bluuebunny/crossref_metadata_2025 huggingface dataset:
Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis
Authors: McAdams Todd A. , Miller William M. , Papoutsakis E. Terry
Published: November 2003

Haemopoietic cultures may experience pH variations of as much as 0.5 units depending on culture duration and cell density. Since pH is a potent modulator of cellular proliferation and differentiation, we examined its effects on the performance of both semisolid and liquid haemopoietic cultures. Culture pH was found to have substantial effects both on progenitor cloning efficiency (as measured in liquid cultures) and on progenitor cell differentiation (as measured in methylcellulose cultures). Liquid cultures were conducted with both peripheral blood (PB) mononuclear cells (MNCs) and cord blood (CB) MNCs using growth factor combinations that promote either erythroid expansion (IL‐3/IL‐6/SCF/Epo) or granulocyte/macrophage expansion (IL‐3/IL‐6/SCF/G‐CSF/GM‐CSF). Reduced pH was found to have either a positive or neutral effect on the expansion and cloning efficiency of progenitors in ex vivo liquid cultures. Cloning efficiencies of PB BFU‐E in the erythroid combination were 9‐fold higher at low pH (7.1) when compared to high pH (7.6). A small pH increase of 0.2 units over physiological values consistently produced significant reductions (42–85%) in cloning efficiencies for all cell types and cytokine combinations tested. Methylcellulose cultures conducted using CB MNC and PB MNC indicated that differentiation of CFU‐GM into progeny was optimal between pH 7.2 and 7.4. The differentiation of erythroid progenitors (BFU‐E) progressively increased as pH was increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cultures using PB CD34+ cells exhibited similar patterns to the MNC cultures. We conclude that even small variations in pH substantially affected the performance of human haemopoietic cultures. The erythroid lineage was particularly sensitive, with its extent of differentiation increasing with increasing pH. PB progenitors are more sensitive to pH variations than CB progenitors.
MYELOPEROXIDASE GENE EXPRESSION IN ACUTE LYMPHOBLASTIC LEUKAEMIA
Authors: Serrano J. , Román J. , Sánchez J. , Torres A.
Published: November 2003

The FAB classification of acute leukaemias is based on the light microscopic detection of myeloperoxidase (MPO) activity in blast cells. Cases with MPO activity in >3% of blasts are classified as acute myeloid leukaemia. We describe two cases of acute leukaemia in which the blast cells had lymphoid morphology, ultrastructure, immunophenotype and molecular rearrangements, but expressed significant levels of the MPO gene (MPO mRNA) by reverse transcription polymerase chain reaction (RT‐PCR), in the absence of translation to protein. Both cases presented a short remission duration. We discuss the incidence, features and outcome of MPO mRNA positive acute lymphoblastic leukaemia (ALL).
A comparison between two activated protein C resistance methods as routine diagnostic tests for factor V Leiden mutation
Authors: ABOUD M. R. , MA D. D. F.
Published: November 2003

The most common commercially available test measuring activated protein C (APC) resistance relies on the the anticoagulant response to added APC in an activated partial thromboplastin time (APTT) based method. Another method is a Russell Viper venom time (RVVT) based system. To improve the specificity for factor V Leiden of the APTT based method, pre‐dilution of test plasma in FV‐deficient plasma has recently been recommended. In this study we tested the relative suitabilities of the APTT‐based system, the RVVT‐based system and their corresponding assays modified by pre‐dilution in FV‐deficient plasma, for screening asymptomatic subjects, a group of thrombophilic patients (in particular those with low APC ratios), patients on oral anticoagulants, and patients with lupus anticoagulant (LAC). We found the RVVT‐based assay to be superior to the APTT‐based method in the separation of normals from those with FV Leiden mutation both in asymptomatic subjects and in the thrombophilic patient group. Both modified assays demonstrated a sensitivity and specificity of 100% for FV Leiden, as verified by genotyping in asymptomatic subjects, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT‐based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid‐dependent coagulation by LAC antibodies rendered the APTT‐based system less suitable than the phospholipid‐rich RVVT‐based one, and as nine of the 20 LAC‐positive patients were on warfarin, we showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.
Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells
Authors: Kukk Eola , Wartiovaara Ulla , Gunji Yuji , Kaukonen Jaakko , Bühring Hans‐Jörg , Rappold Irene , Matikainen Marja‐Terttu , Vihko Pirkko , Partanen Juha , Palotie Aarno , Alitalo Kari , Alitalo Riitta
Published: November 2003

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B‐lymphoblastic leukaemia cell lines were Tie‐positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie‐2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin‐1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long‐term culture of isolated umbilical cord blood CD34+Tie+ and CD34+Tie−cells indicated that the Tie+ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie‐2 and angiopoietin‐1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.
CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection
Authors: Pulik Marc , Lionnet François , Genet Philippe , Petitdidier Charlotte , Jary Laure , Fourcade Christine
Published: November 2003

High‐grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T‐cell‐derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T‐LGL (CD3+, CD4−, CD8+, CD56−, CD57+) in an HIV‐infected, HTLV1/2‐negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.
Oral contraceptives and venous thrombosis: different sensitivities to activated protein C in women using second‐ and third‐generation oral contraceptives
Authors: Rosing J. , Tans G. , Nicolaes G. A. F. , Thomassen M. C. L. G. D. , Van Oerle R. , Van DerPloeg P. M. E. N. , Heijnen P. , Hamulyak K. , Hemker H. C.
Published: November 2003

Epidemiological studies have shown that women who use third‐generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second‐generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor VLeiden), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC.

Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non‐OC users and in individuals that were hetero‐zygous or homozygous for factor VLeiden. The response towards APC was evaluated on basis of the ratio (APC‐sr) of the time integrals of thrombin formation determined in the presence and absence of APC.

Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P < 0.001), independent of the kind of OC used. Women who used third‐generation monophasic OC were significantly less sensitive to APC than women using second‐generation OC (P < 0.001) and had APC‐sr that did not significantly differ from heterozygous female carriers of factor VLeiden who did not use OC. Women who were heterozygous for factor VLeiden and used OC had APC‐sr in the range of homozygous carriers of factor VLeiden. Two women who started OC therapy had significantly elevated APC‐sr within 3 d.

Acquired APC resistance may explain the epidemiol‐ogical observation of increased risk for venous thrombosis in OC users, especially in women using third‐generation OC.
A variant of the EPB3 gene of the anti‐Lepore type in hereditary spherocytosis
Authors: Bianchi Paola , Zanella Alberto , Alloisio Nicole , Barosi Giovanni , Bredi Elena , Pelissero Giovanni , Zappa Manuela , Vercellati Cristina , Baronciani Luciano , Delaunay Jean , Sirchia Girolamo
Published: November 2003

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS‐PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in‐frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt −7 to −3), and the other within exon 13 (nt 1494–1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti‐Lepore type between the two CCTGC sequences.

At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478–499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.
Lupus anticoagulants: strong association with the major histocompatibility complex class II and platelet antibodies
Authors: Panzer Simon , Pabinger Ingrid , Gschwandtner Michael Eduard , Mayr Wolfgang Richard , Hütter Daniela
Published: November 2003

A relationship between the presence of platelet autoantibodies and major histocompatibilty complex class II alleles was determined in 27 patients with lupus anticoagulants. Twenty‐two patients had a ‘primary antiphospholipid syndrome’ and five patients had lupus erythematosus (SLE). Platelet antibodies against the platelet glycoproteins (GP) IIb/IIIa were detectable in 20 patients. Anti‐GPIb/IX or ‐GPIV antibodies were detectable only in patients with anti‐GPIIb/IIIa antibodies. An increased frequency of HLA‐DQB1*06 was demonstrable in the total patient population. The association between the lupus anticoagulants and HLA‐DQB1*06 was even stronger if patients also had detectable platelet antibodies. This association was also seen if patients with a history of thromboembolic disease were considered separately. However, within the patient population there was no difference between frequencies of HLA alleles detectable platelet antibodies.
Interleukin‐4 prevents spontaneous in‐vitro apoptosis in chronic lymphatic leukaemia but sensitizes B‐CLL cells to melphalan cytotoxicity
Authors: Pu Q. Q. , Bezwoda W. R.
Published: November 2003

The effect of IL4 on cell viability, cell growth, apoptotic fraction, melphalan‐induced cytotoxicity and the degree of interstrand DNA cross‐linking after alkylating agent exposure was investigated in peripheral blood B‐cell chronic lymphocytic leukaemia (B‐CLL) cells obtained from 10 patients suffering from chronic lymphocytic leukaemia and in B lymphocytes from five normal individuals. The addition of IL4 to culture medium maintained in‐vitro viability and decreased spontaneous in‐vitro apoptosis in both B‐CLL cells and normal peripheral blood B lymphocytes. IL4 did not, however, stimulate proliferation of either cell type. IL4 sensitized alkylator‐resistant B‐CLL cells to the cytotoxic effects of melphalan (l‐phenylalanine mustard) but had no influence on melphalan‐induced cytotoxicity against normal B lymphocytes. The enhanced cytotoxicity against B‐CLL cells was accompanied by an increase in the amount of interstrand‐DNA cross‐linking in these cells following short‐term exposure to melphalan.
Structural analysis of a missense mutation (Val414Phe) in the catalytic core domain of the factor XIIIA subunit
Authors: Aslam S. , Yee V. C. , Narayanan S. , Duraisamy G. , Standen G. R.
Published: November 2003

