bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient mobilization of haematopoietic progenitors after a single injection of pegylated recombinant human granulocyte colony‐stimulating factor in mouse strains with distinct marrow‐cell pool sizes
Authors: De Haan Gerald , Ausema Albertina , Wilkens Marga , Molineux Graham , Dontje Bert
Published: March 2003

We have compared the efficacy of a single injection of SD/01, a newly engineered, pegylated form of recombinant human granulocyte colony stimulating factor (rhG‐CSF), with a single injection of glycosylated rhG‐CSF (Filgrastim). SD/01 was administered to regular and recombinant inbred strains of mice (AKR, C57L/J, DBA/2, C57BL/6, AKXL) known to have widely distinct marrow‐cell pool sizes and proliferation kinetics. A single injection of G‐CSF was unable to mobilize granulocyte–macrophage colony‐forming units (CFU‐GM). In sharp contrast, a single dose of SD/01 resulted in massive mobilization of progenitors and stem cells. Although all mice strains showed qualitatively similar mobilization responses, large interstrain differences remained. C57L and C57BL/6 mice mobilized relatively poorly, whereas AKR and DBA/2 mice showed threefold to tenfold superior responses. In order to explain these different phenotypes, we studied the effects of SD/01 in nine AKXL recombinant inbred strains, derived from well‐responding AKR and poorly responding C57L parental strains. The best predictor for SD/01 responsiveness in these strains was marrow cellularity prior to mobilization. Comparison of the AKXL strain distribution pattern for marrow cellularity with loci previously mapped in these strains showed complete concordance with Aat, a serine protease inhibitor mapping to chromosome 12.
Bernard–Soulier syndrome in a patient doubly heterozygous for two frameshift mutations in the glycoprotein Ibα gene
Authors: Afshar‐Kharghan Vahid , Craig Fiona E. , López José A.
Published: March 2003

We report here the genetic basis of Bernard–Soulier syndrome in a compound heterozygote for two mutant glycoprotein (GP) Ibα alleles. One allele contained a novel four base‐pair deletion (TGAG) that eliminated the last base of the codon for Ser39 (AGT) and the entire codon for Glu40 (GAG), causing a reading frame shift that yielded a stretch of 51 amino acids before a premature stop codon. The other allele also contained a frame‐shift mutation, caused by deletion of the last two bases of the codon for Tyr492 (TAT). This allele produced a truncated glycoprotein Ibα that, although not expressed on the surface of the patient's platelets, was detectable in the plasma. The second allele has been identified previously by our group and other investigators as the cause of Bernard–Soulier syndrome in patients of northern European ancestry. This allele carried a haplotype identical to those of the previously reported cases, with the following polymorphic markers: two tandem repeats in the VNTR region, C at nucleotide −5 from the ATG start codon and a substitution of G for A in the third base for codon Arg342. These findings suggest that this particular Bernard–Soulier mutation occurred once on the background of a rare haplotype and has spread throughout the northern European population.
Primary myelosarcomas are associated with a high rate of relapse: report on 34 children from the acute myeloid leukaemia–Berlin–Frankfurt–Münster studies
Authors: Reinhardt D. , Pekrun A. , Lakomek M. , Zimmermann M. , Ritter J. , Creutzig U.
Published: March 2003

Primary myelosarcomas are rare manifestations of acute myeloid leukaemia (AML) that precede bone marrow involvement. Out of 744 children observed during the AML–Berlin–Frankfurt–Münster (BFM) studies 87 and 93, 34 children presented with extramedullar myelosarcomas and no blasts (n = 21; 2·8%), or a low blast count (n = 13; 1·7%) in the bone marrow. Owing to the initially mild and variable symptoms, in some children (n = 12) diagnostic procedures were delayed and treatment intensity was reduced. At 0·65 ± 0·13, the cumulative incidence of relapse was significantly higher than in other AML patients (0·28 ± 0·02). The 5‐year event‐free survival was 0·19 ± 0·08 (compared with 0·48 ± 0·02 in AML‐BFM studies 87/93; P(log rank) < 0·03). Overall, 18 out of 34 patients died from disease (estimated 5 year survival 0·44 ± 0·09 compared with 0·55 ± 0·02 in the AML‐BFM‐studies 87/93; P(log rank) = 0·35, n.s.). An early diagnostic workup is needed in children with unusual skin lesions or tumours, considering myelosarcoma as a primary manifestation of AML. Intensive AML‐specific chemotherapy is recommended soon after diagnosis.
Involvement of natural killer cells in patients with myelodysplastic syndrome carrying monosomy 7 revealed by the application of fluorescence in situ hybridization to cells collected by means of fluorescence‐activated cell sorting
Authors: Miura Ikuo , Kobayashi Yoshimi , Takahashi Naoto , Saitoh Kohki , Miura Akira B.
Published: March 2003

Monosomy 7 is the most frequent chromosome abnormality among patients with secondary myelodysplastic syndrome (MDS). We used fluorescence in situ hybridization (FISH) and fluorescence‐activated cell sorting (FACS) in order to clarify the lineage involvement. Four patients, three with de novo MDS and one with secondary MDS, were enrolled in this study. Monosomy 7 was observed in pluripotent stem cells (CD34+Thy‐1+), and in B (CD34+CD19+) and T/natural killer (NK) progenitors (CD34+CD7+). The number of abnormal cells of B (CD19+) and T (CD3+) cells was below the cut‐off value, but approximately 60% of the NK cells (CD3‐CD56+) contained monosomy 7 in three of the patients.
Detection of different Ikaros isoforms in human leukaemias using real‐time quantitative polymerase chain reaction
Authors: Olivero S. , Maroc C. , Beillard E. , Gabert J. , Nietfeld W. , Chabannon C. , Tonnelle C.
Published: March 2003

