bluuebunny/crossref_metadata_2025 huggingface dataset:
Potentiation of dexamethasone-, paclitaxel-, and Ad-p53–induced apoptosis by Bcl-2 antisense oligodeoxynucleotides in drug-resistant multiple myeloma cells
Authors: Liu Qun , Gazitt Yair
Published: May 2003

Overexpression of Bcl-2 in myeloma cells results in resistance to drugs such as dexamethasone (DEX), adenovirus-mediated delivery of p53 (Ad-p53), and paclitaxel (TAX), which work through the intrinsic apoptotic pathway. Bcl-2 antisense oligodeoxynucleotides (Bcl-2-ASO) have been shown to induce apoptosis in cancer cells, as a single agent or, better, in combination with chemotherapy. We hypothesized that down-regulation of Bcl-2 by Bcl-2-ASO will sensitize drug-resistant myeloma cells to undergo apoptosis. In this paper we report a detailed time/dose study of the effect of Bcl-2-ASO on myeloma cells with varying levels of Bcl-2. Treatment of myeloma cells expressing relatively low levels of Bcl-2 with Bcl-2-ASO resulted in a substantial apoptosis concomitant with a substantial depletion of Bcl-2 protein. Maximal apoptosis was observed at 5 to 10 μg/mL Bcl-2-ASO, following 4 days of treatment. Down-regulation of Bcl-2 and apoptosis were time and dose dependent and were sequence specific. In these cell lines, apoptosis was accompanied by activation of caspase-9 and caspase-3 and by release of cytochrome c to the cytosol. In contrast, high Bcl-2–expressing myeloma cells were practically resistant to Bcl-2-ASO. Most important, however, pretreatment of myeloma cells expressing high levels of Bcl-2 with Bcl-2-ASO increased the extent of DEX-, TAX-, and Ad-p53–induced apoptosis from 10%-20% to 70%-90%. Increased apoptosis was accompanied by additional decrease in Bcl-2 protein. Similar results for down-regulation of Bcl-2 and apoptosis were obtained with freshly isolated myeloma cells. These data support development of clinical trials with combinations of Bcl-2-ASO and DEX, TAX, or Ad-p53 in the treatment of refractory myeloma patients.
Transfusion-transmitted cytomegalovirus infection after receipt of leukoreduced blood products
Authors: Nichols W. Garrett , Price Thomas H. , Gooley Ted , Corey Lawrence , Boeckh Michael
Published: May 2003

Leukoreduced blood products are reportedly comparable to cytomegalovirus (CMV)–seronegative products for the prevention of transfusion-transmitted CMV (TT-CMV) infection after stem cell (SC) transplantation. To determine if the incidence of TT-CMV was affected by the increasing use of leukoreduced blood products, we followed a prospective cohort of 807 CMV-seronegative SC transplant (SCT) recipients who underwent weekly surveillance using the pp65 antigenemia assay. The incidence of TT-CMV for 2 time periods was recorded: Period 1 (5/94-11/96), when only CMV-seronegative and/or filtered blood products were provided, and period 2 (12/96-2/00), when leukocyte-reduced platelets obtained by apheresis without filtration were also used. The incidence of TT-CMV was higher during period 2 (18/447, 4%) than period 1 (6/360, 1.7%) (P < .05); this was correlated with higher utilization of both filtered and apheresed products from CMV-positive donors in period 2. Multivariable analysis identified filtered red blood cell (RBC) units (but not apheresis platelet products) from CMV-positive donors as the primary predictor of TT-CMV: each additional filtered RBC unit was associated with a 32% increase in the odds for TT-CMV (95% confidence interval [CI]: 8%-61%, P = .006). Pre-emptive therapy with ganciclovir after detection of antigenemia prevented all but one case of CMV disease prior to day 100. CMV-seronegative products may thus be superior to leukoreduced products (particularly filtered RBCs) for the prevention of TT-CMV. In an era of “universal leukoreduction,” the abandonment of CMV-seronegative inventories appears premature, particularly among populations at high risk of CMV disease that do not receive active surveillance.
Increased sensitivity of Fancc-deficient hematopoietic cells to nitric oxide and evidence that this species mediates growth inhibition by cytokines
Authors: Hadjur Suzana , Jirik Frank R.
Published: May 2003

