bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages
Authors: Zamai Loris , V Marco , Bennett Ian M. , Croce Carlo M. , Perussia Bice
Published: March 2003

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi‐phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.
Long‐term detection of microchimaerism in peripheral blood after pretransplantation blood transfusion
Authors: Vervoordeldonk Susan F. , Doumaid Karim , Remmerswaal Ester B. M. , Ten Berge Ineke J. M. , Wilmink Joep M. , DE Waal Leo P. , Boog Claire J. P.
Published: March 2003

Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA‐B and one HLA‐DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor‐type cells. A sensitive nested PCR technique based on HLA‐DRB1 allele‐specific amplification using sequence‐specific primers (detection level: one donor cell among 105 recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor‐type cells were detected in the first week after transfusion, in 7/8 patients 2–4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed.
Genomic typing of the Kidd blood group locus by a single‐tube allele‐specific primer PCR technique
Authors: Irshaid Nidal M. , Thuresson Britt , Olsson Martin L.
Published: March 2003

The Kidd (JK) blood group system is clinically important in transfusion medicine. Alloantibodies to antigens in this system may be produced following blood transfusion or during pregnancy and can result in serious haemolytic transfusion reactions and haemolytic disease of the newborn (HDN).

JK antigens on erythrocytes are carried by glycoproteins with the capacity to transport urea through cell membranes. cDNA complementary to mRNA transcribed at the JK locus was cloned in 1994. The molecular basis of the Jka/Jkb blood group polymorphism was recently shown to be a single nucleotide substitution predicting an amino acid change (Asp280Asn) in an extracellular loop of the JK glycoprotein.

After confirmation of the JK gene polymorphism we developed a rapid and robust technique for JK genotyping with allele‐specific primers in a single‐tube PCR. In addition, a 217 bp intron located at nucleotides 811–812 in the JK gene was found and sequenced. The genotyping test was validated with samples from 106 Caucasian Swedish and 13 Black South African random blood donors. Complete phenotype–genotype correlations were obtained. However, four Jk(a−b−) samples of Polynesian and Finnish origin typed as JkbJkb.

Potential use of the presented method can be predicted in clinical transfusion medicine including prenatal determination of the JK genotype in a fetus at risk for HDN caused by JK antibodies.
A prospective clinical trial of implantable central venous access in children with haemophilia
Authors: Santagostino  , Gringeri  , MuçA‐Perja  , Mannucci
Published: March 2003

To assess the risks associated with the use of central venous ports in children with haemophilia, 15 HIV‐negative patients were prospectively evaluated. Port insertion was required for immune tolerance in two inhibitor patients and continuous prophylaxis in 13 patients with severe factor VIII deficiency, for whom surgery was covered with recombinant factor VIII (rFVIII), then given daily at home until day 6. One inhibitor patient (titre 7 BU/ml) received high‐dose rFVIII by continuous infusion until day 3, followed by an immune tolerance treatment scheme; the other (titre 12 BU/ml) was given recombinant activated factor VII by continuous infusion until day 7. After training on the use of the port, all patients continued their infusion programme at home. All ports remained in place for a median period of 413 d (range 125–509). The median number of entries into the port was 184 (range 53–567). Port‐site haematoma and infection occurred in one patient on day 7 when an inhibitor became detectable (titre 12 BU/ml). An infectious complication occurred in another patient after 310 d. The port infection rate was 0.42 per 1000 patient‐days (0.33 per 1000 entries into the port). This protocol for port placement with short hospitalization appears feasible and safe.
Type 2N von Willebrand disease due to Arg91Gln substitution and a cytosine deletion in exon 18 of the von Willebrand factor gene
Authors: Casonato  , Gaucher  , Pontara  , Zucchetto  , Zerbinati  , Mazurier  , Girolami
Published: March 2003

Two members of a family previously classified as type 1 von Willebrand disease (VWD), showed a quantitative defect in von Willebrand factor (VWF) antigen and ristocetin cofactor activity and an abnormal capacity of VWF to bind FVIII. Sequencing of the VWF gene region coding for the FVIII binding domain revealed the most frequent type 2N mutation: a single nucleotide change (G2811A) in exon 20, resulting in substitution of glutamine (Gln) for arginine (Arg) 91 in the mature VWF protein in one allele. The other allele contained a cytosine deletion (2680delC) in exon 18, introducing a premature stop codon at position 79 (Val79X) which produced a quantitative defect in VWF levels. The Arg91Gln defect is usually not evident in heterozygotes; however, in these patients it was expressed due to the lack of VWF production from the other allele. This is the first report of type 2N VWD in Italy.
Combined therapy with deferiprone and desferrioxamine
Authors: Wonke  , Wright  , Hoffbrand
Published: March 2003

