bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of human T‐lymphoid progenitor cells in CD34+CD38low and CD34+CD38+ subsets of human cord blood and bone marrow cells using NOD‐SCID fetal thymus organ cultures
Authors: Robin Catherine , Bennaceur‐Griscelli Annelise , Louache Fawzia , Vainchenker William , Coulombel Laure
Published: March 2003

In contrast to myeloid and B‐lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+CD38+ and CD34+CD38low cells from both cord blood and adult bone marrow reproducibly develop into CD4+CD8+T cells when introduced into NOD‐SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC (fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T‐cell potential of human primitive progenitor cells. The frequency of T‐cell progenitors among cord‐blood‐derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD‐SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD4lowCD8−sCD3−TCRαβ–CD5+CD1a+T cells were mixed with CD4+CD8+ cells and more mature CD4+CD8−TCRαβ+cells. However, in FTOC initiated with bone marrow T progenitors, <10% double‐positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T‐cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine‐stimulated CD34+CD38low cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.
Factor VIII inhibitors in haemophiliacs: a single‐centre experience over 34 years, 1964–97
Authors: Yee T. T. , Pasi K. J. , Lilley P. A. , Lee C. A.
Published: March 2003

A retrospective study of the natural history of factor VIII inhibitors in haemophilia A patients experienced in a single comprehensive haemophilia centre over three decades is reported. 431 haemophilia A patients of all severities have been followed‐up for a total of 5626 patient‐years. The frequency of inhibitors was 10% in the severe haemophilia A patients and 37% occurred in children <10 years. The majority of the patients received several products before developing the inhibitors. 59% of patients had <50 exposure days and 48% were high responders (>5 BU). An 8‐year (1987–95) inhibitor‐free period was seen during which all previously untreated patients were treated with an intermediate‐purity factor VIII concentrate. A moderate haemophiliac with a misense mutation that has not been described in association with inhibitor is reported. Six HIV‐positive patients preserved their antibody response to factor VIII even at the advanced stage of their disease.
Prothrombin Carora: hypoprothrombinaemia caused by substitution of Tyr‐44 by Cys
Authors: Sun William Y. , Ruiz‐Saez Arlette , Burkart Melissa C. , Bosch Norma , Degen Sandra J. F.
Published: March 2003

Two members of a family from Carora, Venezuela, were found to have prothrombin activity levels at 4% of normal and undetectable antigen levels. All exons of the prothrombin gene from the proband were sequenced and a mutation at nucleotide 1305 was identified that would result in the substitution of Cys for Tyr at residue 44. Residue 44 is present in the aromatic stack region of the protein. Substitution of a Cys in this region would result in an abnormal folding of the protein which could be the cause for the observed lack of secretion of the abnormal prothrombin.
Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation
Authors: Denning‐Kendall P. A. , Horsley H. , Donaldson C. , Bradley B. , Hows J. M.
Published: March 2003

In‐vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long‐term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (± SD, n = 5) of 93.0 ± 8.0% of the eluted CD34+ cells, 99.6 ± 0.7% of the CFU‐GM and all of the eluted long‐term culture‐initiating cells (LTC‐IC). Cord blood CD34+ cells were then cultured for 14 d with IL‐3, IL‐6, SCF, G‐CSF and GM‐CSF, each at 10 ng/ml. The total cell expansion was 2490 ± 200‐fold and the CD34+ cell expansion was 49 ± 17‐fold. The percentage of CD34+ cells present after expansion culture was 1.2 ± 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 ± 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 ± 8.8% of the eluted CFU‐GM and 79.5 ± 11.0% of the LTC‐IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.
Hepatocyte growth factor in serum after injection of unfractionated and low molecular weight heparin in healthy individuals
Authors: Seidel Carina , Hjorth‐Hansen Henrik , Bendz BjØrn , BØrset Magne , Sandset Per Morten , Hansen John‐Bjarne , Sundan Anders , Waage Anders
Published: March 2003

Soluble heparin displaces the cytokine hepatocyte growth factor (HGF) from heparan sulphate proteoglycans on the cell surface and in the extracellular matrix into the circulation. We examined serum HGF elevation after heparin injections, and whether there is a difference between unfractionated heparin (UH) and low molecular weight heparin (LMWH) in their ability to increase serum HGF. 20 healthy individuals were randomized to a single injection of intravenous or subcutaneous UH or LMWH. There was a significant increase in HGF from pretreatment values. This HGF was bioactive. When these preparations were compared on the basis of their serum concentrations (anti‐factor Xa activity or molar concentrations), the increase in HGF was greater in individuals receiving UH than LMWH. When UH or LMWH were administered over a 5 d period, the increase in HGF, as well as the difference between treatments to induce HGF, remained stable throughout the treatment. In five patients treated with continuous intravenous heparin infusion HGF was increased throughout the treatment period of 5–7 d. In summary, the rise in bioactive HGF after heparin treatment was stable during continued treatment. UH was more potent in inducing HGF increase than LMWH, both after a single injection and after several days of treatment.
Establishment of a near‐triploid human B‐cell lymphoma cell line with t(14;18) and a p53 gene point mutation
Authors: Yamaguchi Hiroki , Inokuchi Koiti , Hanawa Hideki , Tanosaki Sakae , Shinozawa Isao , Matuoka Hiroki , Dan Kazuo
Published: March 2003

