bluuebunny/crossref_metadata_2025 huggingface dataset:
Overall and event-free survival are not improved by the use of myeloablative therapy following intensified chemotherapy in previously untreated patients with multiple myeloma: a prospective randomized phase 3 study
Authors: Segeren Christine M. , Sonneveld Pieter , van der Holt Bronno , Vellenga Edo , Croockewit Alexandra J. , Verhoef Gregor E. G. , Cornelissen Jan J. , Schaafsma Martijn R. , van Oers Marinus H. J. , Wijermans Pierre W. , Fibbe Wim E. , Wittebol Shulamit , Schouten Harry C. , Kooy Marinus van Marwijk , Biesma Douwe H. , Baars Joke W. , Slater Rosalyn , Steijaert Monique M. C. , Buijt Ivon , Lokhorst Henk M.
Published: February 2003

We compared the efficacy of intensified chemotherapy followed by myeloablative therapy and autologous stem cell rescue with intensified chemotherapy alone in patients newly diagnosed with multiple myeloma. There were 261 eligible patients younger than 66 years with stage II/III multiple myeloma who were randomized after remission induction therapy with vincristine, adriamycin, dexamethasone (VAD) to receive intensified chemotherapy, that is, melphalan 140 mg/m2 administered intravenously in 2 doses of 70 mg/m2 (intermediate-dose melphalan [IDM]) without stem cell rescue (n = 129) or the same regimen followed by myeloablative therapy consisting of cyclophosphamide, total body irradiation, and autologous stem cell reinfusion (n = 132). Interferon-α–2a was given as maintenance. Of the eligible patients, 79% received both cycles of IDM and 79% of allocated patients actually received myeloablative treatment. The response rate (complete remission [CR] plus partial remission [PR]) was 88% in the intensified chemotherapy group versus 95% in the myeloablative treatment group. CR was significantly higher after myeloablative therapy (13% versus 29%; P = .002). With a median follow-up of 33 months (range, 8-65 months), the event-free survival (EFS) was not different between the treatments (median 21 months versus 22 months; P = .28). Time to progression (TTP) was significantly longer after myeloablative treatment (25 months versus 31 months; P = .04). The overall survival (OS) was not different (50 months versus 47 months; P = .41). Intensified chemotherapy followed by myeloablative therapy as first-line treatment for multiple myeloma resulted in a higher CR and a longer TTP when compared with intensified chemotherapy alone. However, it did not result in a better EFS and OS.
Granulocyte-colony stimulating factor increases CD123hi blood dendritic cells with altered CD62L and CCR7 expression
Authors: Vuckovic Slavica , Kim Min , Khalil Dailal , Turtle Cameron J. , Crosbie Georgina V. , Williams Noel , Brown Len , Williams Katrina , Kelly Cathryn , Stravos Penny , Rodwell Robyn , Hill Geoff R. , Wright Susan , Taylor Kerry , Gill Devinder , Marlton Paula , Bradstock Ken , Hart Derek N. J.
Published: February 2003

Changes in blood dendritic cell (BDC) counts (CD123hiBDC and CD11c+BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 μg/kg/d subcutaneously) and high-dose G-CSF in patients (30 μg/kg/d) increased CD123hiBDC (2- to 22-fold, mean 3.7 × 106/L-17.7 × 106/L and 1.9 × 106/L-12.0 × 106/L) in healthy donors and MM but decreased CD11c+BDC (2- to 10-fold, mean 5.7 × 106/L-1.6 × 106/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123hiBDC counts remained high, whereas low CD11c+BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123hiBDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.
Hematopoietic origin of glomerular mesangial cells
Authors: Masuya Masahiro , Drake Christopher J. , Fleming Paul A. , Reilly Christopher M. , Zeng Haiqun , Hill William D. , Martin-Studdard Angeline , Hess David C. , Ogawa Makio
Published: February 2003