Fanconi anemia complementation group C (Fancc)–deficient murine bone marrow progenitors demonstrate increased sensitivity to growth inhibition by interferon γ (IFNγ), tumor necrosis factor α (TNFα), and macrophage inflammatory protein 1α (MIP-1α). This property has been proposed as a possible pathogenic factor in the marrow failure seen in Fanconi anemia. Supporting our hypothesis that nitric oxide (NO) production might be a common effector in this sensitivity, we found that cytokine-mediated growth inhibition ofFancc−/− bone marrow cells was prevented by inhibiting NO synthase activity. Interestingly,Fancc−/− hematopoietic cells also exhibited increased growth inhibition on exposure to 2 distinct NO-generating agents, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and diethylenetriamine nitric oxide adduct (DETA/NO). In keeping with the sensitivity of Fancc−/− cells to IFNγ, inducible nitric oxide synthase (iNOS) levels and nitrite release were both increased following stimulation ofFancc−/− macrophages with this cytokine, either alone or in combination with bacterial lipopolysaccharide. Suggesting a plausible mechanism for the increased expression of iNOS, IFNγ-stimulated Fancc−/− macrophages generated higher levels of phospho-Stat1, a positive regulator ofinos (nos2) gene expression. These observations, while confined to C57BL/6 Fancc−/−hematopoietic cells, raise the possibility that nitric oxide has a role in the pathogenesis of Fanconi anemia.
Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon α with an anti-CD20
Authors: Sansonno Domenico , De Re Valli , Lauletta Gianfranco , Tucci Felicia Anna , Boiocchi Mauro , Dammacco Franco
Published: May 2003

A controlled study has been carried out to assess the efficacy of rituximab, a chimeric antibody that binds to the B-cell surface antigen CD20, in 20 patients with mixed cryoglobulinemia (MC) and hepatitis C virus (HCV)–positive chronic active liver disease, resistant to interferon α (IFN-α) therapy. They received an intravenous infusion of 375 mg/m2 rituximab once a week for 4 consecutive weeks. Infusion of rituximab had a good safety profile and no severe side effects were reported. Sixteen patients (80%) showed a complete response (CR), characterized by rapid improvement of clinical signs (disappearance of purpura and weakness arthralgia and improvement of peripheral neuropathy), and decline of cryocrit. CR was associated with a significant reduction of rheumatoid factor (RF) activity and anti-HCV antibody titers. Decline of IgG anti-HCV titers in the cryoprecipitates was usually associated with a favorable response (r = 0.81; P < .005). No differences in the dynamics of B-cell depletion and recovery were found between responders and nonresponders. Molecular monitoring of the B-cell response revealed disappearance/deletion of peripheral clones in the responders and great stability in the nonresponders. Rituximab had a deep impact on hepatitis C viremia; HCV RNA increased approximately twice the baseline levels in the responders, whereas it remained much the same in the nonresponders. Twelve (75%) of 16 responders remained in remission throughout the follow-up. The results indicate that rituximab has clinical and biologic activity in patients with HCV+ MC. However, in view of the increased viremia in the responders, additional modes of application and combination of rituximab with other agents need to be investigated.
Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation
Authors: Oppezzo Pablo , Vuillier Françoise , Vasconcelos Yuri , Dumas Gérard , Magnac Christian , Payelle-Brogard Béatrice , Pritsch Otto , Dighiero Guillaume
Published: May 2003

In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch μ DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.
PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A
Authors: Moon Eun-Yi , Lerner Adam
Published: May 2003

Chronic lymphocytic leukemia (CLL) cells, but not peripheral blood T cells, undergo apoptosis following treatment with inhibitors of type 4 cyclic nucleotide phosphodiesterase (PDE4), a process that correlates dose dependently with elevation of adenosine 3′,5′-cyclic monophosphate (cAMP) in leukemic cells. We show that treatment of CLL cells with rolipram, a prototypic PDE4 inhibitor, and forskolin, an adenylate cyclase activator, induces mitochondrial depolarization, release of cytochrome c into the cytosol, caspase-9 and -3 activation, and cleavage of poly(adenosine diphosphate [ADP]–ribose)polymerase. Inhibitors of caspase-9, but not caspase-8, block rolipram/forskolin-induced CLL apoptosis. In a subset of CLL patients, B-cell lymphoma 2 (Bcl-2)–associated death promoter homolog (Bad), a proapoptotic Bcl-2 family member that when phosphorylated on specific serine residues is sequestered in the cytosol by 14-3-3, was dephosphorylated at Ser112 following rolipram/forskolin treatment of leukemic cells. Rolipram/forskolin treatment also induced Bad to accumulate in CLL heavy-membrane fractions, consistent with Bad translocation to mitochondria. To determine the mechanism for rolipram/forskolin-induced Bad dephosphorylation, we examined CLL phosphatase activity. Rolipram/forskolin treatment augmented protein phosphatase 2A (PP2A) activity, as well as levels of immunoreactive PP2A catalytic subunit. Treatment of CLL cells with a concentration of okadaic acid (5 nM) that selectively inhibits PP2A, reduced both rolipram/forskolin-induced mitochondrial cytochrome c release and mitochondrial depolarization. Okadaic acid restored Bad Ser112 phosphorylation and Bad association with 14-3-3 in rolipram/forskolin-treated CLL cells. These results suggest that PDE4 inhibitors may induce CLL apoptosis by activating PP2A-induced dephosphorylation of proapoptotic BH3-only Bcl-2 family members such as Bad.
Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothrombotic phenotype
Authors: Oury Cécile , Kuijpers Marijke J. E. , Toth-Zsamboki Emese , Bonnefoy Arnaud , Danloy Sophie , Vreys Ingrid , Feijge Marion A. H. , De Vos Rita , Vermylen Jos , Heemskerk Johan W. M. , Hoylaerts Marc F.
Published: May 2003

