, 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the entry of Ca++ and Ba2+ independently of protein kinase C. Thrombin also induced the entry of Ca++ and Ba2+, but thapsigargin, which depletes the stores, induced the entry of only Ca++. Thus, thrombin activated TRPC6 via a SOCE-independent mechanism. In phosphorylation studies, we report that neither TRPC6 nor TRPC1 was a substrate for tyrosine kinases. TRPC6 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK) and associated with other cAMP-PK substrates. TRPC1 was not phosphorylated by cAMP-PK but also associated with other substrates. Activation of cAMP-PK inhibited Ca++ but not Ba2+ entry induced by thrombin and neither Ca++ nor Ba2+entry stimulated by OAG. These results suggest that TRPC6 is a SOCE-independent, nonselective cation entry channel stimulated by thrombin and OAG. TRPC6 is a substrate for cAMP-PK, although phosphorylation appears to not affect cation permeation. TRPC1 is located in IM, suggesting a role at the level of the stores.
The von Willebrand factor–reducing activity of thrombospondin-1 is located in the calcium-binding/C-terminal sequence and requires a free thiol at position 974
Authors: Pimanda John E. , Annis Douglas S. , Raftery Mark , Mosher Deane F. , Chesterman Colin N. , Hogg Philip J.
Published: September 2002

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular glycoproteins consists of 5 members in vertebrates, TSP-1 through TSP-4 and TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca++-binding repeats and C-terminal sequence of TSP-1, but not of TSP-2. Alkylation of Cys974 in the C-terminal TSP-1 construct, which is a serine in TSP-2, ablated VWF reductase activity. These results imply that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence.
Structural requirements for the activation of human factor VIII by thrombin
Authors: Myles Timothy , Yun Thomas H. , Leung Lawrence L. K.
Published: September 2002

The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion-binding exosite-I (Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and anion-binding exosite-II (Arg98Ala), the Na+-binding site (Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the 50-insertion loop (Trp50Ala), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I appears to be important for cleavage at both sites, whereas the anion-binding exosite-II residue Arg98Ala is important for cleavage at Arg372 alone. The Glu229Ala mutant, which contributes to the Na+-binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at Arg372 and Arg1689. This suggests that the integrity of the active site and the Na+-bound form of thrombin are important for its procoagulant activity against FVIII. Detailed mutagenic analysis of thrombin can assist in understanding the pathogenesis of bleeding disorders and may lead to the rational design of selective thrombin inhibitors.
Platelets, circulating tissue factor, and fibrin colocalize in ex vivo thrombi: real-time fluorescence images of thrombus formation and propagation under defined flow conditions
Authors: Balasubramanian Viji , Grabowski Eric , Bini Alessandra , Nemerson Yale
Published: September 2002

Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1Alexa.568 (marker for tissue factor), and anti-T2G1Cy­5 (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s−1. A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury.
Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA)
Authors: Preudhomme Claude , Sagot Christophe , Boissel Nicolas , Cayuela Jean-Michel , Tigaud Isabelle , de Botton Stéphane , Thomas Xavier , Raffoux Emmanuel , Lamandin Charlotte , Castaigne Sylvie , Fenaux Pierre , Dombret Hervé
Published: September 2002