The Ikaros gene is an essential regulator in development and haematopoiesis. Dysregulated Ikaros gene expression participates in leukaemic processes, as evidenced in animal models, and by analyses of blast‐cell populations from leukaemic patients. We used real‐time quantitative polymerase chain reaction (PCR) to evaluate the relative abundance of several Ikaros transcript isoforms in a variety of leukaemic‐cell samples. Total RNA was isolated from bone‐marrow or blood‐cell samples collected at diagnosis in children or adult patients, 18 of whom had acute myeloblastic leukaemia (AML), 61 of whom had acute lymphoblastic leukaemia (ALL) and 11 of whom had chronic myeloid leukaemia (CML). The ratio (Ik1 + Ik2)/(Ik1 + Ik2 + Ik4 + Ik7 + Ik8) ranged from 13·5% to 85% and was lower (P < 0·05) in samples from patients with m‐bcr‐abl ALL. An alternative splicing resulting in the deletion of 30 nucleotides at the end of exon 6 was observed in leukaemic samples, and in normal thymus and bone marrow.

Our results are consistent with previous reports and suggest that the pattern of expression of the different human Ikaros isoforms are not homogeneous among different subsets of leukaemias.
Activation of an ataxia telangiectasia mutation‐dependent intra‐S‐phase checkpoint by anti‐tumour drugs in HL‐60 and human lymphoblastoid cells
Authors: Sugimoto Koichi , Tamayose Kenji , Takagi Masatoshi , Yamada Konagi , Sasaki Makoto , Mizutani Shuki , Oshimi Kazuo
Published: March 2003

In yeast cells, the intra‐S‐phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti‐tumour drugs in HL‐60 and Epstein–Barr virus (EBV)‐transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two‐dimensional flow cytometric analysis, we clearly visualized the cell‐cycle progression of the BrdU‐positive population (cells originally belonging to the S phase) and detected even subtle changes in S‐phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU‐positive HL‐60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti‐metabolite, cytosine β‐ d‐arabinofuranose (Ara‐C; 50 nmol/l), kept them in early‐to‐late S phase after 8 h of incubation. Because 10 μmol/l of caffeine added 2 h later attenuated the S‐phase retardation by these drugs in HL‐60 cells, slowing of the S‐phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)‐derived lymphoblastoid cell lines were impaired in the doxorubicin‐induced S‐phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S‐phase progression elicited by anti‐tumour drugs in HL‐60 and lymphoblastoid cells may therefore correspond to the intra‐S‐phase checkpoint of the yeast cells.
Hereditary non‐spherocytic haemolytic anaemia due to red blood cell glutathione synthetase deficiency in four unrelated patients from Spain: clinical and molecular studies
Authors: Corrons Joan‐Lluis Vives , Alvarez Rodolfo , Pujades Assumpta , Zarza Rocío , Oliva Elvira , Lasheras German , Callis Manuel , Ribes Antonia , Gelbart Terri , Beutler Ernest
Published: March 2003

In four unrelated patients with chronic haemolysis and markedly reduced red blood cell (RBC) glutathione (49·5%, 12·6%, 11·5% and 15% of the normal concentration respectively), a severe glutathione synthetase (GSH‐S, EC 6.3.2.3) deficiency was found. One case exhibited a neonatal haemolytic anaemia associated with oxoprolinuria, but without neurological manifestations. The family study revealed GSH‐S activity in both parents to be around half the normal level, a finding consistent with the presumed autosomal recessive mode of inheritance of this enzymopathy. Two cases exhibited a well‐compensated haemolytic syndrome without anaemia or splenomegaly at steady state. One of these cases was diagnosed after an episode of acute haemolytic anaemia after fava bean ingestion. The remaining patient suffered from moderate to severe chronic non‐spherocytic haemolytic anaemia and splenomegaly, and required occasional blood transfusion for a haemolytic crisis associated with drug ingestion. In this patient, the anaemia was corrected by splenectomy. In addition to GSH‐S, a panel of 16 other RBC enzyme activities was also studied in all the patients. Hexokinase, aldolase, glucose‐6‐phosphate dehydrogenase and pyruvate kinase activities all increased; these increases were to be expected, given the rise in the number of circulating reticulocytes. In two patients, the incubation of RBCs with hydrogen peroxide revealed an enhanced production of malonyldialdehyde. DNA analysis showed a homozygous state for 656 A→G mutation in patients 2 and 3. The GSH‐S gene of patient 1, studied elsewhere, revealed an 808 T→C. The GSH‐S gene of patient 4 was not available for study. The present study demonstrates that GSH‐S deficiency is also present in Spain and further supports the molecular and clinical heterogeneity of this enzymopathy
Prognostic impact of bone marrow erythropoietic precursor cells and myelofibrosis at diagnosis of Ph1+chronic myelogenous leukaemia – a multicentre study on 495 patients
Authors: Kvasnicka Hans Michael , Thiele Juergen , Schmitt‐Graeff Annette , Diehl Volker , Zankovich Rudolf , Niederle Norbert , Leder Lutz‐Dietrich , Schaefer Hans Eckart
Published: March 2003