In a proportion of transfusion‐dependent patients iron chelation with daily doses of deferiprone of 75 mg/kg body weight (b.w.) is inadequate. The effects on iron status of increasing the daily oral dose of deferiprone and/or combining deferiprone therapy with subcutaneous infusions of desferrioxamine have been studied in 13 transfusion‐dependent patients. Raising the daily dose of deferiprone in nine patients from 75 mg/kg to 83–100 mg/kg resulted in a fall in serum ferritin in all nine patients (t test for paired samples, P = 0.0022). Combined therapy of daily deferiprone with subcutaneous desferrioxamine on 2–6 d each week in five patients (with an increased dose of deferiprone in three patients) resulted in a fall in serum ferritin in all five patients studied after 7–15 months (P = 0.0791). No toxic side‐effects attributable to either drug occurred in these five patients or in the nine patients in whom the dose of deferiprone was increased. The effects of the drugs given on the same day on urine iron excretion were additive. These results suggest that increasing the dose of deferiprone or combining subcutaneous desferrioxamine with deferiprone therapy are two methods by which efficacy of iron chelation with deferiprone can be improved in patients inadequately chelated by a daily dose of deferiprone of 75 mg/kg b.w. More extensive trials including full metabolic balance studies are needed to establish the safety and efficacy of long‐term combined therapy.
Coexpression of CD40 and CD40 ligand in B‐cell lymphoma cells
Authors: Clodi Katharina , Asgary Zahra , Zhao Shourong , Kliche Kay‐Oliver , Cabanillas Fernando , Andreeff Michael , Younes Anas
Published: March 2003

CD40 ligand (CD40L) is involved in the T‐cell‐dependent regulation of B‐cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B‐cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B‐cell lymphomas (P  < 0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T–B‐cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.
Increased expression of phosphatidylinositol‐specific phospholipase C resistant prion proteins on the surface of activated platelets
Authors: Holada Karel , Heath Mondoro Traci , Muller Jacqueline , Vostal Jaroslav G.
Published: March 2003

The surface expression of prion protein (PrPC) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two‐fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrPC occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37°C. In comparison, PrPC on the surface of platelets, activated at 22°C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrPC and P‐selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrPC was translocated from internal granules to the plasma membrane during activation, as is P‐selectin. Platelet PrPC was not removed from the surface of platelets by phosphatidylinositol‐specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrPC.
Effect of the interaction between fibronectin and VLA‐4 on the proliferation of human B cells, especially a novel human B‐cell line, OPM‐3
Authors: Yoshida Hitoshi , Nishiura Tetsuo , Karasuno Takahiro , Matsumura Itaru , Ishikawa Jun , Yoshimura Masafumi , Yokota Takafumi , Okajima Yu , Ogawa Megumu , Kanakura Yuzuru , Tomiyama Yoshiaki , Matsuzawa Yuji
Published: March 2003

Very late antigen (VLA)‐4 integrin has been suggested to play an important role in haemopoiesis. However, little is known concerning the roles of the fibronectin (FN)/VLA‐4 interaction in the proliferation of human B cells. In this study we investigated the effect of immobilized FN on the proliferation of various B‐cell lines, including a newly‐established B‐cell line, OPM‐3, and human tonsillar B cells, that primarily express VLA‐4 but not VLA‐5. Immobilized FN significantly promoted the proliferation of OPM‐3 cells and normal B cells via VLA‐4. The cross‐linking of β1 integrins of OPM‐3 cells resulted in the phosphorylation of the focal adhesion kinase (FAK) associated 90 kD protein, an increase in FAK‐associated kinase activity, and the phosphorylation of Raf‐1. Furthermore, the MEK1 inhibitor, PD98059, inhibited the FN‐promoted proliferation of OPM‐3 cells. These results demonstrate that the FN/VLA‐4 interaction transmits the growth signal(s) which may be mediated by Ras pathway in OPM‐3 cells, and suggest that OPM‐3 cells may be of great value in studying the roles of the FN/VLA‐4 interaction in human B‐cell growth.
Factor V Arg306 → Thr (factor V Cambridge) and factor V Arg306 → Gly mutations in venous thrombotic disease
Authors: Franco  , Maffei  , LourenÇo  , Morelli  , Thomazini  , Piccinato  , Tavella  , Zago
Published: March 2003