We report a rare large B‐cell non‐Hodgkin's lymphoma having a characteristic near‐triploid cell population with add(17)(p22) and t(14;18)(q32;q21) translocation. We also established and characterized a new cell line (TK cell) derived from the present lymphoma. A codon 180 mutation (GAG → GAT) in the p53 gene was detected. t(14;18)(q32;q21) was revealed juxtaposition of the bcl‐2 and JH genes. Immunoprecipitation analyses of p53 and bcl‐2 revealed that abnormality of the p53 protein and aberrant bcl‐2 expression, which may protect cells from apoptosis, may be critical to the development of leukaemogenesis exhibiting near‐triploid chromosomes.
A new preconditioning regimen with melphalan, busulphan and total body irradiation followed by low‐dose immunosuppressant in allogeneic haemopoietic stem cell transplantation
Authors: Murata Makoto , Nishida Tetsuya , Haneda Masataka , Kanie Tadaharu , Taji Hirofumi , Iida Hiroatsu , Suzuki Ritsuro , Hamaguchi Motohiro , Minami Saburo , Kodera Yoshihisa
Published: March 2003

Twenty adult patients with high‐risk leukaemia underwent allogeneic haemopoietic stem cell transplantation after melphalan, busulphan and total body irradiation followed by short‐term methotrexate and low‐dose cyclosporine or tacrolimus. Three patients developed veno‐occlusive disease and no patient developed renal dysfunction. Seven patients experienced grade II–IV acute graft‐versus‐host disease (aGVHD) and five patients experienced grade III–IV. The 3‐year probabilities of relapse and leukaemia‐free survival were 22 ± 11% (95% confidence interval) and 50 ± 11%, respectively. These data suggest that this preconditioning regimen followed by a low‐dose immunosuppressant provided a more anti‐leukaemic effect without increased regimen‐related toxicity and aGVHD.
An investigation of the t(12;21) rearrangement in children with B‐precursor acute lymphoblastic leukaemia using cytogenetic and molecular methods
Authors: Kempski Helena , Chalker Jane , Chessells Judith , Sturt Natalie , Brickell Paul , Webb Julie , Clink Jim MacDonald , Reeves Brian
Published: March 2003

The t(12;21) is the commonest recurrent translocation in childhood acute lymphoblastic leukaemia (ALL), the presence of which has been suggested to be a good prognostic feature. We have studied 22 childhood cases of B‐precursor ALL with this rearrangement, and have found no significant differences in event‐free survival between these and a control group of patients with similar phenotypes. Using a variety of cytogenetic and molecular techniques, we have confirmed a strong association with co‐expression of myeloid markers, frequent deletions of the short‐arm of the untranslocated chromosome 12 homologue and duplication of the derivative chromosome 21. Intragenic deletion of the untranslocated ETV6 gene in 3/12 informative patients points to the likelihood of this gene being a target for deletion.
Accelerated telomere shortening and telomerase activation in Fanconi's anaemia
Authors: Leteurtre F. , Li X. , Guardiola P. , Le Roux G. , Sergère J.‐C. , Richard P. , Carosella E. D. , Gluckman E.
Published: March 2003

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure that often evolves towards acute leukaemia. FA also belongs to a group of chromosome instability diseases. Because telomeres are directly involved in chromosomal stability and in cell proliferation capacity, we examined telomere metabolism in peripheral blood mononuclear cells (PBMC). Telomere length was significantly shorter in 54 FA patient samples, compared to 51 controls (P<0.0001). In addition, mean telomere terminal restriction fragment lengths (TRF) in nine heterozygous patient samples did not differ from those of controls. In 14 samples from FA patients with severe aplastic anaemia (SFA), telomere length was significantly shorter than in 22 samples of age‐matched FA patients with moderate haematological abnormalities (NSFA) (P<0.001). However, no correlation was found between TRF length and the presence of bone marrow clonal abnormalities in 16 additional, separately analysed, patient samples. Sequential measurement of TRF in six FA patients showed an accelerated rate of telomere shortening. Accordingly, telomere shortening rate was inversely correlated with clinical status. Telomerase, the enzyme that counteracts telomere shortening, was 4.8‐fold more active in 25 FA patients than in 15 age‐matched healthy controls. A model for the FA disease process is proposed.
Analysis of progenitor cell involvement in B‐CLL by simultaneous immunophenotypic and genotypic analysis at the single cell level
Authors: Gahn Benedikt , Wendenburg Britta , Troff Carmen , Neef Juliane , Grove Doris , Haferlach Torsten , Hiddemann Wolfgang , Wörmann Bernhard
Published: March 2003