The transcription factor C/EBPα is crucial for differentiation of mature granulocytes. Recently, differentCEBPA gene mutations likely to induce differentiation arrest have been described in nearly 10% of patients with acute myeloid leukemia (AML). In the present study, we retrospectively analyzed the prognostic significance of CEBPA mutations in 135 AML patients (French-American-British [FAB]-M3 excluded). All patients were prospectively enrolled between 1990 and 1996 in a multicenter trial of the ALFA (Acute Leukemia French Association) Group (median age 45 years, median follow-up 5.7 years). Mutations were assessed using direct sequencing of the CEBPA gene. Twenty-two mutations were found in 15 (11%) of 135 patients tested. Twelve patients had at least one mutation located in the N-terminal part of the protein leading to the lack of expression of the full-length C/EBPα protein. CEBPA mutations were present only in patients belonging to the intermediate cytogenetic risk subgroup and associated with the FAB-M1 subtype (P = .02). FLT3 internal tandem duplication (ITD) was found in 5 of 15 CEBPA-mutated as compared with 30 of 119 CEBPA-nonmutated cases tested (P = .54). Presence of CEBPA mutations was identified as an independent good prognosis factor for outcome even after adjustment on cytogenetics and FLT3 status (estimated 5-year overall survival 53% vs 25%, P = .04).FLT3-ITD appeared to act as a major bad prognosis factor in patients with CEBPA-mutated AML. We thus propose a risk classification that includes in the favorable subgroup all patients from the intermediate subgroup displaying CEBPA mutations when not associated with FLT3-ITD.
Marginal zone macrophages express a murine homologue of DC-SIGN that captures blood-borne antigens in vivo
Authors: Geijtenbeek Teunis B. H. , Groot Peter C. , Nolte Martijn A. , van Vliet Sandra J. , Gangaram-Panday Shanti T. , van Duijnhoven Gerard C. F. , Kraal Georg , van Oosterhout Antoon J. M. , van Kooyk Yvette
Published: September 2002

Antigen-presenting cells are localized in essentially every tissue, where they operate at the interface of innate and acquired immunity by capturing pathogens and presenting pathogen-derived peptides to T cells. C-type lectins are important pathogen recognition receptors and the C-type lectin, dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), is unique in that, in addition to pathogen capture, it regulates adhesion processes such as DC trafficking and T-cell synapse formation. We have isolated a murine homologue of DC-SIGN that is identical to the previously reported murine homologue mSIGNR1. mSIGNR1 is more closely related to the human DC-SIGN homologue L-SIGN than to DC-SIGN itself because mSIGNR1 is specifically expressed by liver sinusoidal endothelial cells, similar to L-SIGN, and not by DCs. Moreover, mSIGNR1 is also expressed by medullary and subcapsular macrophages in lymph nodes and by marginal zone macrophages (MZMs) in the spleen. Strikingly, these MZMs are in direct contact with the bloodstream and efficiently capture specific polysaccharide antigens present on the surface of encapsulated bacteria. We have investigated the in vivo function of mSIGNR1 on MZMs in spleen. We demonstrate here that mSIGNR1 functions in vivo as a pathogen recognition receptor on MZMs that capture blood-borne antigens, which are rapidly internalized and targeted to lysosomes for processing. Moreover, the antigen capture is completely blocked in vivo by the blocking mSIGNR1-specific antibodies. Thus, mSIGNR1, a murine homologue of DC-SIGN, is important in the defense against pathogens and this study will facilitate further investigations into the in vivo function of DC-SIGN and its homologues.
Current molecular models for NADPH oxidase regulation by Rac GTPase
Authors: Bokoch Gary M. , Diebold Becky A.
Published: September 2002

Reactive oxygen species (ROS) have been increasingly recognized as important components of cell signaling in addition to their well-established roles in host defense. The formation of ROS in phagocytic and nonphagocytic cells involves membrane-localized and Rac guanosine triphosphatase (GTPase)–regulated reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase(s). We discuss here the current molecular models for Rac GTPase action in the control of the phagocytic leukocyte NADPH oxidase. As a mechanistically detailed example of Rac GTPase signaling, the NADPH oxidase provides a potential paradigm for signaling by Rho family GTPases in general.
The triterpenoid CDDO induces apoptosis in refractory CLL B cells
Authors: Pedersen Irene M. , Kitada Shinichi , Schimmer Aaron , Kim Youngsoo , Zapata Juan M. , Charboneau Lula , Rassenti Laura , Andreeff Michael , Bennett Frank , Sporn Michael B. , Liotta Lance D. , Kipps Thomas J. , Reed John C.
Published: September 2002