We investigated the prevalence of two reported mutations of the factor V gene (factor V Arg306 → Thr, or factor V Cambridge, and factor V Arg306 → Gly) in 104 relatively young patients with verified venous thrombosis and in 208 age‐, sex‐ and race‐matched controls, in order to establish whether the two mutations are associated with increased predisposition for venous thrombosis. PCR amplification followed by BstNI and MspI digestion was employed to determine the genotypes, and each mutation was confirmed by DNA sequencing. Among the controls, one individual was found to be heterozygous for the factor V Arg306 → Thr mutation and one heterozygous for the factor V Arg306 → Gly mutation; none of the patients carried either mutation. Our findings do not support factor V Cambridge and factor V Arg306 → Gly as risk factors for venous thrombosis.
Autologous bone marrow transplantation for non‐Hodgkin's lymphoma resulting in long‐term remission of coincidental Crohn's disease
Authors: Kashyap Ashwin , Forman Stephen J.
Published: March 2003

Crohn's disease usually runs a chronic relapsing and remitting course, characterized by a repeated need for immunosuppressive drug therapy or often surgery, and is considered to be incurable by standard treatment. We report a case of successful long‐term disease control of Crohn's disease following autologous bone marrow transplantation. This case provides further support for the concept that some forms of severe immune‐mediated diseases may be amenable to treatment by high‐dose therapy with autologous stem cell support.
Mast cell recruitment after subcutaneous injection of RANTES in the sole of the rat paw
Authors: Conti Pio , Reale Marcella , Barbacane Renato C. , Felaco Mario , Grilli Alfredo , Theoharides Theoharis C.
Published: March 2003

The effect of hrRANTES was studied after the injection in the sole of the rat paw, an area particularly rich in mast cells. Subcutaneous injections of RANTES 50 ng/10 μl produced an erythematous reaction which was inhibited by anti‐RANTES antibody 50 μg/rat injected in the tail vein 30 min before hrRANTES 50 ng/10 μl was injected. In another set of experiments the animals were injected subcutaneously in the sole of the paw with PBS 10 μl (control), LPS (100 ng/10 μl) hrRANTES 50 ng/10 μl or anti‐RANTES 50 μl/rat injected in the tail vein 30 min before hrRANTES 50 ng/10 μl was injected. The biopsies were analysed after 4 h and counted in an optic field. hrRANTES produced a strong recruitment of mast cells selectively coloured with 0.1% toluidine blue and inhibited by anti‐RANTES antibody. In addition to the optical and electron microscope study, in some of the excised tissue Northern blot analysis for histidine decarboxylase (HDC) mRNA was performed to estimate the amount of histamine generation in the tissue of the injection sites. We found that subcutaneous injections of hrRANTES 50 ng/10 μl in the sole of the rat paw produced an accumulation of a great number of mast cells compared to PBS 10 μl (negative control) or LPS 100 ng/10 μl (positive control) after 4 h. The hrRANTES effect was inhibited by anti‐RANTES antibody injected in the tail vein 30 min before hrRANTES exposure. Moreover, hrRANTES increased HDC mRNA and histamine generation.
Role of vascular endothelial growth factor (VEGF) and placenta‐derived growth factor (PlGF) in regulating human haemopoietic cell growth
Authors: Ratajczak Mariusz Z. , Ratajczak Janina , Machalinski Bogdan , Majka Marcin , Marlicz Wojtek , Carter Anna , Pietrzkowski Zbigniew , Gswirtz Alan M.
Published: March 2003

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk‐1/KDR and Flt‐1 respectively. Flk‐1/KDR‐deficient murine embryos manifest failure of blood‐island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt‐1 and Flk‐1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt‐1 and Flk‐1/KDR receptors on stem cell rich CD34+ c‐kit‐R+ Rh123low cells. Further studies revealed that Flt‐1 R mRNA, but not Flk‐1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co‐stimulated HEL cell proliferation as well as CFU‐GM colony formation from ∼15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.
Structural and functional analysis of the cytidine deaminase gene in patients with acute myeloid leukaemia
Authors: Schröder Jan K. , Kirch Christoph , Seeber Siegfried , Schütte Jochen
Published: March 2003

Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro. To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well‐defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF‐AML blasts (P = 0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy (P = 0.043). Structural investigation of the CDD gene by Southern analyses and RT‐PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF‐AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdr1 expression, this result could be useful for the development of risk‐adapted AML treatment strategies and warrants further studies of CDD activity in well‐defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.
P230 BCR/ABL protein may be associated with an acute leukaemia phenotype
Authors: Haškovec Cedrik , Ponzetto Carola , Polák Jaroslav , Maritano Diego , Zemanová Zuzana , Serra Anna , Michalová Kyra , Klamová Hana , Čermák JARoslav , Saglio Giuseppe
Published: March 2003