B‐cell chronic lymphocytic leukaemia (B‐CLL) results from the clonal expansion of mature B lymphocytes. The detection of leukaemia‐associated genetic markers in CD34‐positive progenitor cells in a subset of B‐CLL patients suggests that malignant transformation in B‐CLL occurs in an immature progenitor cell compartment. To further quantify the percentage of B‐CLL patients with genetically aberrant progenitor cells we have investigated CD34+ bone marrow cells in 11 B‐CLL patients at the single cell level by simultaneous genetic and immunophenotypic analysis (FICTION). In five patients with trisomy 12, CD34+ haemopoietic progenitor cells were detectable on bone marrow smears. In one patient with trisomy 12, CD34+ progenitor cells were isolated by FACS sorting. In all six patients trisomy 12 was not found in the CD34+ cells. Progenitor cells were also analysed in three patients with Rb‐deletion and in two patients with deletion of p53. In all patients the genetic marker was not detected in the CD34+ cells. In conclusion, we did not find genetically aberrant progenitor cells in this group of B‐CLL patients. These results suggest that the subset of B‐CLL patients with genetically aberrant CD34+ cells may be very small. This is of significance for our understanding of B‐CLL biology and for future strategies using autologous stem cell transplantation.
Detection of identical T‐cell clonotype expansions in both the donor and recipient after allogeneic bone marrow transplantation
Authors: Trentin Livio , Imberti Luisa , Zambello Renato , Sottini Alessandra , Raimondi Roberto , Facco Monica , Cazzavillan Sefania , Bonoldi Emanuela , Signorini Simona , Bacigalupo Andrea , Semenzato Gianpietro , Rodeghiero Francesco , Primi Daniele
Published: March 2003

Using phenotypic, functional and molecular techniques, this study was performed to compare the complexity of the T‐cell receptor repertoire of a bone marrow transplanted patient with that of his HLA‐matched related donor, both of whom developed a chronic lymphocytosis sustained by CD3+CD8+CD57+CD16−CD56− granular lymphocytes 3 years after transplantation. Although Southern blot analysis revealed the presence of extra bands in both subjects, thus indicating the presence of at least one clonal T‐cell population, the study of the different T‐cell receptor Vβ (TCRBV) usage did not demonstrate discrete overexpression of any TCRBV segments. On the contrary, heteroduplex analysis of TCRBV transcripts suggested the presence of oligoclonal T‐cell expansions in the two subjects. Cloning and sequencing studies demonstrated that T‐cell clones expressing identical TCRBV chains were expanded both in the donor and in the recipient. Furthermore, clones with similar, but not identical, junctional regions were also found in the two subjects. These data indicate that, at the time of the graft, a few cells with a monoclonal/oligoclonal pattern that were present in the donor were transferred to the recipient, where they may have found the same environmental in vivo conditions and/or the antigenic pressure favouring their abnormal expansion.
Improved outcome in haemophagocytic lymphohistiocytosis after bone marrow transplantation from related and unrelated donors: a single‐centre experience of 12 patients
Authors: Dürken Matthias , Horstmann Martin , Bieling Patricia , Erttmann Rudolf , Kabisch Hartmut , Löliger Cornelius , Schneider E. Marion , Hellwege Hans‐Henning , Krüger William , Kröger Nikolaus , Zander Axel R. , Janka Gritta E.
Published: March 2003

Haemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disease with histiocytic and lymphocytic infiltrations in multiple organs. Cure seems possible only by allogeneic bone marrow transplantation (BMT), but matched sibling donors (MSD) are restricted and high mortality rates are associated with BMT from unrelated donors (URD). We report on 12 consecutive HLH patients with an improved outcome following URD transplants. Eight patients received BMT from URD, four from MSD. Five patients had signs of active HLH at the time of BMT. The conditioning regimen consisted of 20 mg/kg busulphan, 60 mg/kg VP‐16 and 120 mg/kg cyclophosphamide and, in case of URD, 90 mg/kg antithymocyte globulin. The doses of busulphan and VP‐16 were reduced during the programme to 16 mg/kg and 30 mg/kg, respectively. Using a fivefold graft‐versus‐host disease (GVHD) prophylaxis, GVHD was absent or mild in 10, and moderate or severe in two patients undergoing unrelated transplants. One patient with URD experienced graft failure and was retransplanted on day 37. Major toxicities were hepatic veno‐occlusive disease in five, capillary leak syndrome in two, pneumonia in three, sepsis in one, severe mucositis in one and seizures in two patients. All patients are alive without HLH after a median follow‐up of 24.5 months. One patient has chronic GVHD, another patient has severe retardation. Three patients show slight to moderate development delay. These results indicate that in HLH, BMT from matched unrelated donors should be performed. Incomplete resolution of disease activity need not impede a successful outcome.
Functional expression of TRAIL by lymphoid and myeloid tumour cells
Authors: Zhao Shourong , Asgary Zahra , Wang Yan , Goodwin Raymond , Andreeff Michael , Younes Anas
Published: March 2003