We have generated transgenic mice overexpressing the human P2X1 ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X1ionotropic activity as determined by more prominent P2X1-mediated Ca2+ influx and platelet shape change. P2X1 overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A2 mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds−1 resulted in increased P2X1-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X1 ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A2-, and shear stress–triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.
Arsenic trioxide–induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL
Authors: Liu Qun , Hilsenbeck Susan , Gazitt Yair
Published: May 2003

Arsenic trioxide (ATO) has been shown to induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells concomitant with down-regulation of the PML-RARα fusion protein, a product of the t(15:17) translocation characteristic of APL leukemic cells. However, ATO is also a potent inducer of apoptosis in a number of other cancer cells lacking the t(15:17) translocation. The exact mechanism of ATO-induced apoptosis in these cells is not yet clear. We tested the effect of ATO on 7 myeloma cell lines with varying p53 status and report that in cells with mutated p53, ATO induced rapid and extensive (more than 90%) apoptosis in a time- and dose-dependent manner concomitant with arrest of cells in G2/M phase of the cell cycle. Myeloma cells with wild-type (wt) p53 were relatively resistant to ATO with maximal apoptosis of about 40% concomitant with partial arrest of cells in G1 and up-regulation of p21. The use of caspase blocking peptides, fluorescence-tagged caspase-specific substrate peptides, and Western immunoblotting confirmed the involvement of primarily caspase-8 and -3 in ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in cells expressing wt p53. We also observed up-regulation by ATO of R1 and R2 APO2/TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) receptors. Most important, however, we observed a synergy between ATO and APO2/TRAIL in the induction of apoptosis in the partially resistant myeloma cell lines and in myeloma cells freshly isolated from myeloma patients. Our results justify the use of the combination of these 2 drugs in clinical setting in myeloma patients.
Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1
Authors: Reca Ryan , Mastellos Dimitrios , Majka Marcin , Marquez Leah , Ratajczak Janina , Franchini Silvia , Glodek Aleksandra , Honczarenko Marek , Spruce Lynn A. , Janowska-Wieczorek Anna , Lambris John D. , Ratajczak Mariusz Z.
Published: May 2003

Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.
Targeting of the collagen-binding site on glycoprotein VI is not essential for in vivo depletion of the receptor
Authors: Schulte Valerie , Rabie Tamer , Prostredna Miroslava , Aktas Barsom , Grüner Sabine , Nieswandt Bernhard
Published: May 2003

Glycoprotein (GP) VI is an essential collagen receptor on platelets and may serve as an attractive target for antithrombotic therapy. We have previously shown that a monoclonal antibody (mAb) against the major collagen-binding site on mouse GPVI (JAQ1) induces irreversible down-regulation of the receptor and, consequently, long-term antithrombotic protection in vivo. To determine whether this unique in vivo effect of JAQ1 is based on its interaction with the ligand-binding site on GPVI, we generated new mAbs against different epitopes on GPVI (JAQ2, JAQ3) and tested their in vitro and in vivo activity. We show that none of the mAbs inhibited platelet activation by collagen or the collagen-related peptide in vitro. Unexpectedly, however, injection of either antibody induced depletion of GPVI with the same efficacy and kinetics as JAQ1. Importantly, this effect was also seen with monovalent F(ab) fragments of JAQ2 and JAQ3, excluding the involvement of the Fc part or the dimeric form of anti-GPVI antibodies in this process. This indicates that anti-GPVI agents, irrespective of their binding site may generally induce down-regulation of the receptor in vivo.
Clonal chromosomal aberrations in bone marrow cells of Fanconi anemia patients: gains of the chromosomal segment 3q26q29 as an adverse risk factor
Authors: Tönnies Holger , Huber Stefanie , Kühl Jörn-Sven , Gerlach Antje , Ebell Wolfram , Neitzel Heidemarie
Published: May 2003

Fanconi anemia (FA) is a condition that induces susceptibility to bone marrow failure, myelodysplastic syndrome (MDS), and leukemia. We report on a high incidence of expanding clonal aberrations with partial trisomies and tetrasomies of chromosome 3q in bone marrow cells of 18 of 53 FA patients analyzed, detected by conventional and molecular cytogenetics. To determine the clinical relevance of these findings, we compared the cytogenetic data, the morphologic features of the bone marrow, and the clinical course of these patients with those of 35 FA patients without clonal aberrations of 3q. The 2 groups did not differ significantly with respect to age, sex, or complementation group. There was a significant survival advantage of patients without abnormalities of chromosome 3q. Even more pronounced was the risk assessment of patients with gains of 3q material with respect to the development of morphologic MDS and acute myeloid leukemia (AML). Thus, our data from 18 patients with 3q aberrations reveal that gains of 3q are strongly associated with a poor prognosis and represent an adverse risk factor in FA.
166Ho-DOTMP plus melphalan followed by peripheral blood stem cell transplantation in patients with multiple myeloma: results of two phase 1/2 trials
Authors: Giralt Sergio , Bensinger William , Goodman Mark , Podoloff Donald , Eary Janet , Wendt Richard , Alexanian Raymond , Weber Donna , Maloney David , Holmberg Leona , Rajandran Joseph , Breitz Hazel , Ghalie Richard , Champlin Richard
Published: May 2003