It was recently reported that crude bone marrow cells have the ability to differentiate into glomerular mesangial cells. However, the exact nature of the engrafting cells in the bone marrow was not known. We tested the hypothesis that hematopoietic stem cells are capable of reconstituting the mesangial cells by transplanting a clonal population of cells derived from a single stem cell. We cultured Lin−, Sca-1+, c-kit+, CD34− bone marrow cells from transgenic enhanced green fluorescent protein (EGFP) mice (C57BL/6-Ly-5.2 background) individually for 1 week in the presence of interleukin-11 and steel factor. We then transplanted viable clones individually into lethally irradiated C57BL/6-Ly-5.1 mice. Kidneys from 5 recipient mice showing high levels (60%-90%) of multilineage hematopoietic reconstitution were examined 2 to 6 months later, using differential interference contrast and epifluorescence microscopy. EGFP+ cells with a morphology characteristic of mesangial cells were evident within the glomeruli. Transplantation of 100 noncultured Lin−, Sca-1+, c-kit+, CD34− bone marrow cells also generated mesangial cells. Cultured EGFP+ glomerular cells from recipient mice contracted in response to angiotensin II. EGFP+ mesangial cells seen in male-to-male transplants revealed only one Y-chromosome. These data demonstrate that a single hematopoietic stem cell is capable of differentiating into glomerular mesangial cells and that the process does not involve cell fusion.
The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana
Authors: Allain Jean-Pierre , Candotti Daniel , Soldan Kate , Sarkodie Francis , Phelps Bruce , Giachetti Cristina , Shyamala Venkatakrishna , Yeboah Francis , Anokwa Margaret , Owusu-Ofori Shirley , Opare-Sem Ohene
Published: February 2003

The risk of hepatitis B virus (HBV) transmission by transfusion in sub-Saharan Africa is considered to be relatively low, and testing of blood donors is often not done or is done relatively poorly. To re-examine this attitude, we identified HBV chronically infected blood donors from a major hospital in Ghana with a range of hepatitis B surface antigen (HBsAg) assays. Test efficacy was estimated using HBV DNA as a gold standard, and the risk of HBV infection in blood recipients was estimated for different testing strategies. Particle agglutination, dipstick, and enzyme immunoassay (EIA) HBsAg screening detected 54%, 71%, and 97% of HBV infectious donors, respectively. The risk of HBV transmission to recipients less than 10 years old ranged between 1:11 and 1:326 with blood unscreened and screened by EIA, respectively. For older recipients, the risk decreased a further 4-fold because of the high frequency of natural exposure to HBV. A total of 98% of HBsAg-confirmed positive samples contained HBV DNA. HBV DNA load was less than 1 × 104 IU/mL in 75% of HBsAg-reactive samples, most of them anti-HBe reactive. Approximately 0.5% of HBsAg-negative but anti-HBc-positive samples contained HBV DNA. The use of sensitive HBsAg tests is critical to prevent transfusion transmission of HBV infection to young children in a population with a 15% prevalence of chronic HBV infection in blood donors. However, this will not have much effect on the prevalence of this infection unless other strategies to protect children from infection are also advanced in parallel.
Ikaros regulates neutrophil differentiation
Authors: Dumortier Alexis , Kirstetter Peggy , Kastner Philippe , Chan Susan
Published: February 2003

The Ikaros gene encodes a zinc finger transcription factor that is selectively expressed by all hematopoietic cells. Although Ikaros is required for lymphocyte differentiation, its role in the myeloid lineage is unclear. We show here that Ikaros expression is temporally regulated during neutrophil differentiation: Ikaros is primarily expressed at immature stages and significantly less so in mature neutrophils. Furthermore IkL/L mice, harboring a hypomorphic mutation at the Ikaros locus, exhibit several defects during neutrophil differentiation. (1) IkL/L fetal livers contain high numbers of neutrophil lineage cells, and this increase is reflected in the number of GM-CSF-dependent progenitor cells. (2) The migratory potential and survival of neutrophil progenitors is altered in vitro. (3) Expression of the Gr-1 marker is delayed and repressed. In contrast, neutrophil function appears normal. These data demonstrate that Ikaros regulates early neutrophil differentiation but is dispensable in mature neutrophils.
High levels of BAX, low levels of MRP-1, and high platelets are independent predictors of response to imatinib in myeloid blast crisis of CML
Authors: Lange Thoralf , Günther Christine , Köhler Thomas , Krahl Rainer , Musiol Scarlet , Leiblein Sabine , Al-Ali Haifa-Kathrin , van Hoomissen Iris , Niederwieser Dietger , Deininger Michael W. N.
Published: February 2003

Imatinib induces remissions in approximately 30% of patients with chronic myeloid leukemia (CML) in myeloid blast crisis (M-BC). Because most patients eventually relapse, allogeneic stem cell transplantation (SCT) in remission offers the best chance for cure. Remission induction with imatinib alone would seem ideal because it is less toxic than conventional chemotherapy. Conversely, patients unlikely to respond may benefit from combination therapy up front. To identify prognostic factors, we studied the mRNA expression of genes related to drug resistance and apoptosis in leukemic cells from patients with M-BC and their in vitro sensitivity to imatinib, and analyzed the results with other baseline factors for their impact on response. We show that high levels of BAX, low levels of MRP-1, and a high platelet count are independently predictive of response to imatinib. Combined into a score, these parameters may be clinically useful for risk-adapted patient stratification.
ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice
Authors: Stocker Jonathan W. , De Franceschi Lucia , McNaughton-Smith Grant A. , Corrocher Roberto , Beuzard Yves , Brugnara Carlo
Published: February 2003