TRAIL is a potent death protein that favours the killing of various types of cancer cells to normal cells, but under the right conditions TRAIL can also kill activated human T cells. TRAIL mRNA is widely expressed by normal cells but its expression by primary tumour cells is not known. In this study, primary tumour cells of haemopoietic origin constitutively expressed TRAIL mRNA and protein and were capable of inducing the apoptosis of target Jurkat cells in a dose‐dependent manner. This killing effect was reversed by anti‐TRAIL antibody. The functional expression of TRAIL by lymphoid and myeloid malignant cells raises the possibility of its involvement in tumour cell evasion of immunosurveillance, and could be related to spontaneous tumour cell death and necrosis.
A novel BCR‐ABL transcript e2a2 in a chronic myelogenous leukaemia patient with a duplicated Ph‐chromosome and monosomy 7
Authors: Leibundgut Elisabeth Oppliger , Jotterand Martine , Rigamonti Veronique , Parlier Valérie , Mühlematter Dominique , Tobler Andreas , Solenthaler Max
Published: March 2003

A novel BCR‐ABL transcript was detected by multiplex RT‐PCR in a patient with Philadelphia chromosome (Ph) positive chronic myelogenous leukaemia (CML) in accelerated phase. Sequencing of the aberrant transcript revealed an in‐frame e2a2 fusion that included a 9 basepairs insertion. Cytogenetic analysis showed t(9;22), an additional Ph chromosome and monosomy 7. The clinical course was dismal: therapy was poorly tolerated, and the patient died in blast crisis 10 months after diagnosis. These data support the association of additional Ph and monosomy 7 with poor prognosis and suggest that the novel e2a2 BCR‐ABL transcript may be related to an aggressive clinical course.
Rabbit antithymocyte globulin (r‐ATG) plus cyclosporine and granulocyte colony stimulating factor is an effective treatment for aplastic anaemia patients unresponsive to a first course of intensive immunosuppressive therapy
Authors: Di Bona E. , Rodeghiero F. , Bruno B. , Gabbas A. , Foa P. , Locasciulli A. , Rosanelli C. , Camba L. , Saracco P. , Lippi A. , Iori A. P. , Porta F. , De Rossi V. , Comotti B. , Iacopino P. , Dufour C. , Bacigalupo A.
Published: March 2003

About 30% of patients with severe aplastic anaemia (SAA) unresponsive to one course of immunosuppressive (IS) therapy with antithymocyte or antilymphocyte globulin can achieve complete or partial remission after a second IS treatment. Among various second‐line treatments, rabbit ATG (r‐ATG) could represent a safe and effective alternative to horse ALG (h‐ALG). In a multicentre study, 30 patients with SAA (17 males and 13 females, median age 21 years, range 2–67) not responding to a first course with h‐ALG plus cyclosporin (CyA) and granulocyte colony stimulating factor (G‐CSF), were given a second course using r‐ATG (3.5 mg/kg/d for 5 d), CyA (5 mg/kg orally from day 1 to 180) and G‐CSF (5 μg/kg subcutaneously from day 1 to 90). The median interval between first and second treatment was 151 d (range 58–361 d). No relevant side‐effects were observed, but one patient died early during treatment because of sepsis. Overall response, defined as transfusion independence, was achieved in 23/30 (77%) patients after a median time of 95 d (range 14–377). Nine patients (30%) achieved complete remission (neutrophils 2.0 × 109/l, haemoglobin 11 g/dl and platelets 100 × 109/l). The overall survival rate was 93% with a median follow‐up of 914 d (range 121–2278). So far, no patient has relapsed. Female gender was significantly associated with a poorer likelihood to respond (P = 0.0006). These data suggest that r‐ATG is a safe and effective alternative to h‐ALG for SAA patients unresponsive to first‐line IS treatment.
Tumour necrosis factor polymorphisms and susceptibility to follicular lymphoma
Authors: Fitzgibbon Jude , Grenzelias Demetrios , Matthews Janet , Lister T. Andrew , Gupta Rajnish K.
Published: March 2003

Follicular lymphoma is characterized in 85% of patients by the presence of a t(14;18) chromosomal translocation that results in overproduction of BCL2. In this study the distribution of high and low expressing TNF alleles at the TNF (−308) and LTα (+252) polymorphic sites in 121 patients with follicular lymphoma and 88 control individuals has been analysed. A reduction in high expressing haplotypes in patients compared to normal controls was found (P = 0.055), with no significant difference observed in response rate or overall survival between patients with high or low expressing haplotypes. These results suggest that the TNF locus, or an adjacent locus within the MHC region, is an important genetic risk factor in this disease.
Antisense‐mediated suppression of Bcl‐2 highlights its pivotal role in failed apoptosis in B‐cell chronic lymphocytic leukaemia
Authors: Pepper Chris , Thomas Alun , Hoy Terry , Cotter Finbarr , Bentley Paul
Published: March 2003