The BCR/ABL rearrangement, the molecular hallmark of chronic myelogenous leukaemia (CML), is rare in acute myeloid leukaemia (AML), being detected in approximately 1% of cases. In the vast majority of CML cases the breakpoint on chromosome 22 falls in the so‐called major breakpoint cluster region of the BCR gene. Only a few cases of CML with breakpoint in the minor or in the micro bcr region have so far been reported. The micro breakpoint position has been associated mainly with a mild form of CML, defined as Philadelphia chromosome‐positive chronic neutrophilic leukaemia (Ph‐positive CNL). Using reverse transcription‐polymerase chain reaction (RT‐PCR) we report a patient with an acute myeloid leukaemia phenotype at diagnosis who showed a BCR/ABL rearrangement with a breakpoint located in the micro bcr region (e19a2 junction). Cytogenetic analysis showed a progression of the malignant clone, finally leading to cells with two Ph chromosomes, trisomy 8, isochromosome 17q and deletion of the long arms of chromosome 7. The findings of chromosomal changes point to a possibility of blast crisis of CML with a clinically silent chronic phase. Immunoprecipitation and auto‐phosphorylation assay revealed the expression, by the patient's blast cells, of an abnormal P230 BCR/ABL protein, which showed for the first time that this protein was constitutively activated in primary cells from patients. This finding may contribute to the understanding of the role of the BCR/ABL rearrangements in determining different leukaemia phenotypes ranging from acute lymphoid and myeloid leukaemias to mild chronic neutrophilic leukaemias.
Interleukin‐1 in multiple myeloma: producer cells and their role in the control of IL‐6 production
Authors: Costes Valérie , Portier Marielle , Lu Zhao‐Yang , Rossi Jean‐François , Bataille Régis , Klein Bernard
Published: March 2003

We studied the role of interleukin (IL)‐1β in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL‐1β gene and produced IL‐1β. Myeloma cells less potently expressed the IL‐1β gene and IL‐1β protein. IL‐1β gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL‐1β gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL‐1 was mainly responsible for IL‐6 production in the tumoural environment through a PGE2 loop. In fact, an IL‐1 receptor antagonist (IL‐1RA) blocked PGE2 synthesis and IL‐6 production by 80%; this blockage could be reversed by adding synthetic PGE2. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE2 synthesis. Taken together, these data emphasize the possibility of blocking IL‐1 by using IL‐1RA or other antagonists in order to block IL‐6 production, which is a major tumoural survival and proliferation factor.
Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant
Authors: Simioni Paolo , Tormene Daniela , Manfrin Davide , Gavasso Sabrina , Luni Sonia , Stocco Domenico , Girolami Antonio
Published: March 2003

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5–10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non‐carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n = 178) or asymptomatic (n = 99) non‐carriers. The percentage of subjects with prothrombin levels above cut‐off values of 1.15 u/ml or 1.30 u/ml was significantly higher in carriers of the prothrombin variant as compared to non‐carriers, regardless of a previous thrombosis. However, among non‐carriers the percentage of those with prothrombin levels above cut‐off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non‐carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.
New alleles of the platelet glycoprotein Ibα gene
Authors: Corral  , González‐Conejero  , Lozano  , Rivera  , Vicente
Published: March 2003

Platelet membrane glycoprotein Ibα (GP Ibα) bears two molecular polymorphisms which are in linkage disequilibrium: the C/T dimorphism at codon 145 (HPA‐2) and the variable number of tandem repeats (VNTR) polymorphism in the macroglycopeptide region. The frequencies of these two polymorphisms, and of another three recently described silent polymorphisms, were investigated by genotypic identification in 729 Caucasian individuals from the south of Spain. Eight different alleles of this gene, including the longest VNTR A allele of the GP Ibα gene, were found in this population. Moreover, we detected an unexpected linkage between the B and A variants of the VNTR polymorphism and the HPA‐2a allele in 5.9% of this population. These results suggest a new evolutionary model of GP Ibα, in which homologous recombination could account for the genetic diversity of the GP Ibα.
Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH‐cytochrome b5 reductase gene
Authors: Higasa Koichiro , Manabe Jun‐Ichi , Yubisui Toshitsuga , Sumimoto Hideki , Pung‐amritt Parichat , Tanphaichitr Varavarn S. , Fukumaki Yasuyuki
Published: March 2003

Hereditary methaemoglobinaemia, caused by deficiency of NADH‐cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C‐to‐T substitution in codon 83 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C‐to‐T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life‐span of erythrocytes.
Study of the association between cytochromes P450 2D6 and 2E1 genotypes and the risk of drug and chemical induced idiosyncratic aplastic anaemia
Authors: Marsh J. C. W. , Chowdry J. , Parry‐Jones N. , Ellis S. W. , Muir K. R. , Gordon‐Smith E. C. , Tucker G. T.
Published: March 2003