Molecular analysis has been performed on a Malaysian patient with a severe bleeding disorder due to factor XIIIA subunit deficiency. Total mRNA was isolated from the patient's leucocytes and four overlapping segments corresponding to the entire coding region of the A subunit cDNA were amplified by RT‐PCR. The cDNA segments amplified efficiently and were of expected size. Direct sequencing of the complete reading frame revealed a single homozygous base change (nt 1327G→T) in exon 10 corresponding to a missense mutation, Val414Phe, in the catalytic core domain of the A subunit monomer. The mutation eliminates a BsaJ1 restriction site and family screening showed that both parents were heterozygous for the defect. The base substitution was absent in 55 normal individuals. Val414 is a highly conserved residue in the calcium‐dependent transglutaminase enzyme family. Computer modelling based on 3D crystallographic data predicts that the bulky aromatic side chain of the substituted phenylalanine residue distorts protein folding and destabilizes the molecule. In addition, conformation changes in the adjacent catalytic and calcium binding regions of the A subunit are likely to impair the enzymatic activity of any protein synthesized.
Central nervous system relapse in acute promyelocytic leukaemia in patients treated with all‐trans retinoic acid
Authors: Evans G. , Grimwade D. , Prentice H. G. , Simpson N.
Published: November 2003

All‐trans retinoic acid (ATRA) is currently recommended as standard treatment for acute promyelocytic leukaemia (APL). However, there has been increasing concern that ATRA is associated with unusual sites of relapse. We present three cases of APL previously treated with ATRA who ultimately relapsed within the central nervous system (CNS) and hypothesize that, by up‐regulating intercellular adhesion molecules, ATRA may facilitate the passage of malignant promyelocytes across the blood–brain barrier.
The prothrombin gene G20210A variant: prevalence in a U.K. anticoagulant clinic population
Authors: CUMMING A. M. , KEENEY S. , SALDEN A. , BHAVNANI M. , SHWE K. H. , HAY C. R. M.
Published: November 2003

We have investigated the prevalence of a recently reported genetic variation in the prothrombin gene (G20210A) in patients with an objectively confirmed history of venous thrombosis. 12/219 patients (5.5%) were found to be heterozygous carriers of the 20210 A allele. The incidence of the 20210 A allele in a group of 164 healthy controls was 1.2% (allele frequency 0.61%, 95% CI 0.08–2.19). When patients with a known alternative hereditary risk factor for venous thrombosis (factor V Leiden mutation or deficiency of antithrombin, protein C or protein S) were excluded, the G20210A variant was found to increase the risk for venous thrombosis by approximately 5‐fold (odds ratio 5.4, 95% CI 1.16–25.0). This prothrombin gene sequence variation adds further to the list of recognized genetic risk factors for thrombophilia.
Thrombocytopenia following liver transplantation is associated with platelet consumption and thrombin generation
Authors: Richards E. M. , Alexander G. J. M. , Calne R. Y. , Baglin T. P.
Published: November 2003

Thrombocytopenia frequently occurs immediately after orthotopic liver transplantation. We have investigated the cause of this phenomenon in a cohort of 45 consecutive liver transplant recipients. The median preoperative platelet count (range) of 129 × 109/l (14–719) fell to 56 × 109/l (23–334) by the fourth postoperative day. The median preoperative reticulated platelet percentage (range) of 6.7% (2.2–23.9) increased to 16.4% (4.6–40.8) on day 7. There was a significant rise in prothrombin fragment F1.2 by the first postoperative day which was followed by rises in fibrinogen and fibrin degradation products. There was no increase in platelet‐associated immunoglobulin or markers of endothelial activation. We conclude that there is an increased rate of platelet consumption associated with thrombin generation that reflects the magnitude of liver transplant surgery.
Immune function in patients with β thalassaemia receiving the orally active iron‐chelating agent deferiprone
Authors: Loebstein Ronen , Dalal Ilan , Nisbet‐Brown Eric , Berkovitch Matitiahu , Meydan Naftaly , Andrews David , Loubser Michael D. , Koren Gideon , Roifman Chaim M. , Olivieri Nancy F.
Published: November 2003

Short‐term deferiprone may reduce body iron in some patients with thalassaemia major. Concerns regarding potential immunosuppressive effects of deferiprone have been raised from results of animal studies and case reports in humans. We studied immune function in 57 thalassaemia patients: 36 treated with deferiprone (L1; CP020) and 21 treated with desferrioxamine (DFO). Circulating B lymphocytes were increased in all patient groups. No differences were detected between treatment groups in percentages of circulating lymphocytes, concentrations of IgG, IgM or IgA, specific antibody titres, complement levels, or in vitro lymphocyte proliferation. No clinically important infections were observed in any patient. These data suggest that no clinical or laboratory changes consistent with immunosuppression or immunodeficiency are observed during deferiprone therapy.
Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM‐CSF and role of the Bcr gene
Authors: Novella Annunziata , Bergamaschi Gaetano , Canale Claudia , Fossati Gianluca , Gritti Donatella , Lucotti Claudia , Mazzone Antonino , Tonon Laura , Cazzola Mario
Published: November 2003