Although advances have been made in the development of more effective treatment modalities, B‐cell chronic lymphocytic leukaemia (B‐CLL) remains incurable due to the development of drug resistance. Defective programmed cell death mechanisms rather than dysregulation of cell cycle appears to predominate in B‐CLL and it is likely that a failure to initiate apoptosis contributes to chemoresistance. Most B‐CLL cells contain high levels of the anti‐apoptotic protein Bcl‐2 and high Bcl‐2/Bax ratios have been associated with in vitro resistance to cytotoxic agents. In this study we evaluated the cellular responses to a Bcl‐2 antisense oligonucleotide in terms of Bcl‐2 mRNA and protein expression and the induction of apoptosis. The antisense molecule induced a specific reduction in Bcl‐2 mRNA and protein expression over the 48 h culture period and was associated with increased apoptosis. The study indicates that Bcl‐2 protein is central to the mediation of resistance to apoptosis in B‐CLL. Therefore Bcl‐2 antisense oligonucleotides might be useful in the treatment of B‐CLL.
Grandpaternal mosaicism in a family with isolated haemophilia A
Authors: Casey Graeme J. , Rodgers Susan E. , Hall Jennifer R. , Rudzki Zbigniew , Lloyd John V.
Published: March 2003

About one third of cases of haemophilia A have no family history of the disorder, and 20% are thought to be due to a new mutation. In the family reported here, a 3 bp deletion was detected in DNA from the proband at the 3′ end of exon 15. Direct sequencing of genomic DNA prepared from blood and buccal cells of the grandfather revealed both normal and mutant sequences, suggesting that he is a mosaic for this mutation. This highlights the usefulness of mutation detection, both for accurate genetic counselling and to determine the origin of new mutations of haemophilia.
Increased serum levels of vascular endothelial growth factor predict risk of progression in early B‐cell chronic lymphocytic leukaemia
Authors: Molica Stefano , Vitelli Gaetano , Levato Domenico , Gandolfo Giuseppe Maria , Liso Vincenzo
Published: March 2003

The present study is the first to evaluate serum levels of vascular endothelial growth factor (VEGF) in B‐cell chronic lymphocytic leukaemia (CLL). All 68 B‐cell CLL patients and 31 control subjects analysed had detectable serum levels of VEGF, with no statistically significant difference between two proups. An aberrant increase of circulating levels of VEGF was found in only 17.6% of cases. B‐cell CLL patients whose serum VEGF levels were higher than the median (i.e. 194.8 pg/ml) or 75th percentile (i.e. 288.5 pg/ml) values were more frequently at an advanced clinical stage. In contrast, no correlation with other clinico‐biological features representative of either tumour mass [bone marrow (BM) histology, peripheral blood (PB) lymphocytosis, beta‐2 microglobulin (β‐2m), LDH, interleukin‐6 (IL‐6)] or disease‐progression (DP) [lymphocyte doubling time (LDT)] was found.  Serum levels of VEGF predicted the risk of DP in early CLL. Among 41 patients in Binet stage A, progression‐free survival (PFS) was significantly shorter in those patients whose VEGF serum concentrations were above the median value. Interestingly, characteristics of stage A patients stratified according to the median value of VEGF were similar with respect to many clinico‐biological features, thus suggesting a possible independent prognostic role for such a marker. Finally, when added to the Rai subclassification, VEGF serum levels identified two groups with different PFS within stages I–II.  We conclude that increased serum levels of VEGF can be considered useful for predicting the risk of DP and add prognostic information to the Rai subclassification of stage A CLL.
Heterozygous factor XI deficiency associated with three novel mutations
Authors: Mitchell Michael , Cutler Jacky , Thompson Simon , Moore Gary , Jenkins ap Rees Elaine , Smith Mark , Savidge Geoffrey , Alhaq Anwar
Published: March 2003

To determine the utility of single‐stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC → TGC (Arg308Cys) mutation in exon 9, a GCT→GTT (Ala412Val) mutation in exon 11 and an AGC → AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.
A time kinetic study of the effect of aminobisphosphonate on murine haemopoiesis
Authors: Nakamura Masanori , Yagi Hideki , Endo Yasuo , Kosugi Hiroshi , Ishi Tadashi , Itoh Tsunetoshi
Published: March 2003

We previously showed that aminobisphosphonates (aminoBPs), potent inhibitors of bone resorption, increased the number of osteoclasts and granulocytes, and enhanced the cell size of osteoclasts in vivo, indicating that aminoBPs have a profound effect on murine haemopoiesis. The possible effect of an aminoBP (4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonate; AHBuBP) on murine haemopoiesis in vivo was examined in more detail. Macroscopically, AHBuBP induced the whitened bone marrow (BM) and splenomegaly. Flow cytometric analysis indicated that in BM, AHBuBP reduced the number of mature monocyte‐macrophage lineage cells and erythroid cells 1 and 2 d after treatment, respectively, whereas it enhanced granulopoiesis on day 4. In the spleen, both erythropoiesis and granulopoiesis were significantly increased. BM haemopoietic progenitors of granulocyte lineage and of monocyte‐macrophage lineage (CFU‐G, CFU‐M and CFU‐GM) were well maintained by the injection of AHBuBP, and even a small increment in these progenitors was observed 2–4 d after treatment. Immunohistochemical examination of BM demonstrated that residential macrophages of erythroblastic islands disappeared. Increased numbers of osteoclasts, as well as enlarged cell size, was confirmed up to 7 d after the treatment, implicating that the inhibition of bone resorption was not due to the reduced generation of osteoclasts by AHBuBP. These results suggest (1) that AHBuBP treatment in vivo rapidly deleted mature residential macrophages from BM, (2) that mature macrophages once deleted did not reappear even when CFU‐M and CFU‐GM increased in number and the number of Mac‐1+/Gr‐1− cells recovered to normal, (3) that BM erythropoiesis was significantly decreased due to the lack of erythroblastic islands, and (4) that compensatory erythropoiesis was evoked in the spleen to induce splenomegaly.
Dominant β‐thalassaemia: a highly unstable haemoglobin is caused by a novel 6 bp deletion of the β‐globin gene
Authors: Vetter  , Neu‐Yilik  , Kohne  , Arnold  , Sinha  , Gaedicke  , Ivančevič  , Kulozik A. E.
Published: March 2003