A multicentre clinicopathological study was performed on 495 patients with chronic‐phase Ph1+chronic myelogenous leukaemia (CML) to determine bone marrow characteristics that exert a significant impact on survival under standard treatment regimens. Immunohistochemical and morphometric techniques were applied to identify nucleated erythroid precursor cells in the bone marrow and to quantify argyrophilic fibre density. Application of the Sokal index and another recently proposed CML score failed to distinguish three clearly defined risk groups. A borderline increase in fibre content (i.e. doubling of the normal density) and a relevant reduction of medullary erythropoiesis proved to be important predictors for survival, even in low‐risk classified patients, according to both clinical scores. With regard to optimal treatment strategies, patients with manifest myelofibrosis showed no significant difference in survival rates under interferon or hydroxyurea treatment. Multivariate analysis confirmed the prognostic value of histological features. A risk model based on three variables (fibre density, erythropoietic precursors and spleen size) was constructed that enabled a distinct discrimination of risk profiles. In conclusion, the presented data provide compelling evidence that bone marrow features at diagnosis exert a significant impact on prognosis in CML. In this context, the generally clinical‐based multivariate risk classification can be improved by consideration of morphological variables that are acting independently of treatment modalities.
bcl‐2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection
Authors: Zuckerman Eli , Zuckerman Tsila , Sahar Dvora , Streichman Sara , Attias Dina , Sabo Edmond , Yeshurun Daniel , Rowe Jacob M.
Published: March 2003

An association between chronic hepatitis C virus (HCV) infection and clonal proliferation of B cells, including B cell lymphoma, has recently been demonstrated. However, the mechanism of malignant transformation is still unknown. It has been shown that B cells from patients with type II mixed cryoglobulinaemia (MC), strongly express the antiapoptotic bcl‐2 oncogene product. Therefore, we investigated a possible mechanism of lymphomagenesis, the occurrence of bcl‐2 and immunoglobulin gene rearrangement (IgH) in HCV‐infected patients. Three groups of patients were studied: (1) 44 patients with HCV and MC (anti‐HCV and HCV RNA positive); (2) 59 patients with chronic HCV infection without MC; (3) 50 patients with chronic liver disease (CLD) not related to HCV infection. The t(14;18) translocation (MBR bcl‐2–JH) and IgH rearrangement (FR3/JH) were detected by polymerase chain reaction (PCR) in peripheral mononuclear cells. bcl‐2 translocation was detected in 17/44 (39%), 7/59 (12%) and in none of the patients of groups 1, 2 and 3 respectively (P < 0·01). Monoclonal IgH rearrangement was detected in 15/44 (34%), 5/59 (8·5%) and 2/50 (4%) patients of groups 1, 2 and 3 respectively (P < 0·05). HCV‐infected patients had a higher prevalence of monoclonal IgH rearrangement and bcl‐2 translocation than patients with CLD of other aetiologies. These data suggest that HCV may play a role in the multistep mechanism of lymphomagenesis by inducing clonal proliferation of B cells and inhibition of apoptosis.
Treatment of steroid refractory acute and chronic graft‐versus‐host disease with daclizumab
Authors: Willenbacher Wolfgang , Basara Nadesta , Blau Igor W. , Fauser Axel A. , Kiehl Michael G.
Published: March 2003

Competitive inhibition of interleukin 2‐dependent lymphocytes by daclizumab demonstrates some beneficial effects in the treatment of graft‐versus‐host disease (GVHD). Sixteen patients with steroid refractory GVHD received daclizumab (1 mg/kg BW) on d 1, 2 (−5), 7, 14 and 21. Twelve patients suffered from grade III–IV acute GVHD and four patients from extensive chronic GVHD. Responses were observed in nine patients (six acute, three chronic GVHD). Fourteen out of 16 patients acquired infections during daclizumab treatment and three deaths were infection related. Daclizumab demonstrates limited activity and is associated with an increased incidence of infectious complications.
Massive immune haemolysis after allogeneic peripheral blood stem cell transplantation with minor ABO incompatibility
Authors: Bolan Charles D. , Childs Richard W. , Procter Jo Lynn , Barrett A. John , Leitman Susan F.
Published: March 2003

Immune haemolysis as a result of minor ABO incompatibility is an underappreciated complication of haematopoietic transplantation. The increased lymphoid content of peripheral blood stem cell (PBSC) transplants may increase the incidence and severity of this event. We observed massive immune haemolysis in 3 out of 10 consecutive patients undergoing HLA‐identical, related‐donor PBSC transplants with minor ABO incompatibility. Non‐ablative conditioning had been given in 9 of these 10 cases, including two with haemolysis. Cyclosporin alone was used as prophylaxis against graft‐vs.‐host disease (GVHD). Catastrophic haemolysis of 78% of the circulating red cell mass led to anoxic death in the first case seen, but severe consequences were avoided by early, vigorous donor‐compatible red cell transfusions in the subsequent two cases. Haemolysis began 7–11 d after PBSC infusion and all patients with haemolysis had a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient antigen. However, neither the intensity of the DAT, the donor isohaemagglutinin titre, nor other factors could reliably be used to predict the occurrence of haemolysis. Our data indicate that haemolysis may be frequent and severe after transplantation of minor ABO‐incompatible PBSCs when utilizing cyclosporin alone to prevent GVHD. Meticulous clinical monitoring and early, vigorous donor‐compatible red cell transfusions should be practiced in all instances.
Safety and complications of interventional radiology for hepatocellular carcinoma in patients with haemophilia and cirrhosis
Authors: Toyoda Hidenori , Fukuda Yoshihide , Yokozaki Shoichi , Hayashi Kazuhiko , Saito Hidehiko , Takamatsu Junki
Published: March 2003