Holmium-166 1, 4, 7, 10-tetraazcyclododecane-1, 4, 7, 10-tetramethylenephosphonate (166Ho-DOTMP) is a radiotherapeutic that localizes specifically to the skeleton and can deliver high-dose radiation to the bone and bone marrow. In patients with multiple myeloma undergoing autologous hematopoietic stem cell transplantation two phase 1/2 dose-escalation studies of high-dose 166Ho-DOTMP plus melphalan were conducted. Patients received a 30 mCi (1.110 Gbq) tracer dose of 166Ho-DOTMP to assess skeletal uptake and to calculate a patient-specific therapeutic dose to deliver a nominal radiation dose of 20, 30, or 40 Gy to the bone marrow. A total of 83 patients received a therapeutic dose of 166Ho-DOTMP followed by autologous hematopoietic stem cell transplantation 6 to 10 days later. Of the patients, 81 had rapid and sustained hematologic recovery, and 2 died from infection before day 60. No grades 3 to 4 nonhematologic toxicities were reported within the first 60 days. There were 27 patients who experienced grades 2 to 3 hemorrhagic cystitis, only 1 of whom had received continuous bladder irrigation. There were 7 patients who experienced complications considered to be caused by severe thrombotic microangiopathy (TMA). No cases of severe TMA were reported in patients receiving in 166Ho-DOMTP doses lower than 30 Gy. Approximately 30% of patients experienced grades 2 to 4 renal toxicity, usually at doses targeting more than 40 Gy to the bone marrow. Complete remission was achieved in 29 (35%) of evaluable patients. With a minimum follow-up of 23 months, the median survival had not been reached and the median event-free survival was 22 months. 166Ho-DOTMP is a promising therapy for patients with multiple myeloma and merits further evaluation. (Blood. 2003;102:2684-2691)
Involvement of CXCR4 and IL-2 in the homing and retention of human NK and NK T cells to the bone marrow and spleen of NOD/SCID mice
Authors: Beider Katia , Nagler Arnon , Wald Ori , Franitza Suzanna , Dagan-Berger Michal , Wald Hanna , Giladi Hilla , Brocke Stefan , Hanna Jacob , Mandelboim Ofer , Darash-Yahana Merav , Galun Eithan , Peled Amnon
Published: May 2003

Human natural killer (NK) and NK T cells play an important role in allogeneic bone marrow (BM) transplantation and graft-versus-leukemia (GVL) effect. The mechanisms by which these cells home to the BM and spleen are not well understood. Here we show that treatment of these cells with pertussis toxin and neutralizing antibodies to the chemokine receptor CXCR4 inhibited homing of the cells to the BM, but not the spleen, of NOD/SCID mice. The retention of NK and NK T cells within the spleen and BM was dependent on Gαi signaling and CXCR4 function. The chemokine receptors CXCR4 and CXCR3 are expressed predominantly on the cell surface of NK T cells. Following activation with interleukin-2 (IL-2), the levels of CXCR4 on NK and NK T cells decreased significantly. Treatment of cells with IL-2 inhibited their migration in response to CXCL12 and their homing and retention in the BM and spleen of NOD/SCID mice. In contrast to CXCR4, the expression levels of the chemokine receptor CXCR3 and the migration of cells in response to CXCL9 and CXCL10 increased after IL-2 treatment. Thus, down-regulation of CXCR4 and up-regulation of CXCR3 may direct the trafficking of cells to the site of inflammation, rather than to hematopoietic organs, and therefore may limit their alloreactive potential.
Transgenic sickle mice have vascular inflammation
Authors: Belcher John D. , Bryant Christopher J. , Nguyen Julia , Bowlin Paul R. , Kielbik Miroslaw C. , Bischof John C. , Hebbel Robert P. , Vercellotti Gregory M.
Published: May 2003