β‐Thalassaemia is inherited as an autosomal recessive trait in most families. Particular interest has recently been focused on the molecular pathology of the rare forms with a dominant mode of inheritance. The index patient and her mother, who are described in this report, displayed typical clinical and haematological features of β‐thalassaemia intermedia with significant ineffective erythropoiesis and additional peripheral haemolysis. Molecular analysis demonstrated a heterozygous genotype for a novel 6 bp (TGGTCT) deletion of the β‐globin gene involving codons 33–35. This deletion results in the removal of two valine residues from the β‐globin chain at position 33/34 (B15/B16) and the substitution of the tyrosine residue at position 35 (C1) by an aspartic acid (β 33–35 [B15‐C1] Val‐Val‐Tyr→0–0‐Asp). According to the index patient's place of birth, this abnormal haemoglobin has been termed Hb Dresden. The stability of the variant and the normal β‐globin chains were similar during the incubation period of in vitro globin chain synthesis analysis. However, Hb Dresden is exquisitely unstable and cannot be detected in the peripheral blood by haemoglobin electrophoresis, high‐performance liquid chromatography (HPLC) or isoelectric focusing. This instability can be explained by the vital structural role of the three affected amino acids that, in normal haemoglobin, establish a total of nine intermolecular bonds (five hydrophobic and four polar) at both the α1β1 (α2β2) and the α1β2 (α2β1) interface.
Effects of antithymocyte globulin on bone marrow CD34+ cells in aplastic anaemia and myelodysplasia
Authors: Killick Sally B. , Marsh Judith C. W. , Gordon‐Smith Edward C. , Sorlin Laurent , Gibson Frances M.
Published: March 2003

The mechanism of action of antithymocyte globulin (ATG) in the treatment of aplastic anaemia (AA) and myelodysplastic syndromes (MDS) is poorly understood and may involve many different mechanisms. The aim of this in vitro study was to investigate further the effect of ATG on haemopoietic progenitor cells. A total of 16 patients (10 AA and 6 MDS) and 12 normal control subjects were studied. Purified bone marrow (BM) CD34+ cells were cultured in committed progenitor assay in the presence of ATG and autologous serum, then scored on day 14 for granulocyte–monocyte colony‐forming units (CFU‐GM) and erythroid colonies. ATG was found to be inhibitory to haemopoietic progenitor cells at high concentrations (1000 μg/ml and 100 μg/ml). This was confirmed by CD34‐FITC and 7AAD staining of purified normal CD34+ cells after overnight incubation with ATG. In contrast, at lower doses (0.1–10 μg/ml), ATG produced an increase in colony growth in most normal, MDS and AA BM CD34+ cells. The greatest effect was in patients with non‐severe AA, in whom the greatest increase in CFU‐GM was seen at 0.5 μg/ml (P < 0.02) and 0.1 μg/ml (P = 0.02) and erythroid colonies at 0.1 μg/ml (P < 0.05). Serum ATG levels peaked during infusion to levels that were found to be toxic to haemopoietic progenitor cells in vitro and fell thereafter to levels that were associated with the highest colony numbers (0.1 and 0.5 μg/ml) in vitro. These results suggest that an increase in haemopoietic progenitor cells by ATG may be one of several important mechanisms for haematological recovery in AA and MDS.
Peripheral blood stem cell (PBSC) mobilization and transplantation after fludarabine therapy in chronic lymphocytic leukaemia (CLL): a report of the European Blood and Marrow Transplantation (EBMT) CLL subcommittee on behalf of the EBMT Chronic Leukaemias Working Party (CLWP)
Authors: Michallet  , Thiébaut  , Dreger  , Remes  , Milpied  , Santini  , Hamon  , Björkstrand  , Kimby  , Belhabri  , Tanguy  , Apperley
Published: March 2003