A prominent feature of sickle cell anemia is the presence of dehydrated red blood cells (RBCs) in circulation. Loss of potassium (K+), chloride (Cl−), and water from RBCs is thought to contribute to the production of these dehydrated cells. One main route of K+ loss in the RBC is the Gardos channel, a calcium (Ca2+)–activated K+ channel. Clotrimazole (CLT), an inhibitor of the Gardos channel, has been shown to reduce RBC dehydration in vitro and in vivo. We have developed a chemically novel compound, ICA-17043, that has greater potency and selectivity than CLT in inhibiting the Gardos channel. ICA-17043 blocked Ca2+-induced rubidium flux from human RBCs with an IC50 value of 11 ± 2 nM (CLT IC50 = 100 ± 12 nM) and inhibited RBC dehydration with an IC50 of 30 ± 20 nM. In a transgenic mouse model of sickle cell disease (SAD), treatment with ICA-17043 (10 mg/kg orally, twice a day) for 21 days showed a marked and constant inhibition of the Gardos channel activity (with an average inhibition of 90% ± 27%, P < .005), an increase in RBC K+ content (from 392 ± 19.9 to 479.2 ± 40 mmol/kg hemoglobin [Hb], P < .005), a significant increase in hematocrit (Hct) (from 0.435 ± 0.007 to 0.509 ± 0.022 [43.5% ± 0.7% to 50.9% ± 2.2%], P< .005), a decrease in mean corpuscular hemoglobin concentration (MCHC) (from 340 ± 9.0 to 300 ± 15 g/L [34.0 ± 0.9 to 30 ± 1.5 g/dL], P < .05), and a left-shift in RBC density curves. These data indicate that ICA-17043 is a potent inhibitor of the Gardos channel and ameliorates RBC dehydration in the SAD mouse.
Induction of C/EBPα activity alters gene expression and differentiation of human CD34+ cells
Authors: Cammenga Jörg , Mulloy James C. , Berguido Francisco J. , MacGrogan Donal , Viale Agnes , Nimer Stephen D.
Published: February 2003

The CCAAT/enhancer binding protein alpha (C/EBPα) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPα−/− mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPα in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPα gene and down-regulation of C/EBPα by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPα in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34+ cells with a retroviral construct that expresses the C/EBPα cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPα function in primary human CD34+ cells, by the addition of β-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPα target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor ofdifferentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.
Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding
Authors: Sun Yong-Hui , Shen Lei , Dahlbäck Björn
Published: February 2003

Protein C is a member of the vitamin K– dependent protein family. Proteins in this family have similar γ-carboxyglutamic acid (Gla)–rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.
Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells
Authors: Yamada Takayuki , Yamazaki Hidetoshi , Yamane Toshiyuki , Yoshino Miya , Okuyama Hiromi , Tsuneto Motokazu , Kurino Tomomi , Hayashi Shin-Ichi , Sakano Seiji
Published: February 2003

Osteoclasts are derived from hematopoietic precursor cells belonging to the monocyte/macrophage lineage. Osteoclast development has been reported to be regulated by several molecules such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor (NF)-κB ligand (RANKL), and a decoy receptor of RANKL, osteoprotegerin (OPG). Recently, it was demonstrated that the Notch signaling pathway regulates myeloid differentiation and antagonizes cell fate determination, however, the effect of Notch signaling on the osteoclast lineage has not been reported. In this study, we examined the effect of signaling via Notch receptors on the differentiation into osteoclasts by using cells from the bone marrow, spleen, and peritoneal cavity, and a cloned macrophagelike cell line. Osteoclastogenesis was inhibited by an immobilized Notch ligand, Delta-1. The dish-adherent bone marrow cells precultured with M-CSF expressed both Mac-1 and M-CSF receptors, c-Fms; osteoclastogenesis of these cells was efficiently inhibited. The immobilized Delta-1 also down-regulated the surface c-Fms expression, while the c-Fms gene expression was not changed. Genes for Notch receptors and Notch ligands are expressed in not only hematopoietic cells but also stromal cells that support osteoclast development. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF gene expression, resulting in reduction of their ability to support osteoclast development. Taken together, these findings indicate that Notch signaling affects both osteoclast precursors and stromal cells and thereby negatively regulates osteoclastogenesis.
The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications
Authors: Mitsiades Nicholas , Mitsiades Constantine S. , Richardson Paul G. , Poulaki Vassiliki , Tai Yu-Tzu , Chauhan Dharminder , Fanourakis Galinos , Gu Xuesong , Bailey Charles , Joseph Marie , Libermann Towia A. , Schlossman Robert , Munshi Nikhil C. , Hideshima Teru , Anderson Kenneth C.
Published: February 2003