Chronic lymphocytic leukemia (CLL) cells develop chemo-resistance over time. Most anticancer agents function through induction of apoptosis, and therefore resistance against these agents is likely to be caused by selection for CLL cells with defects in the particular apoptosis pathway that is triggered by these drugs. Anticancer agents that function through alternative apoptotic pathways might therefore be useful in treating chemo-resistant CLL. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity. We examined the effects of CDDO (triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) on CLL B cells in vitro. CDDO induced apoptosis in a dose-dependent manner in all (n = 30) CLL samples tested, including previously untreated and chemo-resistant CLL specimens. CDDO induced rapid proteolytic processing of caspase-8, but not caspase-9, in CLL B cells, suggesting activation of a mitochondria-independent pathway. CDDO-induced apoptosis of CLL B cells was blocked by cytokine response modifier A (CrmA), a suppressor of caspase-8, but not by X-linked inhibitor of apoptosis protein–baculovirus IAP repeat–3 (XIAP-BIR3), a fragment of XIAP, which selectively inhibits caspase-9. Examination of CDDO effects on expression of several apoptosis-relevant genes demonstrated significant reductions in the levels of caspase-8 homolog Fas-ligand interleukin-1–converting enzyme (FLICE)–inhibitory protein (c-FLIP), an endogenous antagonist of caspase-8. However, reductions of FLIP achieved by FLIP antisense oligonucleotides were insufficient for triggering apoptosis, indicating that CDDO has other targets in CLL B cells besides FLIP. These data suggest that the synthetic triterpenoid CDDO should be further explored as a possible therapeutic agent for treatment of chemo-resistant CLL.
Osteoprotegerin is bound, internalized, and degraded by multiple myeloma cells
Authors: Standal Therese , Seidel Carina , Hjertner Øyvind , Plesner Torben , Sanderson Ralph D. , Waage Anders , Borset Magne , Sundan Anders
Published: September 2002

Multiple myeloma (MM) is a hematologic malignancy characterized by accumulation of plasma cells in the bone marrow (BM). Bone destruction is a complication of the disease and is usually associated with severe morbidity. The balance between receptor activator of nuclear factor-κB (NF-κB) ligand and osteoprotegerin (OPG) is of major importance in bone homeostasis. We have recently shown that serum OPG levels are lower in patients with myeloma than in healthy individuals. Here we show that myeloma cells can bind, internalize, and degrade OPG, thereby providing a possible explanation for the lower levels of OPG in the BM of patients with MM. This process is dependent on interaction of OPG with heparan sulfates on the myeloma cells. The results suggest a novel biologic mechanism for the bone disease associated with MM and that treatment of the bone disease with OPG lacking the heparin-binding domain should be considered.
Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen
Authors: Leo Lorie , Di Paola Jorge , Judd Barbi A. , Koretzky Gary A. , Lentz Steven R.
Published: September 2002

The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRγ. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76−/−) or heterozygous (SLP-76+/−) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76+/− platelets, but not SLP-76−/− platelets (P < .01), and failed to stimulate annexin V binding to either SLP-76+/−or SLP-76−/− platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76+/− or SLP-76−/− platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76+/− and SLP-76−/− platelets (P < .001 versus unstimulated platelets). Similar results were obtained with platelets from FcRγ null mice, for which collagen, but not convulxin, induced procoagulant activity (P < .01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76+/− platelets (P < .05), but not in SLP-76−/− platelets. SLP-76−/− platelets also exhibited less annexin V binding than SLP-76+/−platelets after costimulation with thrombin and convulxin (P < .05). These findings demonstrate that an intact GPVI/FcRγ/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.
Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors
Authors: Galimi Francesco , Noll Meenakshi , Kanazawa Yoshiyuki , Lax Timothy , Chen Cindy , Grompe Markus , Verma Inder M.
Published: September 2002