A genetic susceptibility to drug or chemical toxicity may provide a basis for an increased risk of idiosyncratic aplastic anaemia (AA). The cytochrome P450 enzymes are responsible for the metabolism of many drugs, some of which have been linked to AA. Mutations in the cytochrome P450 CYP2D6 gene result in absent or impaired enzyme activity in about 7% of Caucasians, whereas a specific mutation in the 5′‐regulatory region of the CYP2E1 gene causes overexpression of the gene. We evaluated the frequency of allelic variants of CYP2D6 and CYP2E1 using allele‐specific PCR amplification and restriction enzyme analysis of blood mononuclear cell DNA among 54 Caucasian AA patients. CYP2D6 and CYP2E1 were chosen because of the link between AA and the antipsychotic drug remoxipride (CYP2D6 substrate) and benzene (CYP2E1 substrate), respectively. Results were compared with 53 controls matched for age, sex and ethnicity. The percentage of AA patients homozygous for the CYP2D6*3, CYP2D6*4 alleles (poor metabolizer phenotype) and the CYP2E1 mutant allele (overexpression) was 0%, 4% and 0%, respectively, and the percentage of heterozygotes was 2%, 28% and 15%, respectively. For normal controls the corresponding results for homozygous mutants were 0%, 4% and 0% and for heterozygotes 4%, 25% and 6%, respectively. We concluded that there were no major differences in the frequencies of the genetic polymorphisms between this series of AA patients and controls, but due to the low number of cases with the poor metabolizer phenotype and those with a history of drug exposure, the power of the study was too low to disprove an interaction.
Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects
Authors: SONOKI TAKASHI , HATA HIROYUKI , KURIBAYASHI NORIOMI , YOSHIDA MINORU , HARADA NAOKO , NAGASAKI AKITOSHI , KIMURA TATSUYA , MATSUNO FUMIHIKO , MITSUYA HIROAKI , MATSUZAKI HIROMITSU
Published: March 2003

We analysed PRAD1/cyclin D1 expression in 20 patients with plasma cell malignancy by Northern analysis. 6/17 multiple myeloma patients and 3/3 plasma cell leukaemia patients showed PRAD1/cyclin D1 expression. This incidence appeared to be higher than the expected incidence based on previous studies. Southern analysis did not show rearrangement of the bcl‐1 region. Although there was no statistical difference, the PRAD1/cyclin D1 negative group showed a 1‐year survival of 81.8%, 3‐year survival of 45.5% and 5‐year survival of 22.7%, and those for the PRAD1/cyclin D1 positive group were 63.5%, 16.9% and 16.9%, respectively. Further study is required to determine whether PRAD1/cyclin D1 expression is a prognostic factor.
Development of an EORTC questionnaire module to be used in health‐related quality‐of‐life assessment for patients with multiple myeloma
Authors: STEAD MAXINE , BROWN JULIA , VELIKOVA GALINA , KAASA STEIN , WISLØFF FINN , CHILD J. , HIPPE ERIK , HJORTH MARTIN , SEZER ORHAN , SELBY PETER ,   for the European Organization for Research and Treatment of Cancer Study Group on Quality of Life
Published: March 2003

A multiple myeloma‐specific quality‐of‐life questionnaire module has been designed in collaboration with the EORTC Quality‐of‐Life Study Group to be used in clinical trials with the EORTC QLQ‐C30, a general cancer questionnaire. Strict methodology was employed to ensure thorough and appropriate development of the module. An extensive literature review was performed to identify health‐related quality‐of‐life issues relevant to patients with multiple myeloma. Semi‐structured interviews were then carried out in several European countries with health‐care providers experienced in the treatment of patients with multiple myeloma, and with a group of patients with multiple myeloma, to identify the issues which were most important to patients. A questionnaire was devised from the list of issues, using a 1‐week time‐frame and response categories consistent with the EORTC QLQ‐C30. The provisional questionnaire and the EORTC QLQ‐C30 were administered to patients with multiple myeloma in each participating country with further semi‐structured interviews to refine the content and design of the questionnaire. A review of the results obtained in each stage of development resulted in a 24‐item myeloma‐specific module, the EORTC QLQ‐MY24, which assesses disease‐specific symptoms and their impact on everyday life, treatment side‐effects, social support, and future perspective. The module is currently undergoing further international field‐testing to assess its psychometric properties.
Detection of weak D and DVI red cells in D‐negative mixtures by flow cytometry: implications for feto‐maternal haemorrhage quantification and D typing policies for newborns
Authors: LLOYD‐EVANS P. , GUEST A. R. , VOAK D. , SCOTT M. L.
Published: March 2003

Quantitation of feto‐maternal haemorrhage (FMH) by flow cytometry (FC) has been shown to be more accurate than the Kleihauer‐Bekte test. Fetal cells will be predominately of R1r or R2r phenotype, with antigen site numbers per cell (SPC) of between 9900 and 16 000. If the fetus is of weak D or partial DVI phenotype, fewer SPC will be present.