Many patients with haemophilia have been chronically infected with hepatitis virus owing to multiple transfusions and the prevalence of hepatocellular carcinoma will probably increase in this population. We evaluated the safety and complications of radiological intervention for hepatocellular carcinoma in eight patients with haemophilia and cirrhosis. Radiological interventions can be performed safely in all patients with haemophilia. Unexpectedly, the most common complication was bleeding from the gastrointestinal tract. Attention should be paid to this potential problem in order to take appropriate steps to minimize its occurrence.
Polymorphisms in the non‐coding region of the human mitochondrial genome in unrelated plateletapheresis donors
Authors: Garritsen H. S. P. , Hoerning A. , Hellenkamp F. , Cassens U. , Mittmann K. , Sibrowski W.
Published: March 2003

Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplantation or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non‐coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2–17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty‐six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor–recipient cells after transfusion or transplantation.
Lithuanian haemophilia A and B registry comprising phenotypic and genotypic data
Authors: Ivaskevicius Vytautas , Jurgutis Romualdas , Rost Simone , Müller Alexandra , Schmitt Christoph , Wulff Karin , Herrmann Falko H. , Müller Clemens R. , Schwaab Rainer , Oldenburg Johannes
Published: March 2003

Haemophilia represents the most common hereditary severe bleeding disorder in humans. About 100 families with this condition live in Lithuania, one of the Baltic states with a population of 3·7 million. Haemophilia care and genetic counselling are still rendered difficult owing to limited availability of clotting factor concentrate and molecular genetic diagnosis. In the present study, a haemophilia registry, comprising phenotypic and genotypic data of the majority of Lithuanian haemophilia A and B patients, was established. The phenotype includes the degree of severity, factor VIII:C, factor VIII:Ag, factor IX:C, von Willebrand factor and antigen (VWF:RiCoF, vWF:Ag) and inhibitor status. Genotyping of the factor VIII and IX genes was performed using mutation screening methods and direct sequencing. In 61 out of 63 patients with haemophilia A (96·8%) and all eight patients with haemophilia B (100%), the causative mutations could be detected. Nineteen of the factor VIII gene defects and two of the factor IX gene mutations are reported for the first time. Identified mutations allowed direct carrier diagnosis in 83 female relatives revealing 44 carriers, 38 non‐carriers and one somatic mosaicism. The information provided by this registry will be helpful for monitoring the treatment of Lithuanian haemophilia patients and also for reliable genetic counselling of the affected families in the future.
Relationship between graft‐versus‐host disease and graft‐versus‐leukaemia in partial T cell‐depleted bone marrow transplantation
Authors: Van Der Straaten Hanneke M. , Fijnheer Rob , Dekker Adriaan W. , Nieuwenhuis H. Karel , Verdonck Leo F.
Published: March 2003

The success of allogeneic bone marrow transplantation (BMT) is limited by the major complications, graft‐versus‐host disease (GVHD) and relapse. The very beneficial effect of maximal T‐cell depletion of the graft for prevention of GVHD has been counterbalanced by an increase in graft failure and relapse of disease. Therefore, we started an approach of partial T‐cell depletion of the graft. GVHD and graft‐versus‐leukaemia (GVL) are strongly correlated after non‐T cell‐depleted BMT. Here, we report whether the correlation between GVHD and GVL also exists in partial T cell‐depleted BMT from sibling donors. We retrospectively studied 117 adult patients with early haematological malignancies. Our method of partial T‐cell depletion gave a relapse rate in patients with acute leukaemias similar to that observed in non‐T cell‐depleted BMT. However, patients with chronic myeloid leukaemia had a relapse rate that was similar to that observed in maximal T cell‐depleted BMT. We found a significant correlation between the presence of chronic GVHD and an improved disease‐free survival. Nevertheless, overall survival was lower in patients with chronic GVHD. There was no correlation between the occurrence of acute GVHD and disease‐free or overall survival.
An in situ study of CD34+ cells in human fetal bone marrow
Authors: Allen Janet E. , Henshaw Denis L.
Published: March 2003

The purpose of this study was to characterize the spatial distribution, number and size of CD34+ cells in fetal bone marrow. Thin sections of normal fetal bone marrow from lumbar vertebrae were stained using CD34 antibody QBend/10. Sections were used under light microscopy with various eyepiece graticules to make measurements of CD34+ cells in situ. Results showed that at mid‐ and late gestation, approximately 2% and 0·5% of fetal bone marrow cells were CD34+ respectively. The mean distance of CD34+ cells from the nearest trabecular bone surface was 61 ± 4 and 46 ± 4 μm, respectively, for mid‐ and late gestation. The mean distance to the nearest neighbour was 46 ± 5 and 105 ± 15 μm, and the mean distance to the nearest blood vessel was 13 ± 1 and 17 ± 2 μm respectively. The concentration of CD34+ cells in the peripheral region was 6·5 times greater than that at the centre of the sections. Overall, the percentage number of CD34+ cells decreased with gestational age. The cellular and nuclear diameters of CD34+ cells remained unchanged throughout mid‐ and late gestation at 5·4 ± 0·1 and 3·8 ± 0·1 μm respectively. This information will be used to calculate the natural background alpha‐radiation dose to haemopoietic stem cells.
HFE mutations, iron deficiency and overload in 10 500 blood donors
Authors: Jackson H. A. , Carter K. , Darke C. , Guttridge M. G. , Ravine D. , Hutton R. D. , Napier J. A. , Worwood M.
Published: March 2003