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes(FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca−/− andFancc−/−mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.
BCR-ABL point mutants isolated from patients with imatinib mesylate–resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90
Authors: Gorre Mercedes E. , Ellwood-Yen Katharine , Chiosis Gabriela , Rosen Neal , Sawyers Charles L.
Published: September 2002

Clinical resistance to imatinib mesylate is commonly observed in patients with advanced Philadelphia chromosome– positive (Ph+) leukemias. Acquired resistance is typically associated with reactivation of BCR-ABL due to kinase domain mutations or gene amplification, indicating that BCR-ABL remains a viable target for inhibition in these patients. Strategies for overcoming resistance can be envisioned through exploitation of other molecular features of the BCR-ABL protein, such as its dependence on the molecular chaperone heat shock protein 90 (Hsp90). To determine whether inhibition of Hsp90 could induce degradation of imatinib mesylate–resistant, mutant BCR-ABL proteins, hematopoietic cells expressing 2 mutant BCR-ABL proteins found in imatinib mesylate–resistant patients (T315I and E255K) were examined for sensitivity to geldanamycin and 17-allylaminogeldanamycin (17-AAG). Both compounds induced the degradation of wild-type and mutant BCR-ABL and inhibited cell growth, with a trend indicating more potent activity against mutant BCR-ABL proteins. These data support clinical investigations of 17-AAG in imatinib mesylate–resistant Ph+ leukemias.
Response of Diamond-Blackfan anemia to metoclopramide: evidence for a role for prolactin in erythropoiesis
Authors: Abkowitz Janis L. , Schaison Gerard , Boulad Farid , Brown Deborah L. , Buchanan George R. , Johnson Christine A. , Murray Jeffrey C. , Sabo Kathleen M.
Published: September 2002

A 47-year-old woman with severe macrocytic anemia markedly improved during the second and third trimesters of 3 pregnancies and when breast-feeding her 2 children. Because the serum prolactin level is elevated at these times, we later treated her with metoclopramide (10 mg orally 3 times daily), a medication known to induce prolactin release. Her serum prolactin levels increased from 7 to 133 ng/mL (normal < 20 ng/mL) and hematocrit from 17% to 22% to 35%. With continued therapy (now 10 mg orally daily), her hematocrit has ranged from 30% to 40% for 6 years, although the macrocytosis persists (mean corpuscular volume, 100-112 fL). On the basis of this observation, a pilot study was undertaken of metoclopramide therapy in patients with Diamond-Blackfan anemia who were refractory to low doses of corticosteroids. Fifteen patients were enrolled and 9 completed the planned 16 weeks of therapy. Three individuals responded, suggesting that this therapeutic approach may benefit others. As with the index case, the anemia did not improve until 12 to 15 weeks of therapy had been completed.
Results of consecutive trials for children newly diagnosed with acute myeloid leukemia from the Australian and New Zealand Children's Cancer Study Group
Authors: O'Brien Tracey A. , Russell Susan J. , Vowels Marcus R. , Oswald Cecilia M. , Tiedemann Karin , Shaw Peter J. , Lockwood Liane , Teague Lochie , Rice Michael , Marshall Glenn M.
Published: September 2002