Red cells from 20 adult weak D samples were mixed with rr red cells to give 1% mixes. Mixtures were stained and analysed by FC, using two different monoclonal reagents. The SPC of each sample was measured using SOL‐ELISA with Scatchard plot analysis. 18 samples could not be distinguished and had <1000 SPC. Two samples that could be distinguished had 1350 and 3000 SPC.

Red cells from seven samples of DVI were also analysed. None of these samples could be distinguished; SPC were all <1000. Although one of the reagents used reacts with DVI cells, quantitation of a DVI FMH would not be possible due to low SPC.

The ability of fetal red cells with low Rh D SPC to cause immunization is questionable; failure to measure FMH in these cases is unlikely to cause clinical problems, as long as suitably sensitive serological reagents and techniques are used to type all weak D and D variant babies as Rh D positive, and thus ensure that the mother is given the appropriate dose of anti‐D.
Detection of lymphocytes and granulocytes expressing the mutant WASP message in carriers of Wiskott‐Aldrich syndrome
Authors: Ariga Tadashi , Yamada Masafumi , Wada Takahito , Saitoh Shinji , Sakiyama Yukio
Published: March 2003

Wiskott‐Aldrich syndrome (WAS) is an X‐linked recessive disease caused by mutations in the recently identified WAS protein gene (WASP). In some X‐linked genetic disorders skewed X‐inactivation has been observed in all cell populations or some specific cell lineages of female carriers. Recently, female carriers of WAS were also revealed to present skewed X‐inactivation patterns at the haemopoietic stem cell level. However, it is not clear if all haematological cells expressing the mutant WASP allele are eliminated in WAS carriers. By reverse transcription PCR methods, we studied 14 WAS carriers from 10 different families to assess whether blood cells expressing the mutant WASP message were present in their peripheral blood. The mutations of each WAS patient were known and carrier diagnosis of their female family members was performed using specific mutation analysis. We detected circulating lymphocytes and granulocytes expressing the mutant WASP message in most of the WAS carriers, nevertheless they showed skewed X‐chromosomal inactivation patterns. Interestingly, the presence of blood cells expressing the mutant WASP message seemed to correlate to the WASP genotype and the age of the carriers.
The effect of sample size on fresh plasma thromboplastin ISI determination
Authors: Poller L. , Van Den Besselaar A. M. H. P. , Jespersen J. , Tripodi A. , Houghton D. ,   for European Concerted Action on Anticoagulation
Published: March 2003

The possibility of reduction of numbers of fresh coumarin and normal plasmas has been studied in a multicentre manual prothrombin (PT) calibration of high international sensitivity index (ISI) rabbit and low ISI human reference thromboplastins at 14 laboratories. The number of calibrant plasmas was reduced progressively by a computer program which generated random numbers to provide 1000 different selections for each reduced sample at each participant laboratory. Results were compared with those of the full set of 20 normal and 60 coumarin plasma calibrations. With the human reagent, 20 coumarins and seven normals still achieved the W.H.O. precision limit (3% CV of the slope), but with the rabbit reagent reduction < 50 coumarins with 17 normal plasmas led to unacceptable CV. Little reduction of numbers from the full set of 80 fresh plasmas appears advisable. For maximum confidence, when calibrating the ISI of a new reagent, it would always seem worthwhile to use the full number of patients' and normal plasmas at each centre for the initial calibration, i.e. 60 fresh coumarin and 20 fresh normal plasmas. Smaller numbers should achieve the required precision with the lower ISI reagent.
Platelet glycoprotein IIIa polymorphism HPA 1b (PlA2): no association with platelet fibrinogen binding
Authors: Meiklejohn D. J. , Urbaniak S. J. , Greaves M.
Published: March 2003

The role of the platelet glycoprotein (GP) IIIa polymorphism HPA 1b (PlA2) in the risk of arterial thrombosis is controversial. We investigated the effect of the 1b allele on platelet fibrinogen binding by flow cytometry. Samples from 35 healthy platelet and plasma donors possessing the 1b allele were compared with 35 1a/1a donors. We found no allele‐dependent difference in percentage of platelets binding fibrinogen (P  =  0.6), nor in mean cell fluorescence (P  =  0.3) following stimulation with ADP. These results render it unlikely that any relationship between the 1b polymorphism and arterial thrombosis is mediated by a significant effect on fibrinogen binding.
Familial multiple myeloma: report of fifteen families
Authors: Grosbois B. , Jego P. , Attal M. , Payen C. , Rapp M. J. , Fuzibet J. G. , Maigre M. , Bataille R. ,   for the Intergroupe Francophone du Myélome
Published: March 2003