People with genetic haemochromatosis (GH) accumulate iron from excessive dietary absorption. In populations of northern European origin, over 90% of patients are homozygous for the C282Y mutation of the HFE gene. While about 1 in 200 people in the general population have this genotype the proportion who develop clinical haemochromatosis is not known. The influence of HFE genotype on iron status was investigated in 10 556 blood donors. The allele frequencies of the C282Y and H63D mutations were 8·23% and 15·3% respectively. Heterozygosity for C282Y occurred in 1 in 7·9 donors, for H63D in 1 in 4·2 donors, and 1 in 42 were compound heterozygotes. Homozygosity for H63D occurred in 1 in 42 donors and 1 in 147 (72) were homozygous for C282Y. Mean values increased for transferrin saturation (TS) and serum ferritin (sFn), and decreased for unsaturated iron binding capacity (UIBC) in the order: donors lacking the mutations, H63D heterozygotes, C282Y heterozygotes, H63D homozygotes, compound heterozygotes and C282Y homozygotes, but serum ferritin (sFn) concentrations were no higher in H63D heterozygotes and C282Y heterozygous women than in donors lacking mutations. The percentage of donors failing the screening test for anaemia or of those with sFn < 15 µg/l did not differ among the genotype groups. C282Y and H63D heterozygotes and donors homozygous for H63D were at no greater risk of iron accumulation than donors lacking mutations, of whom 1 in 1200 had both a raised TS and sFn. The risk was higher for compound heterozygotes (1 in 80, P = 0·003) and for C282Y homozygotes (1 in 5, P < 0·0001). There was no correlation between sFn and either age or donation frequency in C282Y homozygotes. None of the 63 C282Y homozygous donors interviewed showed physical signs of overload or were aware of relatives with haemochromatosis. The Welsh Blood Service collects blood from about 140 000 people each year including an estimated 950 who are homozygous for HFE C282Y. They are probably healthy and unaware of any family history of iron overload.
Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage‐induced apoptosis in B‐chronic lymphocytic leukaemia cells
Authors: Wickremasinghe R. Gitendra , Ganeshaguru Kanagasabai , Jones Dylan T. , Lindsay Caroline , Spanswick Victoria J. , Hartley John A. , Wadhwa Meenu , Thorpe Robin , Hoffbrand A. Victor , Prentice H. Grant , Mehta Atul B.
Published: March 2003

We have studied the actions of autologous plasma on both basal and DNA damage‐induced apoptosis in B‐chronic lymphocytic leukaemia (B‐CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis‐specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and γ‐radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons α or γ or stromal cell‐derived factor 1, each of which have been shown to protect B‐CLL cells from apoptosis in vitro. Plasma addition to B‐CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti‐apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3′‐kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B‐CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective‐signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B‐CLL treatment.
Results of autologous stem cell transplant in multiple myeloma patients with renal failure
Authors: Badros Ashraf , Barlogie Bart , Siegel Eric , Roberts Jennifer , Langmaid Candi , Zangari Maurizio , Desikan Raman , Shaver Mary Jo , Fassas Athanasios , McConnell Scott , Muwalla Firas , Barri Yousri , Anaissie Elias , Munshi Nikhil , Tricot Guido
Published: March 2003

Data are presented on 81 multiple myeloma (MM) patients with renal failure (creatinine > 176·8 μmol/l) at the time of autologous stem cell transplantation (auto‐SCT), including 38 patients on dialysis. The median age was 53 years (range: 29–69) and 26% had received more than 12 months of prior chemotherapy. CD34+ cells were mobilized with granulocyte colony‐stimulating factor (G‐CSF) alone (n = 51) or chemotherapy plus G‐CSF (n = 27), yielding medians of 10 and 16 × 106 CD34+ cells/kg respectively (P = 0·003). Sixty patients (27 on dialysis) received melphalan 200 mg/m2 (MEL‐200). Because of excessive toxicity, the subsequent 21 patients (11 on dialysis) received MEL 140 mg/m2 (MEL‐140). Thirty‐one patients (38%) completed tandem auto‐SCT, including 11 on dialysis. Treatment‐related mortality (TRM) was 6% and 13% after the first and second auto‐SCT. Median times to absolute neutrophil count (ANC) > 0·5 × 109/l and to platelets > 50 × 109/l were 11 and 41 d respectively. Non‐haematological toxicities included mucositis, pneumonitis, dysrhythmias and encephalopathy. At a median follow up of 31 months, 30 patients have died. Complete remission (CR) was achieved in 21 patients (26%) after first SCT and 31 patients (38%) after tandem SCT. Two patients discontinued dialysis after SCT. Median durations of complete remission (CR) and overall survival (OS) have not been reached; probabilities of event‐free survival (EFS) and OS at 3 years were 48% and 55% respectively. Dialysis dependence and MEL dose did not affect EFS or OS. Sensitive disease prior to SCT, normal albumin level and younger age were independent prognostic factors for better OS. In conclusion, renal failure had no impact on the quality of stem cell collections and did not affect engraftment. MEL‐140 had an acceptable toxicity and appeared equally effective as MEL‐200. In the setting of renal failure, the role of auto‐SCT early in the disease course and benefits of tandem SCT require further evaluation.
A comparison of the efficacy and rate of response to oral and intravenous Vitamin K in reversal of over‐anticoagulation with warfarin
Authors: Watson Henry G. , Baglin Trevor , Laidlaw Stuart L. , Makris Michael , Preston F. Eric
Published: March 2003