Despite improvements in the treatment of acute myeloid leukemia (AML), approximately 50% of children die of the disease. Clinical trials in adult patients with AML indicate that idarubicin may have superior efficacy when compared to daunorubicin in the remission-induction phases of chemotherapy. We conducted consecutive clinical trials in children with newly diagnosed AML in which daunorubicin (group 1, n = 102) or idarubicin (group 2, n = 160) was used during the remission-induction (RI) and the early consolidation phases of chemotherapy. Idarubicin was given at a dose of either 10 mg/m2 (group 2A, n = 106) or 12 mg/m2 (group 2B, n = 53). A high rate of RI was achieved for all groups (95% group 1, 90% group 2A, 94% group 2B). There were no significant differences in 5-year event-free survival (EFS) or in overall survival (OS) when the 3 groups were compared (group 1: EFS 50%, OS 56%; group 2A: EFS 50%, OS 60%; group 2B: EFS 34%, OS 50%). RI deaths resulting from treatment toxicity were low—2% for group 1 and 5% for group 2. More gastrointestinal, pulmonary, and renal toxicity but fewer infections were observed in patients receiving idarubicin (P < .001, P = .04,P = .03, respectively). Following RI chemotherapy, all patients received 3 to 4 more courses of identical chemotherapy and then underwent either autologous (n = 156) or an allogeneic bone marrow transplantation (BMT) (n = 35). OS was higher in allogeneic BMT patients than in autologous BMT patients (79% vs 63%;P = .23). We conclude that daunorubicin is as effective as idarubicin for remission-induction therapy for childhood AML and has reduced toxicity.
Gene transfer improves erythroid development in ribosomal protein S19–deficient Diamond-Blackfan anemia
Authors: Hamaguchi Isao , Ooka Andreas , Brun Ann , Richter Johan , Dahl Niklas , Karlsson Stefan
Published: September 2002

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion–dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing theRPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34+ bone marrow (BM) cells from 4 patients with DBA withRPS19 gene mutations. Overexpression of theRPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19gene is feasible.
Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture
Authors: Huygen Sandra , Giet Olivier , Artisien Vincent , Di Stefano Ivano , Beguin Yves , Gothot André
Published: September 2002

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34+ progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of α4 and α5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34+ cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-α4 but not by anti-α5 antibodies. In mitotically activated CD34+ cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on α5 integrin and to a lesser extent on α4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant α4 to a preferential α5 integrin–dependent interaction with Fn.
Myeloid blood CD11c+ dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes
Authors: Osugi Yuko , Vuckovic Slavica , Hart Derek N. J.
Published: September 2002

Dendritic cells (DCs) initiate and direct immune responses. Recent studies have defined different DC populations, therefore we undertook this study comparing 2 types of myeloid DCs: blood CD11c+DCs and in vitro monocyte-derived DCs (Mo-DCs), which are both candidates as cellular adjuvants for cancer immunotherapy. Blood CD11c+ DCs were prepared by cell sorting from peripheral blood mononuclear cells cultured overnight in RPMI 1640 medium supplemented with autologous or pooled AB serum. Mo-DCs were prepared in the same medium using granulocyte macrophage–colony-stimulating factor (GM-CSF)/interleukin 4 (IL-4) and differentiated/activated with lipopolysaccharide or monocyte-conditioned medium (ActMo-DCs). Morphologically, differences between the DC preparations were noted both at a light and and electron microscopic level. Blood CD11c+ DCs expressed similar levels of HLA-DR, CD40, CD86, and CD83 as Mo-DCs. CD209 was present on Mo-DCs but not on blood CD11c+ DCs. Blood CD11c+ DCs generated a lower proliferative mixed leukocyte response (MLR) than Mo-DCs. Blood CD11c+ DCs loaded with 0.1 μg/mL tetanus toxoid (TT)–generated greater T lymphocyte proliferative responses than did Mo-DCs or ActMo-DCs, but when loaded with higher TT concentrations no difference in T lymphocyte proliferative response was observed. Keyhole limpet hemocyanin (KLH)–loaded blood CD11c+ DCs generated greater T lymphocyte proliferative responses than Mo-DCs or ActMo-DCs. Allogeneic MLR- or KLH-specific responses induced by blood CD11c+ DCs generated more Th1 effectors than the responses induced by Mo-DCs or ActMo-DCs. These data establish several differences in the properties of blood CD11c+ DCs, Mo-DCs, and ActMo-DCs, which suggest that blood DCs merit further consideration as DC preparations for clinical programs are evolved.
Induction of apoptosis of human B-CLL and ALL cells by a novel retinoid and its nonretinoidal analog
Authors: Zhang Yuxiang , Dawson Marcia I. , Mohammad Ramzi , Rishi Arun K. , Farhana Lulu , Feng Kai-Chia , Leid Mark , Peterson Valerie , Zhang Xiao-kun , Edelstein Mark , Eilander David , Biggar Sandra , Wall Nathan , Reichert Uwe , Fontana Joseph A.
Published: September 2002