We performed a survey from 122 centres of the European Group of Blood and Marrow Transplantation (EBMT) concerning peripheral blood stem cell (PBSC) mobilization after fludarabine treatment of patients with chronic lymphocytic leukaemia (CLL). A total of 101 leucaphereses from 29 patients was performed. The median cell numbers collected were: CD34+ cells, 2.2 × 106/kg (0.1–15.3); granulocyte–macrophage colony‐forming units (GM‐CFU), 4.29 × 104/kg (0.4–177); and mononuclear cells, 6.4 × 108/kg (1.3–63). In univariate and multivariate analyses, the numbers of cells collected were not significantly influenced by the nature of mobilizing regimen and there was a trend towards the collection of a higher number of CD34+ cells from patients who received fludarabine only before mobilization. There was a significant correlation between the median number of CD34+ cells collected and the number of courses of fludarabine (higher CD34+ cell numbers were related to more than six courses) and the interval between the last dose of fludarabine and the start of mobilizing therapy (higher CD34+ cell numbers were related to a delay ≥ 2 months). Sixteen patients have subsequently undergone autologous transplantation and showed rapid engraftment. In conclusion, the results reported favour early stem cell mobilization in CLL patients who are in remission after first‐line therapy. However, attention should be given to the timing of mobilization with respect to the time since the last dose of fludarabine.
Conditioning regimens in autologous stem cell transplantation for multiple myeloma: a comparative study of efficacy and toxicity from the Spanish Registry for Transplantation in Multiple Myeloma
Authors: Lahuerta Juan José , Martinez‐Lopez Joaquín , Grande Carlos , Bladé Joan , Serna Javier De La , Alegre Adrián , García‐Laraña José , Caballero Dolores , Sureda Ana , Rubia Javier De La , Alvarez Ana M. , Marín Julian , Escudero Antonio , Conde Eulogio , Perez‐Equiza Katy , García Ruiz Juan C. , Moraleda José M. , León Angel , Bargay Juan , Cabrera Rafael , Hernandez‐García Miguel T. , Diaz‐Mediavilla Joaquín , Miguel Jesús San
Published: March 2003

High‐dose chemoradiotherapy conditioning regimens for autologous stem cell transplantation (ASCT) are generally held to give similar results in multiple myeloma (MM), but no specific comparative study has been published. We addressed this issue by comparing the main high‐dose chemoradiotherapy regimens used in the Spanish Registry. Patient cohorts included 315 cases treated with 200 mg/m2 melphalan (MEL200), 127 patients with 140 mg/m2 melphalan plus total body irradiation (MEL140 + TBI) and 121 cases with 12 mg/kg busulphan plus 140 mg/m2 melphalan (BUMEL). After ASCT, granulocyte and platelet recovery time was similar in all conditioning groups. There were no differences in transplant‐related mortality. All regimens yielded a similar response in reference to pre‐ASCT MM status, although BUMEL produced a slightly better overall response when compared with the other regimens (97% vs. 89% and 92%, P = 0·003). The 5‐year overall survival (OS) with BUMEL was 47% [95% confidence interval (CI) 26–68] compared with 43% (CI 31–54) for MEL140 + TBI and 37% (CI: 18–56) for MEL200. The median survival for the BUMEL group was 64 months compared with 45 and 37 months for the MEL200 and MEL140 + TBI groups respectively. These differences were non‐significant (P = 0·2). The median event‐free survival (EFS) was better for BUMEL (32 months) than for MEL200 (22 months) or for MEL140 + TBI (20 months). The differences in EFS between BUMEL and the other conditioning regimens reached statistical significance (P = 0·01). Nevertheless, the adjusted multivariate analysis for OS and EFS revealed that the conditioning regimens had no independent prognostic value. We concluded that three different conditioning regimens, commonly used for ASCT in MM, have a similar antimyeloma effect. However, the trend for better results observed in our series with BUMEL requires a prospective trial.
Role of BCL‐2 and cell cycle regulatory proteins for corticosensitivity assessment in childhood acute lymphoblastic leukaemia
Authors: Baghdassarian N. , Bertrand Y. , Ffrench P. , Duhaut P. , Bryon P. A. , Ffrench M.
Published: March 2003

Results of treatment in childhood acute lymphoblastic leukaemia (ALL) remain unsatisfactory because relapses occur even after high‐dose chemotherapy. Corticosensitivity is used in numerous therapeutic trials as a prognostic factor for treatment choice. The aim of this study was to evaluate the role of cell cycle regulatory protein expression before and during the first 48 h of corticotherapy for predicting corticosensitivity. Fifty‐two children presenting with ALL were studied at diagnosis and during the first 48 h of treatment for cell proliferation and apoptosis level by measurement of DNA content, and for expression of several cell proliferation regulatory proteins by means of Western blot. Glucocorticoids induced a significant decrease in the percentage of cells in S‐phase and in CDK1, CDK4 and CDK6 expression and an increase in the percentage of cells in subG1 peak. Two criteria for corticosensitivity were used: (i) the number of blast cells after 7 d of treatment with a threshold at 1 × 109/l (usual criterion), (ii) the J8/J1 blast cell ratio, which is independent from initial leucocytosis. Bcl‐2 expression at diagnosis was the best predictive variable for the usual corticosensitivity criterion in B‐ and T‐cell ALL. For the second criterion, in B‐cell ALL, p21waf1 expression at diagnosis was the sole (albeit poorly) predictive variable, whereas bcl‐2 remained of high interest in T‐cell ALL. Interestingly, these proteins, bcl‐2 and p21waf1, are associated with prolonged cell lifespan and their increased expression is often linked to poor response to cytotoxic drugs. Such preliminary results call for subsequent studies on large independent sets of T‐cell and B‐cell lineage ALL in order to confirm the J8/J1 blast cell ratio value as well as the role of bcl‐2 and p21waf1 expression in predicting corticosensitivity.
Re‐exposure to recombinant (r)‐hirudin in antihirudin antibody‐positive patients with a history of heparin‐induced thrombocytopenia
Authors: Harenberg J. , Huhle G. , Wang L. C. , Hoffmann U. , Song X. H.
Published: March 2003