Although devoid of proliferative capacity, polymorphonuclear neutrophils (PMN) express receptors for haemopoietic growth factors and need growth factors for survival and functional stimulation. This study showed that in vitro treatment of human PMN with GM‐CSF for up to 48 h increases cell surface expression of the β2‐integrin molecules CD11b/CD18 and CD11c/CD18 and of the receptor for the chemotactic peptide fMLP. Such modifications are usually expression of PMN activation. PMN treated with GM‐CSF also displayed increased phagocytosis of latex particles and enhanced oxidative burst and superoxide anion release. Since integrins mediate PMN adhesion to endothelium, homotypic adhesion, chemotaxis/phagocytosis and the triggering of respiratory burst, our results suggested that functional stimulation of PMN persisted following prolonged exposure of PMN to growth factors and that it was not a temporary phenomenon which lasted only for the first 12–24 h of treatment. We also used oligonucleotides antisense to the Bcr gene mRNA to inhibit expression of the gene and evaluate its function in PMN, following the recent observation that PMN from Bcr‐null mutant mice produced increased amounts of reactive oxygen metabolites upon activation. The antisense oligonucleotides had no effect on the parameters investigated. This may indicate that increased production of O−2 by neutrophils in which the Bcr gene is not expressed requires either that gene expression is absent in the earlier stages of myeloid differentiation/maturation, so that when inhibition occurs in the terminally differentiated neutrophils their functional status is no longer influenced, or that the residual low‐level expression of the gene which may be present in the antisense‐treated cells is sufficient to provide a normal response to stimulation.
Activation of neutrophil function via CD66: differential effects upon β2 integrin mediated adhesion
Authors: Ruchaud‐Sparagano Marie‐Hélène , Stocks S. Craig , Turley Helen , Dransfield Ian
Published: November 2003

To further define the role of CD66 glycoproteins in the regulation of neutrophil function, we analysed the effects of a CD66 monoclonal antibody, Kat4c, which recognizes an epitope present on AB domains of CD66a, CD66b and CD66c. Intact Kat4c and F(ab′)2 fragments were found to augment fMLP‐induced oxidation of 1,2,3‐dihydrorhodamine (oxidant species production) and β2 integrin‐mediated adhesion to fibrinogen but did not promote β2 integrin‐mediated binding of albumin coated latex beads. Since the latter assay is a sensitive indicator of neutrophil CD11b/CD18 functional activation, these results imply CD66 may exert differential effects upon β2 integrin activity. Neutrophil oxidant species production and spreading on fibrinogen substrates were further potentiated by cross‐linking of Kat4c F(ab′)2, in keeping with the suggestion that ligation of CD66 regulates neutrophil function. However, although intact Kat4c promoted β2 neutrophil adhesion, F(ab′)2 fragments were without effect, implying a role for Fc receptors in this effect which has previously been attributed to CD66. Together these data define more clearly the role of CD66 in regulation of neutrophil function and further suggest that augmented β2 integrin‐mediated adhesion following CD66 ligation occurs independently of affinity regulation.
Two new mutations of the glucose‐6‐phospate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships
Authors: Zarza R. , Pujades A. , Rovira A. , Saavedra R. , Fernandez J. , Aymerich M. , Vives Corrons J. Ll.
Published: November 2003

In two unrelated Spanish males with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid406 C→T and G6PD Madrid1155 C→G .

In vitro biochemical characterization of both mutant enzymes showed important differences in their molecular properties according to their different clinical behaviour. In G6PD Valladolid, the mutation of which is located in exon 5, the normal in vitro heat stability may explain its mild clinical expression (low‐grade haemolysis interrupted by an acute haemolytic crisis at age 70). In G6PD Madrid, the mutation, located in exon 10, results in a deficient variant associated with neonatal jaundice and life‐long chronic nonspherocytic haemolytic anaemia (CNSHA). This finding further emphasizes the importance of this specific region of the G6PD gene in the stabilization of the G6PD molecule. Putative relationships between these single point mutations and the molecular properties of the mutant enzymes are also discussed.
Endothelial cell precursors are normal components of human umbilical cord blood
Authors: Nieda Mie , Nicol Andrew , Denning‐Kendall Patricia , Sweetenham John , Bradley Ben , Hows Jill
Published: November 2003

Endothelial cells are part of the normal bone marrow stroma. We have previously shown human umbilical cord blood (UCB) does not produce stroma in standard long‐term cultures. Highly enriched (93–98%) UCB CD34+ cells were cultured for 6 weeks with interleukin‐2 and conditioned medium from the 5637 carcinoma cell line (n = 4). The resulting ‘fibroblast like’ cells were shown to be endothelial by expression of von Willebrand factor (VWF), ICAM‐1 (CD54), E‐selectin (CD62E) and PECAM (CD31). Endothelial monolayers seeded with CD34+ UCB cells supported expansion of colony forming cells and CD34+ cells. We conclude that endothelial cell precursors circulate in UCB, and may be derived from the CD34+ cell fraction.
A sensitive and specific functional flow cytometric assay for the diagnosis of heparin‐induced thrombocytopenia
Authors: Tomer Aaron
Published: November 2003