Inflammation may play an essential role in vaso-occlusion in sickle cell disease. Sickle patients have high white counts and elevated levels of serum C-reactive protein (CRP), cytokines, and adhesion molecules. In addition, circulating endothelial cells, leukocytes, and platelets are activated. We examined 4 transgenic mouse models expressing human α- and sickle β-globin genes to determine if they mimic the inflammatory response seen in patients. These mouse models are designated NY-S, Berk-SAntilles, NY-S/SAntilles (NY-S × Berk-SAntilles), and Berk-S. The mean white counts were elevated 1.4- to 2.1-fold (P ≤ .01) in the Berk-SAntilles, NY-S/SAntilles, and Berk-S mice, but not in the NY-S mice compared with controls. Serum amyloid P-component (SAP), an acute-phase response protein with 60% to 70% sequence homology to CRP, was elevated 8.5- to 12.1-fold (P ≤ .001) in transgenic sickle mice. Similarly, serum interleukin-6 (IL-6) was elevated 1.6- to 1.9-fold (P ≤ .05). Western blots, confirming immunohistochemical staining, showed vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and platelet-endothelial cell adhesion molecule (PECAM) were up-regulated 3- to 5-fold (P ≤ .05) in the lungs of sickle mice. Ribonuclease protection assays (RPAs) demonstrated VCAM mRNA also was elevated in sickle mice 1.2- to 1.4-fold (P ≤ .01). Nuclear factor κB (NF-κB), a transcription factor critical for the inflammatory response, was elevated 1.9-fold (P ≤ .006) in NY-S sickle mouse lungs. We conclude that transgenic sickle mice are good models to study vascular inflammation and the potential benefit of anti-inflammatory therapies to prevent vaso-occlusion in sickle cell disease.
The heat-shock protein receptor CD91 is up-regulated in monocytes of HIV-1–infected “true” long-term nonprogressors
Authors: Stebbing Justin , Gazzard Brian , Kim Louise , Portsmouth Simon , Wildfire Adrian , Teo Ian , Nelson Mark , Bower Mark , Gotch Frances , Shaunak Sunil , Srivastava Pramod , Patterson Steve
Published: May 2003

A small proportion of patients with human immunodeficiency virus type 1 (HIV-1) remains asymptomatic for a long period after infection. It is thought that a vigorous immune response may contribute to long-term nonprogression, though studies are confounded by heterogeneity among patients. We studied the levels of HIV-1 receptors, costimulatory T-cell molecules, and dendritic cell (DC) numbers in 18 patients with long-term infection, CD4 counts greater than 400 cells/mm3, and HIV-1 viral loads lower than 50 copies/mL. These patients were further differentiated through the presence or absence of 2-LTR DNA circles, a possible marker for residual ongoing HIV-1 replication. A statistically significant increase in levels of CD91, the heat-shock protein (HSP) receptor, was observed in therapy-naive patients who had no evidence of ongoing viral replication (P = .01). This difference was most notable on their monocytes. High levels of CD91 may be a host factor that contributes to the maintenance of long-term nonprogression. The ability of CD91 to internalize α-defensins and to cross-present exogenous antigen to cytotoxic T lymphocytes through major histocompatibility complex (MHC) class 1 may maintain CD8+ responses in these patients.
β2-Microglobulin as a negative regulator of the immune system: high concentrations of the protein inhibit in vitro generation of functional dendritic cells
Authors: Xie Jin , Wang Ying , Freeman Muta E. , Barlogie Bart , Yi Qing
Published: May 2003

Two common features in human immunodeficiency virus infection and acquired immunodeficiency syndrome, rheumatoid arthritis, and hematologic malignancies including multiple myeloma are elevated serum levels of β2-microglobulin (β2M) and activation or inhibition of the immune system. We hypothesized that β2M at high concentrations may have a negative impact on the immune system. In this study, we examined the effects of β2M on monocyte-derived dendritic cells (MoDCs). The addition of β2M (more than 10 μg/mL) to the cultures reduced cell yield, inhibited the up-regulation of surface expression of human histocompatibility leukocyte antigen (HLA)–ABC, CD1a, and CD80, diminished their ability to activate T cells, and compromised generation of the type-1 T-cell response induced in allogeneic mixed-lymphocyte reaction. Compared with control MoDCs, β2M-treated cells produced more interleukin-6 (IL-6), IL-8, and IL-10. β2M-treated cells expressed significantly fewer surface CD83, HLA-ABC, costimulatory molecules, and adhesion molecules and were less potent at stimulating allospecific T cells after an additional 48-hour culture in the presence of tumor necrosis factor-α and IL-1β. During cell culture, β2M down-regulated the expression of phosphorylated mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), and mitogen-induced extracellular kinase (MEK), inhibited nuclear factor-κB (NF-κB), and activated signal transducer and activator of transcription-3 (STAT3) in treated cells, all of which are involved in cell differentiation and proliferation. Thus, our study demonstrates that β2M at high concentrations retards the generation of MoDCs, which may involve down-regulation of major histocompatibility complex class I molecules, inactivation of Raf/MEK/ERK cascade and NF-κB, and activation of STAT3, and it merits further study to elucidate the underlying mechanisms.
Cell context–specific effects of the BCR-ABL oncogene monitored in hematopoietic progenitors
Authors: Wong Stephane , McLaughlin Jami , Cheng Donghui , Witte Owen N.
Published: May 2003