To further define the frequency, clinical and biological features of familial multiple myeloma we performed a retrospective study of related patients who presented with multiple myeloma. Most cases of familial multiple myeloma were observed in siblings (10/15), in whom the mean age at diagnosis was similar to unrelated multiple myeloma. In successive generations the mean age at diagnosis was lower. Monoclonal component was identical (IgG kappa) in seven families. Familial history of monoclonal gammopathy of undetermined significance was observed in three families. Five other prospective studies of 1263 patients identified four affected families (3.2 per 1000 cases of multiple myeloma), and raise the question of a genetic background in multiple myeloma.
Gly319 → Arg substitution in the dysfunctional prothrombin Segovia
Authors: Akhavan Sepideh , Rocha Eduardo , Zeinali Sirous , Mannucci Pier Mannuccio
Published: March 2003

The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Segovia, was identified in a patient with a severe bleeding tendency, reduced prothrombin coagulant activity, and normal antigen level. Nucleotide sequencing of amplified DNA revealed a G → A change at nucleotide 7539 of exon 9 of the prothrombin gene. This resulted in the substitution of Gly319 by Arg. The proband was homozygous for this mutation, his father and brother were heterozygous. We surmised that the substitution, which occurs near the site of cleavage of prothrombin by factor Xa (Arg320–Ile321), altered the conformation of the protein making the cleavage site inaccessible.
Spontaneous and cytokine‐induced thrombocytopenia in myelodysplastic syndromes: serum thrombopoietin levels and bone marrow morphology
Authors: Hellström‐Lindberg Eva , Kanter‐Lewensohn Lena , Nichol Janet , Öst Åke ,   for the Scandinavian MDS Group, Sweden and Norway
Published: March 2003

Thrombocytopenia is a substantial clinical problem for patients with myelodysplastic syndromes (MDS). Cytokine treatment for granulocytopenia and anaemia may further reduce the platelet counts. We studied serum thrombopoietin levels (S‐TPO) in 52 patients with MDS and 96 healthy controls and related the results to clinical and morphological variables. S‐TPO was also assessed after treatment with granulocyte‐CSF (G‐CSF) and erythropoietin (EPO) in 30 of these patients. S‐TPO in MDS was not a normally distributed variable; mean value was 394 pg/ml, SD ±831 and median value 123 (12–5000 pg/ml). The controls showed lower S‐TPO levels than the patients (median 78 pg/ml, P = 0.003) whereas no differences between the MDS subgroups were observed (P = 0.86). Patients with ringed sideroblastic anaemia (RARS) showed the highest platelet counts and higher S‐TPO levels than the controls (P = 0.005). No association between platelet counts and S‐TPO levels was found in the patients (P = 0.67). TPO levels were generally low in patients with refractory anaemia with an excess of blasts (RAEB), but very high levels were found in five patients. Patients with a high transfusion need had higher S‐TPO levels, whereas bone marrow blast counts, cellularity or megakaryocytes showed no correlation with S‐TPO. Patients with 5q− showed lower TPO levels than the other patients, indicating that thrombopoietin is not a mediator of thrombocytosis in these cases. Treatment with G‐CSF + EPO significantly reduced the platelet counts (P = 0.0002), but this change was not related to significant changes in S‐TPO levels or morphology. Patients with RARS and thrombocytosis who normalized their platelet counts showed a concomitant reduction in S‐TPO. This may suggest that the increased platelet counts observed in RARS may be caused by increased S‐TPO levels. In conclusion, our study shows that platelet, megakaryocyte and thrombopoietin regulation is rather complex in myelodysplastic syndromes and that spontaneous or induced thrombocytopenia are not usually mirrored by increased S‐TPO levels.
A randomized controlled trial of itraconazole versus fluconazole for the prevention of fungal infections in patients with haematological malignancies
Authors: Morgenstern Godfrey R. , Prentice Archibald G. , Grant Prentice H. , Ropner Janet E. , Schey Stephen A. , Warnock David W. ,   on behalf of the U.K. Multicentre Antifungal Prophylaxis Study Group
Published: March 2003

Fluconazole is widely used as antifungal prophylaxis but it is ineffective against Aspergillus. Itraconazole has a broader spectrum of activity but the capsules give erratic bioavailability in neutropenic patients. We compared itraconazole oral solution (which has an improved pharmacokinetic profile) with fluconazole for antifungal prophylaxis.