A functional flow cytometric assay (FCA) for the immediate diagnosis of heparin‐induced thrombocytopenia (HIT), with simultaneous compatibility testing for alternative anticoagulant therapies, has been developed to provide rapid and reliable results which effectively support patient management. The assay provides results within 1–2 h, uses readily available non‐radioactive reagents, and employs standard equipment. Using the highly sensitive annexin V protein probe, the method detects activated platelets induced by heparin immune‐complexes, with 300‐fold increased binding to activated platelets. Twenty‐five samples from patients clinically‐suspected of having HIT (131 tests) and 10 normal control (NC) samples (36 tests) were simultaneously tested with unfractionated heparin (UH) and low‐molecular‐weight heparin (LMWH), and by the radioactive serotonin‐release assay (SRA) (62 and 16 tests respectively). The FCA highly correlated with the SRA, showing 100% specificity and 95% sensitivity. Moreover, the FCA exhibited higher resolution between positive and negative samples (an average value of 8.6‐fold the NC versus 4.0‐fold the NC by SRA). The LMWH showed concordant results with UH (r=0.95). We conclude that the functional FCA for HIT is practical, specific and sensitive, thereby permitting the rapid diagnosis of HIT and the suitability of alternative therapies.
Inhibition of autophagy abrogates tumour necrosis factor α induced apoptosis in human T‐lymphoblastic leukaemic cells
Authors: Jia Li , Dourmashkin Robert R. , Allen Paul D. , Gray Alan B. , Newland Adrian C. , Kelsey Stephen M.
Published: November 2003

The pattern and the sequence of tumour necrosis factor‐α (TNFα) induced cell death in the acute T‐lymphoblastic leukaemic cell line CCRF‐CEM and its vinblastine‐resistant subline CEM/VLB100 have been studied. Previously, we found that the CEM/VLB100 cell line was more sensitive to TNFα‐induced killing than its parental CCRF‐CEM cell line. TNFα‐induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and condensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3‐methyladenine (3MA), inhibited the formation of autophagosomes. TNFα‐induced DNA fragmentation and cytolysis were completely inhibited by 10 mM 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFα‐induced apoptosis. In addition, amino acid and protein deprivation enhanced TNFα‐induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNFα‐induced apoptosis.
Expression and functional analysis of two isoforms of the human GM‐CSF receptor α chain in myeloid development and leukaemia
Authors: Crosier Kathryn E. , Hall Louanne R. , Vitas Maria R. , Crosier Philip S.
Published: November 2003

The human granulocyte‐macrophage colony‐stimulating factor receptor (GM‐CSFR) α chain RNA is alternatively spliced to yield receptor isoforms. Two of these, α1 and α2, differ in their cytoplasmic domains. Because the GM‐CSFR β chain (βc) is shared with the receptors for interleukins 3 and 5 it is possible that the α chain confers specificity on the GM‐CSF response and that the different isoforms might refine this response further. Studies have been directed at determination of the respective biological roles of the α1 and α2 isoforms. Expression of the isoforms was examined by RNase protection analysis in normal granulocytes and a variety of cell lines of haemopoietic origin, at different stages of differentiation and activation. Expression was also analysed in cells from patients with a variety of leukaemic subtypes. Results demonstrated that the relative abundance of the isoforms was similar in all cell populations examined. The human GM‐CSFR α1 or α2 receptors were independently expressed in the murine factor‐dependent cell line FDC‐P1, so that the properties of the receptors could be compared. Cell lines that expressed either receptor could be converted to growth in response to human GM‐CSF and assumed a more differentiated phenotype when compared with the parental cell line. However, the morphology, expression of cell surface antigens and dose–growth response characteristics did not differ significantly between cells that expressed either the α1 or α2 receptor. These studies demonstrate that the α1 and α2 subunits of the GM‐CSF receptor are co‐ordinately regulated in both normal and malignant haemopoiesis. Furthermore, each receptor is able to deliver both proliferative and differentiative signals to myeloid cells.
Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug‐induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance
Authors: Jia Li , Allen Paul D. , Macey Marion G. , Grahn Michael F. , Newland Adrian C. , Kelsey Stephen M.
Published: November 2003

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine‐resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF‐CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine‐induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1F0‐ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy‐dependent P‐glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine‐induced cell cycle changes in the CCRF‐CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB‐induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over‐expression of Pgp by drug‐resistant cells may up‐regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down‐regulation of mitochondrial ETC activity may confer resistance to vinblastine‐induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine‐induced apoptosis downstream of microtubule disruption and cell cycle changes.
Bone marrow innervation regulates cellular retention in the murine haemopoietic system
Authors: Afan A. M. , Broome C. S. , Nicholls S. E. , Whetton A. D. , Miyan J. A.
Published: November 2003