Acute BCR-ABL expression during in vitro hematopoietic development of embryonic stem (ES) cells causes expansion of multipotent and myeloid progenitors with a concomitant reduction in differentiation toward erythroblasts. Progenitor cell expansion is due to a rapid, cell autonomous, suppression of programmed cell death with an increase in expression of the antiapoptotic molecule BCL-XL. Other antiapoptotic effectors, including AKT, STAT5, and BCL-2 are not up-regulated by BCR-ABL in this system. In addition, the proapoptotic p38 mitogen–activated protein kinase (MAPK) pathway is suppressed by BCR-ABL expression in ES-derived hematopoietic progenitors. Inhibition of p38 MAPK by the small molecule inhibitor SB203580 expanded ES-derived hematopoietic progenitors by an antiapoptotic mechanism and is sufficient to expand ES-derived hematopoietic progenitors to levels approaching 80% of that seen following BCR-ABL expression. In the cellular context of ES-derived hematopoietic progenitors, BCR-ABL expression expands cells by suppressing programmed cell death with a set of antiapoptotic pathways distinct from those previously reported in continuous cell line studies.
Leukocyte antiadhesive actions of annexin 1: ALXR- and FPR-related anti-inflammatory mechanisms
Authors: Gavins Felicity N. E. , Yona Simon , Kamal Ahmad M. , Flower Roderick J. , Perretti Mauro
Published: May 2003

Recent investigations conducted with human neutrophils have indicated an involvement for the receptor for formylated peptides, termed FPR, and its analog FPRL1 (or ALXR because it is the receptor for the endogenous ligand lipoxin A4) in the in vitro inhibitory actions of the glucocorticoid-regulated protein annexin 1 and its peptidomimetics. To translate these findings in in vivo settings, we have used an ischemia/reperfusion (I/R) procedure to promote leukocyte-endothelium interactions in the mouse mesenteric microcirculation. In naive mice, the annexin 1 mimetic peptide Ac2-26 (20 to 100 μg administered intravenously prior to reperfusion) abolished I/R-induced cell adhesion and emigration, but not cell rolling. In FPR-deficient mice, peptide Ac2-26 retained significant inhibitory actions (about 50% of the effects in naive mice), and these were blocked by an FPR antagonist, termed butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe, or Boc2. In vitro, neutrophils taken from these animals could be activated at high concentrations of formyl-Met-Leu-Phe (30 μM; fMLP), and this effect was blocked by cell incubation with peptide Ac2-26 (66 μM) or Boc2 (100 μM). FPR-deficient neutrophils expressed ALXR mRNA and protein. Both ALXR agonists, lipoxin A4 and peptide Ac2-26, provoked detachment of adherent leukocytes in naive as well as in FPR-deficient mice, whereas the CXC chemokine KC or fMLP were inactive. The present findings demonstrate that endogenous regulatory autocoids such as lipoxin A4 and annexin 1–derived peptides function to disengage adherent cells during cell-cell interactions.
Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells
Authors: Waller Karena L. , Nunomura Wataru , An Xiuli , Cooke Brian M. , Mohandas Narla , Coppel Ross L.
Published: May 2003

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.
Molecular sequelae of histone deacetylase inhibition in human malignant B cells
Authors: Mitsiades Nicholas , Mitsiades Constantine S. , Richardson Paul G. , McMullan Ciaran , Poulaki Vassiliki , Fanourakis Galinos , Schlossman Robert , Chauhan Dharminder , Munshi Nikhil C. , Hideshima Teru , Richon Victoria M. , Marks Paul A. , Anderson Kenneth C.
Published: May 2003

Histone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid–based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor–mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion–mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.
Synergistic effect of sphingosine 1-phosphate on thrombin-induced tissue factor expression in endothelial cells
Authors: Takeya Hiroyuki , Gabazza Esteban C. , Aoki Shinya , Ueno Hikaru , Suzuki Koji
Published: May 2003

Sphingosine 1-phosphate (S1P), a bioactive lipid, is produced and stored in platelets and is released from activated platelets during blood coagulation activation. Thrombin, which is also generated during blood coagulation, has been shown to induce tissue factor (TF), the initiator of blood coagulation, in endothelial cells (ECs); however, the effect of S1P on this process is not evaluated. Here we demonstrated that S1P strongly potentiated thrombin-induced TF expression in ECs and that S1P itself did not induce TF expression. Among signaling lipids, platelet-activating factor slightly enhanced thrombin-induced TF expression; other lipids, including lysophosphatidic acid, lysophosphatidylcholine, sphingosine, and C2-ceramide exert no effect on TF expression. S1P enhanced TF expression at the transcriptional level, possibly via promoting the activation of transcription factors nuclear factor–κB (NF-κB) and Egr-1. Thrombin weakly and S1P strongly activated extracellular signal–regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase and, in the presence of both stimulants, enhanced and sustained activation of this kinase was observed. The ERK1/2-specific inhibitor PD98059 significantly inhibited enhanced TF expression induced by both stimulants but only weakly inhibited thrombin-induced TF expression, thus indicating the requirement of the ERK1/2 pathway in synergistic induction of TF expression. In addition, we found that thrombin and S1P rapidly up-regulated the expression of S1P receptors, endothelial differentiation gene-1 (EDG-1) and EDG-3, thereby suggesting that the effect of S1P on TF expression and other EC functions may be enhanced by thrombin and S1P itself. The present data reveal the synergistic effect of S1P on thrombin-induced TF expression in ECs, which may promote further thrombin and S1P generation, thus propagating a positive feedback reaction.
Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3
Authors: Teixeira Christine , Stang Stacey L. , Zheng Yong , Beswick Naomi S. , Stone James C.
Published: May 2003