The proteasome inhibitor PS-341 inhibits nuclear factor-κB (NF-κB) activation, induces apoptosis in cancer cells, including multiple myeloma (MM) cells, and has marked clinical activity as a monotherapy for MM. In this study, we found that subtoxic concentrations of PS-341 potently sensitized MM cell lines and patient cells to DNA-damaging chemotherapeutic agents such as doxorubicin and melphalan, including cells resistant to these drugs and those isolated from a patient who had relapsed after PS-341 monotherapy. Moreover, PS-341 abolished cell adhesion–mediated drug resistance. Using gene expression profiling and proteomic analysis, we demonstrate that PS-341, among its other proapoptotic effects, down-regulates the expression of several effectors involved in the cellular response to genotoxic stress. These data suggest that, in addition to down-regulating the expression of apoptosis inhibitors, PS-341 inhibits genotoxic stress response pathways and thereby restores sensitivity to DNA-damaging chemotherapeutic agents. These studies, therefore, provide the framework for clinical use of this agent in combination with conventional chemotherapy.
Differential regulation of CXCR2 trafficking by Rab GTPases
Authors: Fan Guo-Huang , Lapierre Lynne A. , Goldenring James R. , Richmond Ann
Published: February 2003

Intracellular trafficking of chemokine receptors plays an important role in fine-tuning the functional responses of neutrophils and lymphocytes in the inflammatory process and HIV infection. Although many chemokine receptors internalize through clathrin-coated pits, regulation of the receptor trafficking is not fully understood. The present study demonstrated that CXCR2 was colocalized with transferrin and low-density lipoprotein (LDL) after agonist treatment for different periods of time, suggesting 2 intracellular trafficking pathways for this receptor. CXCR2 was colocalized with Rab5 and Rab11a, which are localized in early and recycling endosomes, respectively, in response to agonist stimulation for a short period of time, suggesting a recycling pathway for the receptor trafficking. However, overexpression of a dominant-negative Rab5-S34N mutant significantly attenuated CXCR2 sequestration. The internalized CXCR2 was recycled back to the cell surface after removal of the agonist and recovery of the cells, but receptor recycling was inhibited by overexpression of a dominant-negative Rab11a-S25N mutant. After prolonged (4-hour) agonist treatment, CXCR2 exhibited significantly increased colocalization with Rab7, which is localized in late endosomes. The colocalization of CXCR2 with LDL and LAMP-1 suggests that CXCR2 is targeted to lysosomes for degradation after prolonged ligand treatment. However, the colocalization of CXCR2 with Lamp1 was blocked by the overexpression of a dominant-negative Rab7-T22N mutant. In cells overexpressing Rab7-T22N, CXCR2 was retained in the Rab5- and Rab11a-positive endosomes after prolonged (4-hour) agonist treatment. Our data suggest that the intracellular trafficking of CXCR2 is differentially regulated by Rab proteins.
Donor T cell–derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation
Authors: Schmaltz Cornelius , Alpdogan Onder , Muriglan Stephanie J. , Kappel Barry J. , Rotolo Jimmy A. , Ricchetti Eric T. , Greenberg Andrew S. , Willis Lucy M. , Murphy George F. , Crawford James M. , van den Brink Marcel R. M.
Published: February 2003

Previous studies in murine bone marrow transplantation (BMT) models using neutralizing anti-tumor necrosis factor (TNF) antibodies or TNF receptor (TNFR)–deficient recipients have demonstrated that TNF can be involved in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). TNF in these GVHD and GVL models was thought to be primarily produced by activated monocytes and macrophages, and the role of T cell–derived TNF was not determined. We used TNF−/− mice to study the specific role of TNF produced by donor T cells in a well-established parent-into-F1 hybrid model (C57BL/6J→C3FeB6F1/J). Recipients of TNF−/− T cells developed significantly less morbidity and mortality from GVHD than recipients of wild-type (wt) T cells. Histology of GVHD target organs revealed significantly less damage in thymus, small bowel, and large bowel, but not in liver or skin tissues from recipients of TNF−/− T cells. Recipients of TNF−/−T cells which were also inoculated with leukemia cells at the time of BMT showed increased mortality from leukemia when compared with recipients of wt cells. We found that TNF−/− T cells do not have intrinsic defects in vitro or in vivo in proliferation, IFN-γ production, or alloactivation. We could not detect TNF in the serum of our transplant recipients, suggesting that T cells contribute to GVHD and GVL via membrane-bound or locally released TNF.
The degree of phenotypic correction of murine β-thalassemia intermedia following lentiviral-mediated transfer of a human γ-globin gene is influenced by chromosomal position effects and vector copy number
Authors: Persons Derek A. , Hargrove Phillip W. , Allay Esther R. , Hanawa Hideki , Nienhuis Arthur W.
Published: February 2003