The role of oral Vitamin K administration in the reversal of anticoagulation is not yet clear because of a paucity of data on the early effects of treatment, apparent differences in efficacy between preparations and a lack of data comparing oral with intravenous administration. We have compared the effects on the International Normalized Ratio (INR) and activities of the Vitamin K‐dependent clotting factors II, VII, IX and X at 4 h and 24 h after administration of three oral Vitamin K preparations and of intravenous Vitamin K in 64 anticoagulated patients who required non‐urgent partial correction of anticoagulation. Our data confirm that correction of anticoagulation is more rapid after intravenous administration of Vitamin K than after oral administration of similar or larger doses. At 24 h, satisfactory correction of INR can be achieved using low‐dose Vitamin K given by either the intravenous or oral route. Our data, and that from previous studies, suggest that there may be differences in efficacy between orally administered products. Administration of Vitamin K by either route was accompanied by changes in the activities of the Vitamin K‐dependent clotting factors that reflected their respective biological half‐lives. In the 24 h after treatment, the relationship between the INR and the individual Vitamin K‐dependent clotting factors was similar to that described previously in stable anticoagulated patients. We conclude that the reversal of anticoagulation with warfarin is achieved more rapidly by intravenous administration of Vitamin K. Satisfactory, but slower, reversal of anticoagulation can be effected using oral Vitamin K, but there may be differences in efficacy between the products tested in our study.
Primary prophylaxis with lamivudine of hepatitis B virus reactivation in chronic HbsAg carriers with lymphoid malignancies treated with chemotherapy
Authors: Rossi Giuseppe , Pelizzari Annamaria , Motta Maddalena , Puoti Massimo
Published: March 2003

Hepatitis B virus (HBV) reactivation of various degrees of severity, including fulminant hepatitis, may develop in 20–50% of hepatitis B virus surface antigen (HbsAg)‐positive patients undergoing immunosuppressive or cytostatic treatment. Lamivudine is a nucleoside analogue that can directly suppress HBV replication. We have performed a pilot study to test the efficacy and tolerability of lamivudine as a primary prophylaxis of HBV reactivation in 20 consecutive patients treated for haematological malignancies, mainly of lymphoid origin. Lamivudine, 100 mg/d, was given orally from the start until 1 month after the end of chemotherapy, which included corticosteroids and/or purine analogues in 85% of cases. It was well tolerated and did not cause any unexpected reduction of cytostatic drugs dosages. The chemotherapy programme was completed in all patients without modifications. A transient threefold increase in serum amylase was observed in one case. HBV‐DNA levels decreased in six out of six patients (P = 0·039) and ALT levels in five out of six patients (P = 0·057) whose serum levels were abnormal at the onset of therapy. Two patients developed transient hepatitis. HBV reactivation was documented in only one of these patients who had stopped lamivudine 1 month before. No signs of HBV reactivation were detected both during and after treatment in 18 patients with a median follow‐up of 6 months (range 3–12). Thus, primary prophylaxis with lamivudine may be a well tolerated and effective method to reduce the frequency of chemotherapy‐induced HBV reactivation in chronic HBsAg carriers.
The methylenetetrahydrofolate reductase C677T gene polymorphism decreases the risk of childhood acute lymphocytic leukaemia
Authors: Franco Rendrik F. , Simões Belinda P. , Tone Luiz G. , Gabellini Sérgio M. , Zago Marco A. , Falcão Roberto P.
Published: March 2003

We have determined the prevalence of methylenetetrahydrofolate reductase (MTHFR) mutations C677T and A1298C in 71 children ( 15 years) with acute lymphoblastic leukaemia (ALL) and in 71 control subjects. Odds ratio (OR) for ALL linked to MTHFR C677T was 0·4 (95% CI 0·2–0·8); for heterozygotes it was 0·5 (95% CI 0·2–0·9) and for homozygotes it was 0·3 (95%CI 0·09–0·8). MTHFR A1298C yielded an overall OR for ALL of 1·3 (95% CI: 0·7–2·6); for heterozygotes it was 1·3 (95% CI: 0·7–7·6) and for homozygotes it was 2·8 (95% CI 0·5–15·6). In conclusion, MTHFR C677T was linked to a significant 2·4‐fold decreased risk of developing childhood ALL, whereas MTHFR A1298C did not significantly affect the risk of ALL in our population.
Abnormal growth factor modulation of β1‐integrin‐mediated adhesion in chronic myelogenous leukaemia haematopoietic progenitors
Authors: Bhatia Ravi , Munthe Heidi A. , Forman Stephen J.
Published: March 2003

Abnormal progenitor circulation and extramedullary haematopoiesis are characteristic features of chronic myelogenous leukaemia (CML). Growth factor (GF) and β1‐integrin interactions play an important role in regulation of progenitor trafficking to and from the marrow space. CML progenitors demonstrate abnormal β1‐integrin‐mediated adhesion to fibronectin (FN). In the present study we investigated whether GF modulation of β1‐integrin‐mediated adhesion and migration was altered in CML progenitors. Culture with low concentrations of GF enhanced normal progenitor adhesion to FN compared with no GF, but failed to enhance CML progenitor adhesion to FN. Similarly, high concentrations of selected GF rapidly enhanced β1‐integrin‐mediated adhesion of normal progenitors to FN through a phosphotidylinositol‐3 (PI‐3) kinase‐dependent mechanism, but failed to increase CML progenitor adhesion. Exposure to a BCR–ABL tyrosine kinase inhibitor restored GF modulation of CML progenitor adhesion. CML colony‐forming cells (CFC) demonstrated increased migration across FN‐coated transwells compared with normal CFC in the absence of GF. The addition of stem cell factor resulted in enhanced migration of CML and normal CFC on FN. In conclusion, GF stimulation failed to enhance integrin‐mediated adhesion but enhanced migration in CML progenitors on FN. BCR–ABL induced abnormalities in GF–integrin interactions could contribute to abnormal circulation and microenvironmental localization of CML progenitors.
Interferon γ and tumour necrosis factor α are overexpressed in bone marrow T lymphocytes from paediatric patients with aplastic anaemia
Authors: Dufour Carlo , Corcione Anna , Svahn Johanna , Haupt Riccardo , Battilana Nicoletta , Pistoia Vito
Published: March 2003