Adults with haematological malignancies receiving chemotherapy or bone marrow transplants were randomly allocated 5 mg/kg/d itraconazole (itra) solution (288 episodes) or 100 mg fluconazole suspension (flu) (293 episodes) from before the onset of neutropenia until neutrophil recovery or suspected fungal infection. Outcomes were assessed by independent reviewers unaware of the prophylaxis allocation.

More proven systemic fungal infections occurred in flu (Aspergillus four, Candida tropicalis one, C. krusei one) than itra (C. albicans one) and more of these were fatal (four versus nil). This difference reached statistical significance when first study episodes were considered separately (six flu versus nil itra, P = 0.03). Significantly more deaths of presumed fungal origin occurred in flu than itra (seven versus nil, P = 0.024). There were significantly more cases of proven aspergillosis in flu than itra (six versus nil, P = 0.038, 5/6 cases were fatal) if those occurring outside the study period are included. Significantly more patients receiving flu required amphotericin B (58 v 39, P = 0.043) but this may have been affected by the fact that the study was not blinded. There were 11 proven mucosal candidal infections in flu and four in itra.

Itraconazole solution and fluconazole provide effective prophylaxis against Candida but itraconazole affords greater protection against fatal aspergillosis.
Heparin lowers plasma levels of activated factor VII
Authors: Pernerstorfer Thomas , Jilma Bernd , Eichler Hans‐Georg , Aull Susanne , Handler Sylvia , Speiser Wolfgang
Published: March 2003

Based on heparin's antithrombin and anti‐FXa activity and its in vitro inhibition of activated factor VII (FVIIa) activity, we hypothesized that unfractionated heparin (UFH) may decrease plasma levels of FVIIa in humans. Therefore, 10 healthy young male volunteers received an intravenous UFH infusion over 24 h. Heparin decreased FVIIa levels by 30% (95% CI 14–47%) at 12 h, which was sustained until 24 h. In contrast, neither the substrate pool (i.e. total factor VII) as measured by FVII antigen nor FVII activity were affected by UFH. These results may improve our understanding of the regulation of FVIIa levels and heparin's mode of action.
Human osteoclasts derive from CD14‐positive monocytes
Authors: Massey  , Flanagan
Published: March 2003

Osteoclasts have been defined as calcitonin (CT) and vitronectin (VN) receptor (R) positive, and CD14‐, CD11b‐ and CD11c‐negative cells which resorb bone. The aim of this study was to identify the phenotype of the osteoclast precursor. Osteoclasts were generated by co‐culturing peripheral blood mononuclear cells (PBMNCS) with the rat osteoblastic UMR 106 cell line. On days 2–4 at least 80% of CTR‐positive cells co‐expressed CD14, CD11b and CD11c (monocyte markers), but by day 14 < 3.3% expressed these markers. Selection of CD14‐positive monocytes from PBMNCS enhanced osteoclastic bone resorption 2–4‐fold compared to unfractionated PBMNCS.

This study demonstrates that osteoclasts derive largely from CD14‐positive monocytes.
Oversecretion of IL‐18 in haemophagocytic lymphohistiocytosis: a novel marker of disease activity
Authors: Takada Hidetoshi , Ohga Shouichi , Mizuno Yumi , Suminoe Aiko , Matsuzaki Akinobu , Ihara Kenji , Kinukawa Naoko , Ohshima Koichi , Kohno Keizo , Kurimoto Masashi , Hara Toshiro
Published: March 2003

We investigated the significance of interleukin (IL)‐18 levels in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). IL‐18 levels were significantly elevated in all nine patients with active HLH compared with those of healthy controls. Serial determination of IL‐18 levels in three cases, showed a gradual decrease compared with those of IL‐12, interferon (IFN)‐γ or soluble Fas ligand (sFasL) in the course of clinical improvement, and seemed to be elevated until complete disappearance of disease activity. IL‐18 and IFN‐γ (CC 0.711, P = 0.018), and IFN‐γ and sFasL (CC 0.849, P = 0.0049) levels were significantly correlated. On the other hand, correlation between IL‐12 and IFN‐γ, IL‐18 and sFasL, or IL‐18 and IL‐12 was not observed. IL‐18, IFN‐γ and sFasL levels significantly correlated with disease activity such as fever and alanine transaminase (ALT) levels. IL‐18 mRNA expression was enhanced in spleen, but not in peripheral blood mononuclear cells (MNC), bone marrow MNC, liver from patients of active HLH, or the tumour from a patient with lymphoma‐associated haemophagocytic syndrome (LAHS). These results suggest that IL‐18 may play important roles in the pathogenesis of HLH, particularly through induction of Th1 cells. IL‐18 measurement may be useful for the diagnosis and for the detection of smouldering disease activity.