An anatomical analysis of the innervation of murine femora revealed intimate association of haemopoietic and stromal cells with nerve fibres. The mechanical denervation of these femora resulted in significant mobilization of cells into the peripheral blood within 24 h. There was a decrease in femoral cellularity and analysis of the type of cells mobilized also revealed that there was an increase in progenitor cells in the peripheral blood. In non‐splenectomized mice these progenitor cells were quickly cleared from the circulation. Chemical sympathectomy of mice with 6‐hydroxydopamine resulted in decreased bone marrow cellularity without a change in bone marrow or peripheral blood progenitor cell numbers, nor the sustained rise in peripheral leucocytes observed with whole nerve denervation. These observations argue for selective control of mobilization by the nervous system and also indicate possible control of proliferation within the bone marrow. We conclude that the innervation has an important role in the maintenance of the blood–marrow interface, control of peripheral blood cell numbers, and mobilization of colony forming cells into the periphery.
An erythroid and megakaryocytic common precursor cell line (B1647) expressing both c‐mpl and erythropoietin receptor (Epo‐R) proliferates and modifies globin chain synthesis in response to megakaryocyte growth and development factor (MGDF) but not to erythropoietin (Epo)
Authors: Bonsi Laura , Grossi Alberto , Strippoli Pierluigi , Tumietto Fabio , Tonelli Roberto , Vannucchi Alessandro M. , Ronchi Antonella , Ottolenghi Sergio , Visconti Giovannella , Avanzi Gian Carlo , Pegoraro Luigi , Paolo Bagnara Gian
Published: November 2003

A human megakaryocyte cell line (B1647) has been established from bone marrow cells obtained from a patient with acute myelogenous leukaemia (FAB M2). The cells were CD34−, CD33+, HLA‐DR+, CD38+, and expressed the immunophenotypic markers of the megakaryocyte lineage (CD41 and von Willebrand factor). Moreover the cells expressed the c‐mpl (thrombopoietin receptor) mRNA and protein. On the other hand, the B1647 cells also possessed erythroid lineage characteristics: the vast majority of cells were glycophorin positive, and about 10% of unstimulated cells stained with an anti‐globin γ chain MoAb. In addition, S1 protection analysis demonstrated expression of β‐globin mRNA, and Epo receptor (Epo‐R) protein was detected by cytofluorimetric assay. Several growth factors, when tested alone or in combination, failed to influence the B1647 cell growth. A significant increase of cell proliferation was observed only after the addition, in serum‐free culture, of recombinant human megakaryocyte growth development factor (MGDF), a recombinant c‐mpl ligand encompassing the receptor‐binding domain and identical to thrombopoietin (TPO), at concentrations ranging from 0.01 to 1 ng/ml. Interestingly, MGDF failed to induce megakaryocytic differentiation of the B1647 cells, but significantly increased the synthesis of the globin γ‐chain.

B1647 cells could be a useful model for studying the biological effect of TPO on common megakaryocyte and erythroid progenitors.
The successful use of protein C concentrate during pregnancy in a patient with type 1 protein C deficiency, previous thrombosis and recurrent fetal loss
Authors: Richards E. M. , Makris M. , Preston F. E.
Published: November 2003

The risks of venous thrombosis and fetal loss are increased in patients with protein C deficiency. We describe a patient with a history of thrombosis and recurrent fetal loss who was found to have type 1 protein C deficiency. In view of her history and intolerance of heparin preparations, she was treated with protein C concentrate during the first and third trimesters of her seventh pregnancy. The patient suffered no thromboembolic event during this pregnancy and gave birth to a healthy infant. The implications of the success of this therapeutic strategy are discussed.
Platelet activation by a novel solid‐phase agonist: effects of VWF immobilized on polystyrene beads
Authors: STEWART MICHAEL W. , ETCHES WAI S. , BOSHKOV LYNN K. , MANT MICHAEL J. , GORDON PHILIP A. , SHAW ANDREW R. E.
Published: November 2003

The interaction between platelets stirred in suspension and VWF immobilized on polystyrene beads was studied. Platelets aggregated and released ATP in response to stirring with VWF beads. Closer examination of the interaction using transmission electron microscopy revealed that the platelets did not simply aggregate with one another but initially adhered to the beads and spread. Platelets in suspension then bound to the bead‐adherent platelets forming layers of platelets associated with each bead. The VWF bead‐induced platelet activation was com‐pletely inhibited by addition of monoclonal antibody (mAb) to GPIb or GPIIb/IIIa. In addition, the activation response was inhibited in the presence of aspirin, indomethacin or the thromboxane receptor antagonist BM13.177, demonstrat‐ing a dependence on an intact cyclo‐oxygenase pathway.  Platelet function studies were carried out on 30 patients with a history of mild bleeding using conventional optical aggregation and VWF bead‐induced platelet activation. 12 patients were abnormal by conventional optical aggregometry, whereas 27 patients showed depressed ATP release in response to VWF beads. The results suggest that easily‐bruised patients may have a platelet function defect rather than a vascular‐based abnormality and that VWF bead‐induced platelet activation is a more sensitive test for detecting platelet dysfunction.
The anti‐neoplastic drug 5‐fluorouracil produces echinocytosis and affects blood rheology
Authors: Baerlocher G. M. , Beer J. H. , Owen G. Rh. , Meiselman H. J. , Reinhart W. H.
Published: November 2003