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of β-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell–targeted gene transfer of a γ-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based γ-globin vectors for expression in transduced erythroid cell lines. Compared with γ-globin, oncoretroviral vectors containing either a β-spectrin or β-globin promoter and the α-globin HS40 element, a γ-globin lentiviral vector utilizing the β-globin promoter and elements from the β-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human γ-globin was observed with levels of vector-encoded γ-globin mRNA ranging from 9% to 19% of total murine α-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of β-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric mα2hγ2molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. γ-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a γ-globin lentiviral vector for gene therapy of β-thalassemia.
IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes
Authors: Verhoeyen Els , Dardalhon Valerie , Ducrey-Rundquist Odile , Trono Didier , Taylor Naomi , Cosset François-Loı̈c
Published: February 2003

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.
Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia
Authors: Molina-Arcas Mı́riam , Bellosillo Beatriz , Casado F. Javier , Montserrat Emili , Gil Joan , Colomer Dolors , Pastor-Anglada Marçal
Published: February 2003

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.
Hematologic effects of inactivating the Ras processing enzymeRce1
Authors: Aiyagari Abigail L. , Taylor Brigit R. , Aurora Vikas , Young Stephen G. , Shannon Kevin M.
Published: February 2003

Posttranslational processing of Ras proteins has attracted considerable interest as a potential target for anticancer drug discovery. Rce1 encodes an endoprotease that facilitates membrane targeting of Ras and other prenylated proteins by releasing the carboxyl-terminal 3 amino acids (ie, the -AAX of the CAAX motif). Homozygous Rce1 mutant embryos(Rce1−/−) die late in gestation. To characterize the role of Rce1 in hematopoiesis, we performed adoptive transfers and investigated cells from the recipients. Rce1−/− fetal liver cells rescued lethally irradiated recipients and manifested normal long-term repopulating potential in competitive repopulation assays. The recipients of Rce1−/− cells developed modest elevations in mature myeloid cells (neutrophils + monocytes), but remained well. Bone marrow cells from mice that received transplants of Rce1−/− activated extracellular signal-related kinase (ERK) normally in response to granulocyte-macrophage colony-stimulating factor. These data suggest that pharmacologic inhibitors of Rce1 will have minimal effects on normal hematopoietic cells.
Side effects of retroviral gene transfer into hematopoietic stem cells
Authors: Baum Christopher , Düllmann Jochen , Li Zhixiong , Fehse Boris , Meyer Johann , Williams David A. , von Kalle Christof
Published: February 2003

Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of “transgene toxicology,” in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.
Potentiation of TRAIL-induced apoptosis in primary effusion lymphoma through azidothymidine-mediated inhibition of NF-κB
Authors: Ghosh Subrata K. , Wood Charles , Boise Lawrence H. , Mian Abdul M. , Deyev Vadim V. , Feuer Gerold , Toomey Ngoc L. , Shank Nicole C. , Cabral Lisa , Barber Glen N. , Harrington William J.
Published: February 2003

The survival of viral mediated lymphomas depends upon constitutive nuclear factor kappa B (NF-κB) activity. AIDS-related human herpesvirus type 8–associated primary effusion lymphoma (PEL) responds poorly to chemotherapy and is almost invariably fatal. We have previously demonstrated that the antiviral combination of interferon alpha (IFN-α) and azidothymidine (AZT) induces apoptosis in PEL cell lines. We therefore used these agents as therapy for an AIDS patient with PEL. The patient had a dramatic response, with complete resolution of his malignant effusion in 5 days. In PEL cells, the death receptor ligand known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is markedly up-regulated by IFN-α; however, signals transduced by death receptors may also activate an antiapoptotic response mediated by NF-κB. In both the primary tumor cells from our patient and PEL cell lines, AZT selectively blocked nuclear entry of the NF-κB heterodimer p50 and p65, an effect not seen with other nonthymidine antiviral nucleosides. AZT monophosphate, the principal intracellular metabolite, inhibited phosphorylation and degradation of IκB by the IκB kinase complex. AZT- and IFN-α-mediated apoptosis was blocked by expression and nuclear localization of an IκB-resistant form of NF-κB (the p50 subunit linked to the transactivation domain of herpes simplex virus VP16). The proapoptotic effect of AZT and IFN-α in PEL occurs through the concomitant activation of TRAIL and blockade of NF-κB and represents a novel antiviral therapy for a virally mediated tumor.
Inhibition of microsomal glucose-6-phosphate transport in human neutrophils results in apoptosis: a potential explanation for neutrophil dysfunction in glycogen storage disease type 1b
Authors: Leuzzi Rosanna , Bánhegyi Gábor , Kardon Tamás , Marcolongo Paola , Capecchi Piero-Leopoldo , Burger Hans-Joerg , Benedetti Angelo , Fulceri Rosella
Published: February 2003