Twelve paediatric patients with aplastic anaemia and two groups of normal control subjects underwent flow cytometric analysis for intracytoplasmic expression of γ interferon (γ‐IFN) and tumour necrosis factor α (TNF‐α) in bone marrow and peripheral blood CD4+ and CD8+ cells. The same cytokines were tested, by immunoassay, in culture supernatants from unstimulated bone marrow mononuclear cells (MNCs). Marrow CD4+ and CD8+ cells expressing γ‐IFN and TNF‐α were significantly increased in the patients in comparison with normal control subjects (P from < 0·05 to < 0·0001 in the different cellular subsets). Patients' marrow CD4+ and CD8+ cells containing γ‐IFN and TNF‐α were significantly increased when compared with the same cell fractions from paired peripheral blood samples (P from < 0·05 to < 0·001 in the various cellular subsets). In the supernatant of marrow MNCs, γ‐IFN and TNF‐α were detected in four out of eight and five out of eight cases, respectively, whereas neither cytokine was traceable in the control subjects. Patients' peripheral blood CD4+ and CD8+ cells containing γ‐IFN and TNF‐α were not significantly increased in comparison with those from normal control subjects. Whereas patients with favourable and unfavourable outcomes had no significantly different proportions of marrow γ‐IFN+/CD4+ and γ‐IFN+/CD8+ cells, the percentages of marrow CD4+ and CD8+ cells containing TNF‐α were significantly lower in subjects with favourable than in those with unfavourable outcome. Overall, these findings show that, in aplastic patients, T cells overexpressing γ‐IFN and TNF‐α concentrate in the bone marrow and that intracytoplasmic expression of TNF‐α in marrow CD4+ and CD8+ cells is associated with an unfavourable clinical course.
Colour Doppler ultrasonography of a segmental branch of the portal vein is useful for early diagnosis and monitoring of the therapeutic course of veno‐occlusive disease after allogenic haematopoietic stem cell transplantation
Authors: Yoshimoto Kohji , Yakushiji Kazuaki , Ijuin Hiroyasu , Ono Naofumi , Hashiguchi Michitoshi , Imamura Rie , Ogata Hideaki , Okamura Takashi , Sata Michio , Hashimoto Hiroshi
Published: March 2003

We report two cases in which visualization of the segmental branch of the hepatic portal vein with the colour Doppler ultrasonography (US) technique was useful for the early diagnosis of veno‐occlusive disease. The change in blood flow in the segmental branch of the portal vein occurred 5 and 6 d before the clinical criteria were fulfilled in the two cases. Reverse flow in the segmental branch began partially in the liver at first, and then spread to the whole liver several days later. All the US findings in both cases disappeared after thrombolytic therapy.
A new approach to evaluating intestinal acute graft‐versus‐host disease by transabdominal sonography and colour Doppler imaging
Authors: Klein S. A. , Martin H. , Schreiber‐Dietrich D. , Hermann S. , Caspary W. F. , Hoelzer D. , Dietrich C. F.
Published: March 2003

An objective and non‐invasive method to assess the extent, severity, course and prognosis of acute graft‐versus‐host disease (aGvHD) of the gastrointestinal tract would be of great importance. As high‐resolution ultrasonography (HRU) and colour Doppler imaging (CDI) are used in inflammatory and ischaemic bowel diseases, we introduced these techniques to evaluate acute intestinal GvHD. In this study, 12 patients with suspected intestinal and/or severe cutaneous aGvHD were investigated by HRU and CDI. The structure and thickness of the bowel wall and the blood flow pattern in the superior mesenteric artery (SMA) as well as in the bowel wall were evaluated. All patients showed thickened bowel wall segments, especially in the ileocaecal region. Even in patients without any clinical signs of intestinal involvement (3/12), sonographic changes were observed. In six patients with severe diarrhoea, sonographic signs of secretory diarrhoea were observed. In 4/12 patients, an increased arterial blood flow in the bowel wall was demonstrated as a typical characteristic of an inflammatory bowel process. In contrast, in 4/12 patients, ischaemic bowel wall lesions with high‐resistance flow pattern in the SMA were found. These patients did not respond to immunosuppressive therapy and died. In conclusion, HRU is a useful tool for detecting acute bowel GvHD even before clinical symptoms. Moreover, HRU and CDI can be applied to define the severity of aGvHD and to identify patients with a poor prognosis.
The TEL‐AML1 real‐time quantitative polymerase chain reaction (PCR) might replace the antigen receptor‐based genomic PCR in clinical minimal residual disease studies  in children with acute lymphoblastic leukaemia
Authors: De Haas V. , Breunis W. B. , Dee R. , Verhagen O. J. H. M. , Kroes W. , Van Wering E. R. , Van Dongen J. J. M. , Van Den Berg H. , Van Der Schoot C. E.
Published: March 2003