Members of the RasGRP family of Ras activators have C1 domains that bind diacylglycerol (DAG) and DAG analogs such as the tumor-promoting phorbol esters. RasGRP members could be responsible for some of the DAG signaling processes that have previously been attributed to protein kinase C (PKC). We found that RasGRP3 is selectively expressed in B cells, suggesting that RasGRP3 might function downstream of the B-cell receptor (BCR). Indeed, stimulation of Ramos B cells with the DAG analog phorbol ester myristate (PMA) results in the association of RasGRP3 with the membrane fraction. However, we also made the unexpected observation that RasGRP3 is phosphorylated, coincident with Ras activation after stimulation. When inhibitors of PKC are present, Ras activation is attenuated, and this attenuation correlates with an inhibition of RasGRP3 phosphorylation. RasGRP3 is phosphorylated in vitro by PKC-θ and PKC-β2. When ectopically coexpressed in HEK-293 cells, a dominant-activated mutant of PKC-θ phosphorylates RasGRP3 and enhances Ras-Erk signaling. These results provide the first indication for a functional interaction between a RasGRP family member and a dissimilar DAG binding protein. A convergent DAG signaling system could be important in fine-tuning Ras signaling during B-cell development or during the humoral immune response.
Microsatellite polymorphism in heme oxygenase-1 gene promoter is associated with susceptibility to oxidant-induced apoptosis in lymphoblastoid cell lines
Authors: Hirai Hisao , Kubo Hiroshi , Yamaya Mutsuo , Nakayama Katsutoshi , Numasaki Muneo , Kobayashi Seiichi , Suzuki Satoshi , Shibahara Shigeki , Sasaki Hidetada
Published: May 2003

Heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. A (GT)n dinucleotide repeat in the 5′-flanking region of human HO-1 gene shows length polymorphism, which was classified into S (< 27 GT), M (27-32 GT), and L alleles (≥ 33 GT). Polymorphism in the HO-1 gene promoter was shown to be associated with susceptibility to pulmonary emphysema and restenosis after angioplasty. However, the biologic mechanism underlying these associations is still unclear. To examine this issue, we established lymphoblastoid cell lines (LCLs) from subjects possessing S/S or L/L genotypes. HO-1 mRNA expressions and HO activities induced by oxidative stress were significantly higher in LCLs with S/S than those with L/L. Furthermore, LCLs with S/S were significantly more resistant to oxidant-induced apoptosis than those with L/L. These findings suggested that the polymorphism of the HO-1 gene is associated with the strength of antiapoptotic effects of HO-1, resulting in an association with susceptibility to oxidative stress–mediated diseases.
Cyclin D1 overexpression is a favorable prognostic variable for newly diagnosed multiple myeloma patients treated with high-dose chemotherapy and single or double autologous transplantation
Authors: Soverini Simona , Cavo Michele , Cellini Claudia , Terragna Carolina , Zamagni Elena , Ruggeri Deborah , Testoni Nicoletta , Tosi Patrizia , de Vivo Antonio , Amabile Marilina , Grafone Tiziana , Ottaviani Emanuela , Giannini Barbara , Cangini Delia , Bonifazi Francesca , Neri Antonino , Fabris Sonia , Tura Sante , Baccarani Michele , Martinelli Giovanni
Published: May 2003

We used a sensitive real-time reverse transcription–polymerase chain reaction assay to quantify cyclin D1 mRNA levels in bone marrow samples collected at diagnosis from 74 newly diagnosed multiple myeloma (MM) patients who were randomized to undergo either single or double autologous peripheral blood stem cell transplantation as part of first-line therapy for their malignancy. In 46 cases, fluorescence in situ hybridization (FISH) analysis and/or conventional cytogenetics were performed to detect chromosome 11 abnormalities. Patients with the t(11;14) or trisomy 11 significantly overexpressed cyclin D1 (P < .0001) in comparison with patients without 11q abnormalities, who had cyclin D1 mRNA levels similar to healthy donors. Overall, 32 (43%) of 74 patients showed cyclin D1 overexpression. No difference was found between cyclin D1–positive (group A) and cyclin D1–negative (group B) patients with respect to presenting clinical and laboratory characteristics, including chromosome 13 abnormalities, as well as to response to therapy and overall survival, both of which were calculated on an intent-to-treat basis. Patients who overexpressed cyclin D1 had significantly longer duration of remission in comparison with patients who did not (41 vs 26 months, respectively; P = .02). As a result, median event-free survival (EFS) was longer in group A than in group B (33 vs 24 months, respectively; P = .055). We concluded that cyclin D1 overexpression is closely associated with 11q abnormalities and identifies a subset of MM patients who are more likely to have prolonged duration of remission and EFS following autologous transplantation.
The nonenzymatic subunit of pseutarin C, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V
Authors: Rao Veena S. , Swarup Sanjay , Kini R. Manjunatha
Published: May 2003