Mutations in the gene of the hepatic glucose-6-phosphate transporter cause glycogen storage disease type 1b. In this disease, the altered glucose homeostasis and liver functions are accompanied by an impairment of neutrophils/monocytes. However, neither the existence of a microsomal glucose-6-phosphate transport, nor the connection between its defect and cell dysfunction has been demonstrated in neutrophils/monocytes. In this study we have characterized the microsomal glucose-6-phosphate transport of human neutrophils and differentiated HL-60 cells. The transport of glucose-6-phosphate was sensitive to the chlorogenic acid derivative S3483,N-ethylmaleimide, and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, known inhibitors of the hepatic microsomal glucose-6-phosphate transporter. A glucose-6-phosphate uptake was also present in microsomes from undifferentiated HL-60 and Jurkat cells, but it was insensitive to S3483. The treatment with S3484 of intact human neutrophils and differentiated HL-60 cells mimicked some leukocyte defects of glycogen storage disease type 1b patients (ie, the drug inhibited phorbol myristate acetate–induced superoxide anion production and reduced the size of endoplasmic reticulum Ca2+ stores). Importantly, the treatment with S3484 also resulted in apoptosis of human neutrophils and differentiated HL-60 cells, while undifferentiated HL-60 and Jurkat cells were unaffected by the drug. The proapoptotic effect of S3483 was prevented by the inhibition of nicotinamide adenine dinucleotide phosphate oxidase or by antioxidant treatment. These results suggest that microsomal glucose-6-phosphate transport has a role in the antioxidant protection of neutrophils, and that the genetic defect of the transporter leads to the impairment of cellular functions and apoptosis.
Identification of an MIP-1α–binding heparan sulfate oligosaccharide that supports long-term in vitro maintenance of human LTC-ICs
Authors: Stringer Sally E. , Nelson Matthew S. , Gupta Pankaj
Published: February 2003

We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1α (MIP-1α), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1α binding ability. A specific MIP-1α-binding HS oligosaccharide preparation of Mr 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1α binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.
A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expression but maintains anIGH/MMSET fusion transcript
Authors: Santra Madhumita , Zhan Fenghuang , Tian Erming , Barlogie Bart , Shaughnessy John
Published: February 2003

Previous studies have revealed that that approximately 10% to 15% of multiple myelomas (MMs) are characterized by a reciprocal t(4;14)(p16;q32) translocation that activates expression ofFGFR3 and creates an IGH/MMSET fusion transcript. Current data suggest that activation of FGFR3is the oncogenic consequence of this rearrangement. Using a combination of microarray profiling, reverse transcriptase–polymerase chain reaction (RT-PCR), and interphase fluorescence in situ hybridization (FISH), we show that 32 (18%) of 178 newly diagnosed cases of MM harbor the t(4;14)(p16;q32). Importantly, 32% of these cases lack expression of FGFR3, yet express MMSET and have an IGH/MMSET fusion transcript. Interphase FISH showed that whereas the IGH/MMSET fusion was present in more than 80% of the clonotypic plasma cells in these novel cases, there was typically a complete loss of one copy of FGFR3. These data indicate that the t(4;14)(p16;q32) and loss of FGFR3occurred at a very early stage and suggest that activation ofMMSET, not FGFR3, may be the critical transforming event of this recurrent translocation.
Antithymocyte globulin has limited efficacy and substantial toxicity in unselected anemic patients with myelodysplastic syndrome
Authors: Steensma David P. , Dispenzieri Angela , Moore S. Breanndan , Schroeder Georgene , Tefferi Ayalew
Published: February 2003

Antithymocyte globulin (ATG) has recently been popularized as an effective treatment in myelodysplastic syndrome (MDS). We treated 8 anemic MDS patients (refractory anemia [RA] and refractory anemia with excess blasts [RAEB-1]) with ATG (40 mg/kg/d for 4 days) and prednisone in a phase 2 trial. The study was stopped early according to a preset termination rule because of lack of efficacy. There were no salutary responses. Toxicities included serum sickness (in all patients), transient neutropenia and thrombocytopenia, diarrhea, vomiting, and syncope with a generalized seizure. At least 3 patients had the HLA-DR15 (DR2) allele. We conclude that the risk-benefit ratio of ATG in an unselected population of MDS patients may be unfavorable, and more work is needed to define the subset of patients who will respond to ATG before its widespread use can be recommended.
Residual effect of nCPAP applied for part of the night in patients with obstructive sleep apnoea
Authors: Hers V , Liistro G , Dury M , Collard P , Aubert G , Rodenstein DO
Published: February 2003