We have recently described a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437/AHPN) that induces apoptosis in a number of malignant cell types. We now describe our studies examining the effects of CD437 and a nonretinoidal analog (MM002) on the in vitro proliferation of the ALL-REH cell line, the in vitro and in vivo growth of a novel Epstein-Barr virus–negative (EBV−) B-cell chronic lymphocytic leukemia (B-CLL) cell line (WSU-CLL), and primary cultures of human B-CLL and acute lymphoblastic leukemia (ALL) cells. CD437 and MM002 induce apoptosis in both cell lines, as indicated by the activation of caspase-2 and caspase-3, cleavage of poly(adenosine diphosphate–ribose) (poly(ADP-ribose)) polymerase, increase in annexin V binding, and subsequent nuclear fragmentation. CD437-mediated apoptosis was not associated with the modulation of Bcl-2, Bax, or Mcl-1 levels, but was associated with the cleavage of the antiapoptotic protein Bcl-XL to a proapoptotic 18-kD form. This cleavage of Bcl-XL was dependent on caspase-3 activation since Bcl-XL cleavage and apoptosis were inhibited by the caspase-3 inhibitor Z-DVED-fmk. CD437 markedly inhibited the growth of WSU-CLL cells in severe combined immunodeficiency (SCID) mice. Tumor growth inhibition, growth delay, and log cell kill were 85.7%, 21 days, and 2.1, respectively, in the treated mice. Moreover, 1 of the 5 treated mice was tumor-free longer than 150 days and thus was considered cured. Exposure of primary cultures of both B-CLL and ALL cells obtained from patients to CD437 and MM002 resulted in their apoptosis. These results suggest that CD437 and MM002 analogs may have a potential role in the treatment of B-CLL and ALL.
Expression of Skp2, a p27Kip1 ubiquitin ligase, in malignant lymphoma: correlation with p27Kip1 and proliferation index
Authors: Lim Megan S. , Adamson Ann , Lin Zhaosheng , Perez-Ordonez Bayardo , Jordan Richard C. K. , Tripp Sheryl , Perkins Sherrie L. , Elenitoba-Johnson Kojo S. J.
Published: September 2002

Reduced levels of p27Kip1 are frequent in human cancers and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27Kip1 degradation. Increased Skp2 levels are seen in some solid tumors and are associated with reduced p27Kip1. We examined the expression of these proteins using single and double immunolabeling in a large series of lymphomas to determine if alterations in their relative levels are associated with changes in cell proliferation and lymphoma subgroups. We studied the expression of Skp2 in low-grade and aggressive B-cell lymphomas (n = 86) and compared them with p27Kip1 and the proliferation index (PI). Fifteen hematopoietic cell lines and peripheral blood lymphocytes were studied by Western blot analysis. In reactive tonsils, Skp2 expression was limited to proliferating germinal center and interfollicular cells. Skp2 expression in small lymphocytic lymphomas (SLLs) and follicular lymphomas (FCLs) was low (mean percentage of positive tumor cells, less than 20%) and was inversely correlated (r = −0.67;P < .0001) with p27Kip1 and positively correlated with the PI (r = 0.82;P < .005). By contrast, whereas most mantle cell lymphomas (MCLs) demonstrated low expression of p27Kip1 and Skp2, a subset (n = 6) expressed high Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression was highest in diffuse large B-cell lymphomas (DLBCLs) (mean, 22%) and correlated with Ki-67 (r = 0.55;P < .005) but not with p27Kip1. Cytoplasmic Skp2 was seen in a subset of aggressive lymphomas. Our data provide evidence for p27Kip1 degradative function of Skp2 in low-grade lymphomas. The absence of this relationship in aggressive lymphomas suggests that other factors contribute to deregulation of p27Kip1 expression in these tumors.