The anti‐neoplastic agent 5‐fluorouracil (5‐FU) in high therapeutic doses can induce angina pectoris and myocardial infarction. The pathophysiological mechanism of this side‐effect has not yet been elucidated. We analysed the influence of 5‐FU on blood rheology in vitro. Whole blood, blood cell suspensions and plasma were incubated with increasing concentrations of 5‐FU (final concentrations 0, 0.08, 0.4, 2, 10 and 25 mg/ml 5‐FU) at 37°C. Erythrocyte morphology was analysed after fixation with glutaraldehyde. Viscosity was measured at high and low shear rates (94 and 0.1 s−1). Erythrocyte aggregation and the cell transit times of erythrocytes through 5 μm pores and polymorphonuclear leucocytes through 8 μm pores were determined. 5‐FU induced a dose‐dependent formation of echinocytes within minutes and was reversible upon removal of 5‐FU, which reflected a preferential intercalation of the drug in the outer hemileaflet of the cell membrane. High shear blood viscosity was increased at the highest 5‐FU concentration (148 ± 12%), and at low shear rate a dose‐dependent decrease was found (0 mg/ml: 100%, 0.08 mg/ml: 87 ± 10%, 0.4 mg/ml: 80 ± 19%, 2 mg/ml: 70 ± 15%, 10 mg/ml: 40 ± 19%, 25 mg/ml: 33 ± 5%). Erythrocyte aggregation was decreased by the 5‐FU‐induced echinocytosis. The transit time of erythrocytes through narrow pores was increased in a dose‐dependent manner by 5‐FU, whereas the transit time of polymorphonuclear leucocytes was initially decreased at 10 mg/ml and returned to control after 60 min incubation. We conclude that 5‐FU interacts with the cell membrane, induces echinocytosis and vesiculation and affects blood rheology in several ways which may contribute to cardiovascular complications.
Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases
Authors: Criel Arnold , Verhoef Gregor , Vlietinck Robert , Mecucci Cristina , Billiet Johan , Michaux Lucienne , Meeus Peter , Louwagie Andries , Van Orshoven Angeline , Van Hoof Achiel , Boogaerts Mark , Van den Berghe Herman , De Wolf‐Peeters Chris
Published: November 2003

We analysed a group of 390 patients, diagnosed with chronic lymphocytic leukaemia (CLL). Cases were subclassified as morphologically typical and atypical CLL according to the criteria of the FAB proposal. Typical CLL cases were mostly diagnosed at a low‐risk stage (Binet A/Rai 0), required no immediate treatment and expected a long survival; atypical CLL cases mostly presented at a more advanced risk stage (Binet B/Rai I–II), usually required immediate treatment and their survival was shorter. Moreover, clinical staging was of prognostic significance in typical but not in atypical cases.  In typical CLL, del(11q) was the most common chromosomal abnormality (21%) whereas in atypical CLL trisomy 12 was found in about 65% of the cases documented with an abnormal karyotype. Although chromosomal abnormalities were associated with a poor survival in typical CLL, they are of no prognostic significance in atypical CLL.  Based on these data, we conclude that subtyping CLL by morphology enables the identification of two groups of cases, each characterized by a specific clinical presentation, different cytogenetic abnormalities and prognostic parameters. We speculate that these two groups may represent two related, but different, diseases with different prognostic parameters and a different survival.
The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous β‐thalassaemia and in glucose‐6‐phosphate dehydrogenase deficiency
Authors: Sampietro Maurizio , Lupica Loredana , Perrero Luca , Comino Alessia , Martinez di Montemuros Franco , Cappellini Maria Domenica , Fiorelli Gemino
Published: November 2003

We evaluated the effect of Gilbert's syndrome, the most common defect of bilirubin conjugation, on the bilirubin levels of subjects with inherited haematological disorders which cause increased bilirubin production. 57 patients heterozygous for β‐thalassaemia, 21 with G6PD deficiency and 44 controls were examined by typing the TATA‐box in the promoter of the gene uridine diphosphate glucuronosyltransferase 1A. Nearly 80% of patients with increased bilirubin levels were heterozygous or homozygous for the UGT1A TA(7) variant associated with Gilbert's syndrome. These findings indicate that Gilbert's syndrome accounts for a large proportion of the variability of bilirubin levels in β‐thalassaemia and G6PD deficiency.
Deletion of a consensus oestrogen response element half‐site in the glucocorticoid receptor of human multiple myeloma
Authors: Karkera Jayaprakash D. , Taymans Susan E. , Turner Gordon , Yoshikawa Takeo , Detera‐Wadleigh Sevilla D. , Wadleigh Robert G.
Published: November 2003