The aim of the present study was to assess whether nasal continuous positive airway pressure (nCPAP) treatment, applied for only a few hours at the beginning of the night, has any residual effect on sleep and breathing during the ensuing hours of unassisted sleep in patients with obstructive sleep apnoea syndrome (OSAS). In 27 patients with newly-diagnosed OSAS, effective nCPAP was applied during the first part of the night and then withdrawn. Polysomnographic parameters after nCPAP withdrawal were compared with those of the corresponding part of the diagnostic polysomnography performed a few days or weeks before and with those of the first part of night on nCPAP. After 255+/-63 (mean+/-SD) min of sleep with normalization of sleep and breathing parameters under nCPAP, there was partial improvement of OSAS severity during the remaining 124+/-56 min of nocturnal sleep without treatment; mean oxygen saturation, desaturation index (equivalent to the apnoea/hypopnoea index) and movement arousal index all improved significantly with respect to the diagnostic night (p=0.001). This improvement was not accounted for by a change in sleep architecture. We conclude that there is an improvement in severity of obstructive sleep apnoea syndrome after only 4 h of nasal continuous positive airway pressure. This carryover effect could explain why a number of patients with obstructive sleep apnoea syndrome apply nasal continuous positive airway pressure for only part of the night or not every night.
Respiratory muscle function and control of breathing in patients with acromegaly
Authors: Iandelli I , Gorini M , Duranti R , Bassi F , Misuri G , Pacini F , Rosi E , Scano G
Published: February 2003

Increase in lung size has been described in acromegalic patients, but data on respiratory muscle function and control of breathing are relatively scarce. Lung volumes, arterial blood gas tensions, and respiratory muscle strength and activation during chemical stimulation were investigated in a group of 10 patients with acromegaly, and compared with age- and sex-matched normal controls. Inspiratory muscle force was evaluated by measuring pleural (Ppl,sn) and transdiaphragmatic (Pdi,sn) pressures during maximal sniffs. Dynamic pleural pressure swing (Ppl,sw) was expressed both as absolute value and as percentage of Ppl,sn. Expiratory muscle force was assessed in terms of maximal expiratory pressure (MEP). In 8 of the 10 patients, ventilatory and respiratory muscle responses to hyperoxic progressive hypercapnia and to isocapnic progressive hypoxia were also evaluated. Large lungs, defined as total lung capacity (TLC) greater than predicted (above 95% confidence limits), were found in five patients. Inspiratory or expiratory muscle force was below normal limits in all but three patients. During unstimulated tidal breathing, respiratory frequency (fR) and mean inspiratory flow (tidal volume/inspiratory time (VT/tI)) were greater, while inspiratory time (tI) was shorter than in controls. Minute ventilation (V'E) and mean inspiratory flow response slopes to hypercapnia were normal In contrast, four patients had reduced delta(VT/tI)/arterial oxygen saturation (Sa,O2) and three had reduced deltaV'E/Sa,O2. Ppl,sw(%Ppl,sn) response slopes to increasing end-tidal carbon dioxide tension (PET,CO2) and decreasing Sa,O2 did not differ from the responses of the normal subjects, suggesting normal central chemoresponsiveness. At a PET,CO2 of 8 kPa or an Sa,O2 of 80%, patients had greater fR and lower tI compared with controls. Pdi,sn and Ppl,sn related both to deltaV'E/deltaSa,O2 (r=0.729 and r=0.776, respectively) and delta(VT/tI)/deltaSa,O2 (r=0.860 and r=0.90, respectively). Pdi,sn also related both to deltaV'E/deltaPET,CO2 (r=0.8) and delta(VT/tI)/deltaPET,CO2 (r=0.76). In conclusion, the data suggest the relative independence of pneumomegaly and respiratory muscle strength. Peripheral (muscular) factors appear to modulate a normal central motor output to give a more rapid pattern of breathing.
A noninvasive electromyographic study on threshold and intensity of cough in humans
Authors: Fontana GA , Pantaleo T , Lavorini F , Boddi V , Panuccio P
Published: February 2003