Summary. Prospective studies in children with B‐precursor acute lymphoblastic leukaemia (ALL) have shown that polymerase chain reaction (PCR)‐based detection of minimal residual disease (MRD) using immunoglobin (Ig) and T‐cell receptor (TCR) gene rearrangements as targets can be used to identify patients with a high relapse risk. The disadvantage of this approach is that for each patient preferably two different targets have to be identified. The t(12;21)(p13;q22) with the TEL‐AML1 fusion gene is present in approximately 25% of children with B‐precursor ALL. In these patients, sensitive reverse transcription (RT)‐PCR analysis of the TEL‐AML1 fusion transcript might be a more simple and less laborious alternative approach. However, it is unknown how stable the mRNA is and whether the number of transcripts per leukaemic cell remains constant during follow‐up. We investigated whether the MRD results obtained using RT‐PCR of TEL‐AML1 transcripts correlated with the clinically validated genomic PCR for Ig and TCR gene rearrangements. Therefore, we used real‐time quantitative (RQ)‐PCR analysis for both types of targets and assessed the MRD levels in 36 follow‐up bone marrow samples (obtained during the first 1·5 years after diagnosis) from 13 patients with B‐precursor ALL. In 34/36 bone marrow samples the Ig/TCR RQ‐PCR and TEL‐AML1 RQ‐PCR revealed equal levels of MRD and these results had a strong correlation (P < 0·0001, R2 = 0·84). Therefore, we conclude that the TEL‐AML1 RQ‐PCR can, in principle, replace Ig/TCR RQ‐PCR in B‐precursor ALL with t(12;21).
Expression of the Ly‐6A (Sca‐1) lacZ transgene in mouse haematopoietic stem cells and embryos
Authors: Ma Xiaoqian , De Bruijn Marella , Robin Catherine , Peeters Marian , Kong‐A‐San John , De Wit Ton , Snoijs Corne , Dzierzak Elaine
Published: March 2003

Summary. The Sca‐1 surface glycoprotein is used routinely as a marker for haematopoietic stem cell enrichment. Two allelic genes, Ly‐6A and Ly‐6E, encode this marker and appear to be differentially regulated in haematopoietic cells and haematopoietic stem cells. The Sca‐1 protein has been shown to be expressed at a greater frequency in these cells from Ly‐6A strains of mice. To study the specific expression pattern and haematopoietic regulation of the Ly‐6A gene, we constructed a 14 kb cassette from a genomic Ly‐6A fragment, inserted a lacZ reporter gene and created transgenic mice. We found that the Ly‐6A lacZ transgene was expressed in the haematopoietic tissues and predominantly in the T‐lymphoid lineage. Some expression was also found in the B‐lymphoid and myeloid lineages. We demonstrated functional haematopoietic stem cell enrichment by sorting for β‐galactosidase‐expressing cells from the bone marrow. In addition, we found an interesting embryonic expression pattern in the AGM region, the site of the first haematopoietic stem cell generation. Surprisingly, when compared with data from Ly‐6E lacZ transgenic mice, our results suggest that the Ly‐6A cassette does not improve lacZ marker gene expression in haematopoietic cells.
Differential expression of CD56 and CD44 in the evolution  of extramedullary myeloma
Authors: Dahl Inger Marie S. , Rasmussen Thomas , Kauric Goran , Husebekk Anne
Published: March 2003

Summary. We report on the different expression of CD56 and CD44 in plasma cells (PCs) simultaneously collected from bone marrow, extramedullary locations and peripheral blood in seven patients with multiple myeloma. Extramedullary PCs showed absence of CD56. In the bone marrow, however, subsets with varying CD56 expression were found in five out of seven patients, with one subset corresponding to that of extramedullar PCs. This differs from the de novo downregulation of CD56 in PC leukaemia, and suggests different mechanisms of spread of myeloma cells. CD44 expression was generally upregulated on extramedullary PCs. In three of the patients we investigated the clonal origin of extramedullary myeloma cells by sequencing the variable portion of the heavy chain immunoglobulin gene in phenotypically defined PCs isolated from different locations. In each patient we found malignant PCs with different homing behaviour originating from a common precursor cell.
Liposomal encapsulation diminishes daunorubicin‐induced generation of reactive oxygen species, depletion of ATP and necrotic cell death in human leukaemic cells
Authors: Liu Feng‐Ting , Kelsey Stephen M. , Newland Adrian C. , Jia Li
Published: March 2003

Summary. In this study, we tested the mechanisms of daunorubicin (DNR)‐ and the liposomal encapsulated daunorubicin (DaunoXome or DNX)‐induced killing in three human leukaemic cell lines, K562, K/Bax and CEM. DNX showed less cytotoxicity in leukaemic cells than conventional DNR. The intracellular accumulation of DNX was 10 times less than conventional DNR during exposure to drugs for up to 5 h. Cell cycle analysis indicated that DNR induced concentration‐dependent G2/M arrest, apoptosis and necrosis. However, DNX induced G2/M arrest and apoptosis but not necrotic cell death, even at a higher concentration. DNR‐ or DNX‐induced activation of caspase‐9 and ‐3 was detected at concentrations that induced apoptosis and necrosis. The sensitivity of leukaemic cells to DNR‐ and DNX‐induced apoptosis correlated with the activation of caspases and the reduction of mitochondrial membrane potential (ΔΨm), but not the depletion of ATP and the generation of reactive oxidative species (ROS). DNX did not provoke ROS generation and ATP depletion in leukaemic cells. We conclude that the liposomal encapsulation of DNR restricts the intracellular accumulation speed and therefore diminishes ROS generation, ATP depletion and necrotic cell death. This may have implications for the cause of cardiotoxicity seen with DNR, its main dose‐limiting step.