We have carried out molecular scanning of the glucocorticoid receptor (GR) of the glucocorticoid resistant multiple myeloma cell line U266. An amplified fragment from the 3′ untranslated region displayed an aberrant migration by PCR–single‐stranded conformational polymorphism (PCR‐SSCP) analysis. The mutant allele had a deletion of an 8 base pair sequence containing a half‐site of an oestrogen response element. This motif was found conserved in rat GR. This same allele lacked four As in an upstream region with 18 consecutive As in the normal allele. These mutations may affect mRNA stability or alter interactions with regulatory factors.
Dapsone for chronic autoimmune thrombocytopenic purpura: a report of 66 cases
Authors: Godeau Bertrand , Durand Jean‐Marc , Roudot‐Thoraval Françoise , Tennezé Anne , Oksenhendler Eric , Kaplanski Gilles , Schaeffer Annette , Bierling Philippe
Published: November 2003

Sixty‐six adults with chronic autoimmune thrombocytopenic purpura AITP and platelet count <50 ×109 l were treated with dapsone (75–100 mg orally). A response was observed in 33 patients. The median duration of treatment required to obtain a response was 21 d (range 8–90). The median maximal platelet count on treatment was 130 × 109 l (range 71–355). Dapsone was continued in 20/33 responders for a median of 12.5 months (range 1–48) and the response persisted in 19. Treatment was intentionally withdrawn in the other 13 responders and thrombocytopenia immediately recurred in 12. Reversible side‐effects required cessation of treatment in seven patients. These results demonstrate that dapsone is an effective, inexpensive, and well‐tolerated treatment for chronic AITP.
Hepatitis G virus infection in patients with bleeding disorders
Authors: Wilde Jonathan T. , Ahmed Monzur M. , Collingham Kathryn E. , Skidmore Susan J. , Pillay Deenan , Mutimer David
Published: November 2003

Eighty‐two patients with bleeding disorders registered with our centre were screened for infection with hepatitis G virus (HGV). 80 patients were positive for hepatitis C (HCV) antibodies, 66 of whom (83%) were HCV PCR positive. 11 patients (13%) were HGV RNA‐positive, a similar prevalence rate to that of other studies of patients with bleeding disorders who received factor concentrates prior to the introduction of viral inactivation procedures. There was no significant difference in histological activity index (HAI) between the 10 HGV RNA‐positive and the 31 HGV RNA‐negative patients who underwent liver biopsy for assessment of HCV infection (median HAI scores 5.5, range 2–10 and four, range 0–10 respectively, P = 0.07). One patient in each group had established cirrhosis. In patients who underwent HCV quantitation there was no significant difference in HCV viral titre between HGV RNA‐positive and negative patients (median HCV titre in HGV RNA‐positive patients 2.10 × 105 DNA copies /ml (n = 8) range 4.17 × 102 to 4.17 × 106, median HCV titre in HGV RNA‐negative patients 3.33 × 105 (n = 31) range 1.00 × 103 to 6.67 × 106, P = 0.68). In this study there was no evidence that individuals co‐infected with HGV and HCV have more severe liver disease than those infected with HCV alone.
The role for liver biopsy in haemophiliacs infected with the hepatitis C virus
Authors: SHIM WONG VOI , BAGLIN TREVOR , BEACHAM EMMA , WIGHT DEREK D. G. , PETRIK JURAJ , ALEXANDER GRAEME J. M.
Published: November 2003

Assessment of chronic hepatitis C virus infection requires a liver biopsy in most circumstances. There is a reluctance to perform liver biopsy in haemophiliacs because of a perceived risk of haemorrhage, although with adequate factor concentrate replacement in patients without factor concentrate inhibitors it should be safe. We report a 4‐year experience of liver biopsy in patients with haemophilia infected with chronic hepatitis C virus. Of 55 patients seropositive for anti‐HCV, 35 have undergone liver biopsy; the median age of this group was 33 years (range 13–68). Seven patients had a normal liver. 22 had portal tract inflammation, four with lymphoid aggregates. Mild piecemeal necrosis was observed in only two and no bile duct injury was found. 11 patients had mild mixed micro‐ and macro‐vesicular fat. 19 patients had no evidence of fibrosis despite an estimated median duration of disease of 20 years (range 8–43). In the remaining 16 patients the maximum degree of fibrosis achieved was stage III. Patients with more significant fibrosis could not be identified on the basis of ALT or HCV RNA. There were no complications of liver biopsy in this series. Liver biopsy following a well‐defined protocol in chronic hepatitis C virus haemophiliac carriers is safe in the absence of factor concentrate inhibitors. In this young group of patients without HIV infection there was no evidence of significant liver disease despite a considerable duration of disease. Performing liver biopsy allows accurate information to be given to the patient and avoids unnecessary therapy. The relative youth of this group may be important in the light of the benign histology.