Pseutarin C is a group C prothrombin activator from the venom of the eastern brown snake Pseudonaja textilis. It is a multi-subunit protein complex consisting of catalytic and nonenzymatic subunits similar to coagulation factor Xa and factor Va, respectively. Here we describe the complete sequence of the nonenzymatic subunit. Based on the partial amino acid sequence of the nonenzymatic subunit, degenerate primers were designed. Using a “walking” strategy based on sequentially designed primers, we determined the complete cDNA sequence of the nonenzymatic subunit. The cDNA encodes a protein of 1461 amino acid residues, which includes a 30-residue signal peptide, a mature protein of 1430 amino acid residues, and a stop codon. cDNA blot analysis showed a single transcript of approximately 4.6 kb. The deduced amino acid sequence shows approximately 50% identity to mammalian factor V and by homology has a similar domain structure consisting of domains A1-A2-B-A3-C1-C2. Interestingly, the B domain of pseutarin C is shorter than that of mammalian factor V (FV). Although most of the proteolytic activation sites are conserved, 2 of 3 proteolytic sites cleaved by activated protein C are mutated, and thus activated protein C is not able to inactivate this procoagulant toxin. The predicted posttranslational modifications, including disulfide bonds, N-glycosylation, phosphorylation, and sulfation, in pseutarin C are significantly different compared with bovine factor V. Thus, our data demonstrate that the nonenzymatic subunit of group C prothrombin activators is structurally similar to mammalian FV.
Murine GPVI stimulates weak integrin activation in PLCγ2–/– platelets: involvement of PLCγ1 and PI3-kinase
Authors: Suzuki-Inoue Katsue , Inoue Osamu , Frampton Jon , Watson Steve P.
Published: May 2003

Collagen stimulates platelet activation through a tyrosine kinase–based pathway downstream of the glycoprotein VI (GPVI)–Fc receptor (FcR) γ-chain complex. Genetic ablation of FcR γ-chain results in a complete inhibition of aggregation to collagen. In contrast, a steady increase in light transmission is induced by collagen in phospholipase Cγ2–deficient (PLCγ2–/–) platelets in a Born aggregometer, indicating a weak level of activation. This increase is inhibited partially in the presence of an α2β1-blocking antibody or an αIIbβ3 antagonist and completely by a combination of the 2 inhibitors. It is also abolished by the Src kinase inhibitor PP1 and reduced in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. The GPVI-specific agonists convulxin and collagen-related peptide (CRP) also stimulate weak aggregation in PLCγ2–/– platelets, which is inhibited by wortmannin and PP1. Collagen and CRP stimulate tyrosine phosphorylation of PLCγ1 at its regulatory site, Tyr 783, in murine but not in human platelets through a Src kinase–dependent pathway. Adhesion of PLCγ2–/– platelets to a collagen monolayer is severely reduced at a shear rate of 800 s–1, relative to controls, whereas it is abolished in FcR γ-chain–/– platelets. These results provide strong evidence that engagement of GPVI stimulates limited integrin activation in PLCγ2–/– platelets via PLCγ1 and PI3-kinase.
Double jeopardy from a single translocation: deletions of the derivative chromosome 9 in chronic myeloid leukemia
Authors: Huntly Brian J. P. , Bench Anthony , Green Anthony R.
Published: May 2003

Chronic myeloid leukemia (CML) is characterized by formation of a BCR-ABL fusion gene, usually as a consequence of the Philadelphia (Ph) translocation between chromosomes 9 and 22. Recently the development of new fluorescence insitu hybridization (FISH) techniques has allowed identification of unexpected deletions of the reciprocal translocation product, the derivative chromosome 9, in 10% to 15% of patients with CML. These deletions are large, span the translocation breakpoint, and occur at the same time as the Ph translocation. Such deletions therefore give rise to previously unsuspected molecular heterogeneity from the very beginning of this disease, and there is mounting evidence for similar deletions associated with other translocations. Several studies have demonstrated that CML patients who carry derivative chromosome 9 deletions exhibit a more rapid progression to blast crisis and a shorter survival. Deletion status is independent of, and more powerful than, the Sokal and Hasford/European prognostic scoring systems. The poor prognosis associated with deletions is seen in patients treated with hydroxyurea or interferon, and preliminary evidence suggests that patients with deletions may also have a worse outcome than nondeleted patients following stem cell transplantation or treatment with imatinib. Poor outcome cannot be attributed to loss of the reciprocal ABL-BCR fusion gene expression alone, and is likely to reflect loss of one or more critical genes within the deleted region. The molecular heterogeneity associated with the Philadelphia translocation provides a new paradigm with potential relevance to all malignancies associated with reciprocal chromosomal translocations and/or fusion gene formation.