The assessment of cough threshold and intensity is important in respiratory medicine. We have developed a method for objectively and noninvasively assessing cough threshold and intensity of expiratory muscle efforts in response to inhalation of ultrasonically nebulized distilled water (UNDW). Thirty (83%) out of 36 volunteers studied coughed in response to UNDW inhalation. Cough threshold was taken as the lowest nebulizer output (mL x min(-1)) that induced cough in two challenges performed at a 30 min interval. At threshold level, repeatability of peak and slope of the integrated electromyographic (IEMG) activity of abdominal muscles was evaluated. Short- and long-term repeatability of cough threshold were evaluated in 15 subjects following a 3 h and a 6-9 month interval, respectively. Dose-response relationships between nebulizer outputs and IEMG-related variables were also investigated, as were the correlations between the latter and expiratory flow during voluntary coughing. The median (1st and 3rd quartile) cough threshold value was 0.89 (0.40 and 1.54) mL x min(-1). At threshold level, peak and slope of IEMG activity were highly reproducible. Cough threshold displayed a high degree of short- and long-term repeatability. Peak and slope of IEMG activity displayed a clear trend to increase (p<0.01) following inhalation of progressively higher UNDW outputs. Maximum flow during voluntary coughs of varying intensity correlated with the peak (p<0.05) and, more closely, with the slope (p<0.01) of abdominal IEMG activity. The assessment of cough threshold as well as the evaluation of the intensity of cough efforts by abdominal integrated electromyographic recordings may represent useful and reliable tools for cough research in humans.
Histochemical properties of upper airway muscles: comparison of dilator and nondilator muscles
Authors: Bracher A , Coleman R , Schnall R , Oliven A
Published: February 2003

The upper airway dilator muscles (UADMs) represent a subgroup of muscles in the pharyngeal area which, in addition to their roles in mastication, vocalization, etc., also have an important respiratory function. Failure of these muscles to maintain upper airway patency during sleep is important in the development of the obstructive sleep apnoea syndrome. In the present study, we evaluated the histochemical properties of the UADMs and compared them to those of neighbouring muscles without respiratory functions, and to the diaphragm, to determine whether the UADMs are specifically adapted to their respiratory role. Our results, both in dogs and rats, indicate that the dilator and nondilator upper airway muscles are similar and differ from the diaphragm. In rats, there were significantly less type I fibres (<12% as compared to 42% for the diaphragm) and more type IIb fibres (39-67% as compared to 27% for the diaphragm). A similar pattern was seen in dogs: type I fibres <38% as compared to 46% for the diaphragm, and type IIb fibres, 29-35% as compared to 10% for the diaphragm. These findings suggest that the upper airway dilator muscles are not specifically designed for their respiratory role. They may fail in the presence of increased loads, often encountered in patients with obstructive sleep apnoea, unless appropriate adaptive structural changes take place.
Effect of roxithromycin on airway responsiveness in children with bronchiectasis: a double-blind, placebo-controlled study
Authors: Koh YY , Lee MH , Sun YH , Sung KW , Chae JH
Published: February 2003

Increased airway responsiveness (AR) is frequently associated with bronchiectasis. Roxithromycin is a new semisynthetic macrolide antibiotic that also has anti-inflammatory activities. This study was designed to see whether roxithromycin could favourably alter the degree of AR in patients with bronchiectasis and increased AR. Twenty five children with bronchiectasis, who had an increased AR (defined as a provocative concentration of methacholine causing a 20% fall in forced expiratory volume in one second (FEV1) (PC20) <25 mg x mL(-1) evaluated by the dosimeter method), were randomized, double-blind into two parallel groups. Thirteen of the children were treated with roxithromycin (4 mg x kg(-1) b.i.d.) for 12 weeks and 12 received placebo. FEV1, sputum purulence and leucocyte scores were assessed every 3 weeks. To estimate AR, high-dose methacholine challenge tests were performed before and after treatment. On the dose-response curve to methacholine, PD20 and maximal response (two indices of AR) were measured. Changes in FEV1 were not observed during the course of the study in both groups. A significant improvement in sputum features was noted after 6 weeks of treatment in the roxithromycin group. After 12 weeks of roxithromycin therapy, the geometric mean (range of 1 SD) of provocative cumulative dose producing a 20% fall in FEV1 (PD20) increased significantly (p<0.01) to 169.2 (83.2-344.2) breath units (BU) (1 BU denotes one inhalation of 1 mg x mL(-1) methacholine) and the mean+/-SD of maximal response decreased significantly (p<0.01) to 32.5+/-6.8%, as compared with the initial values (PD20 87.1 (47.3-160.4) BU; maximal response 40.9+/-7.4%). No significant changes in either parameter were observed in the placebo group. Our results indicate that roxithromycin may decrease the degree of airway responsiveness in patients with bronchiectasis and increased airway responsiveness. Further study is necessary to determine the mechanism by which roxithromycin reduces airway responsiveness in bronchiectasis and its clinical impact.