Patients with a history of heparin‐induced thrombocytopenia (HIT) and antibodies to hirudin were re‐exposed to recombinant (r)‐hirudin for prophylaxis of thromboembolism. Four patients were re‐exposed to 2 × 25 mg of subcutaneous r‐hirudin for 8–27 d. Two patients were re‐exposed once, one patient twice and one four times. Re‐exposure was well tolerated in all patients and no thromboembolism occurred. Antihirudin IgG (4/4 patients), IgA and IgM (1/4 patients) antibody levels increased. Baseline ecarin clotting times showed high variability. Patients with antibodies to hirudin may be re‐exposed but anticoagulant monitoring is mandatory.
In vivo depletion of B cells using a combination of high‐dose cytosine arabinoside/mitoxantrone and rituximab for autografting in patients with non‐Hodgkin's lymphoma
Authors: Voso Maria Teresa , Pantel Gitta , Weis Mirjam , Schmidt Petra , Martin Simona , Moos Marion , Ho Anthony D. , Haas Rainer , Hohaus Stefan
Published: March 2003

We performed a pilot study including rituximab (Mabthera; IDEC‐C2B8, Hoffmann‐La Roche) with a sequential high‐dose therapy protocol in 15 patients with follicular and three patients with mantle cell lymphoma and studied the potential of the chemoimmunotherapy to induce depletion of malignant B cells in vivo. Our treatment protocol included induction with three cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy, followed by peripheral blood stem cell (PBSC) mobilization using high‐dose cytosine arabinoside (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3) (HAM), preceeded by rituximab (375 mg/m2). The proportion of CD19+ B cells in blood and bone marrow decreased from 1·2 ± 0·4% to 0·13 ± 0·1% (P = 0·01) and from 2·7 ± 0·8% to 0·8 ± 0·5% (P = 0·03) respectively. The number of t(14;18)‐positive cells in blood and bone marrow progressively decreased with treatment, as assessed by the quantitative real‐time PCR assay in four patients. Conversion to PCR‐negativity was achieved in the peripheral blood (PB) of seven informative patients. Leucaphereses were performed during the granulocyte colony‐stimulating factor (G‐CSF)‐supported leucocyte recovery phase. In 17 of 18 patients, a median of 15·1 × 106 CD34+ cells/kg body weight (BW) could be harvested by a single procedure for enrichment by an immunomagnetic method. Leucapheresis products contained 51·3 ± 28·8 × 104 CD19+ B cells/kg BW (mean) and were t(14;18) PCR negative in all seven informative patients. These data compare favourably with results obtained in patients treated with the same regimen without rituximab. The high‐dose therapy (n = 12 patients), including total body irradiation (14·4 Gy) and cyclophosphamide (200 mg/kg BW), was also preceeded by rituximab. Recovery of neutrophils to > 0·5 × 109/l and of platelets to > 20 × 109/l required a median of 13·5 and 11·5 d (range 11–24 and 9–24 d) respectively. In conclusion, the addition of the CD20 antibody to chemotherapy ensured tumour depletion in vivo and allowed the collection of PBSCs devoid of tumour cells and with conserved engraftment capability.
JAK2 tyrosine kinase inhibitor tyrphostin AG490 downregulates the mitogen‐activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) pathways and induces apoptosis in myeloma cells
Authors: De Vos John , Jourdan Michel , Tarte Karin , Jasmin Claude , Klein Bernard
Published: March 2003

Cytokines of the interleukin 6 (IL‐6) family, which activates the signal transducer gp130, are major survival and growth factors for human multiple myeloma (MM) cells. The signal transduction of gp130 involves the Janus tyrosine kinases (JAK) JAK1, JAK2 and Tyk2 and then the downstream effectors comprising the signal transducer and activator of transcription 3 (STAT3) and mitogen‐activated protein kinase (MAPK) pathways. We evaluated the effects of the JAK2 inhibitor tyrphostin AG490 on MM cells. We found that AG490 suppressed cell proliferation and induced apoptosis in IL‐6‐dependent MM cell lines. JAK2 kinase activity, ERK2 and STAT3 phosphorylation were inhibited. These results suggest that the chemical blocking of the gp130 signalling pathway at the JAK level could be a relevant therapeutic approach to MM.
Therapy‐related acute myeloid leukaemia after successful therapy for acute promyelocytic leukaemia with t(15;17): a report of two cases and a review of the literature
Authors: Zompi S. , Legrand O. , Bouscary D. , Blanc C. M. , Picard F. , Casadevall N. , Dreyfus F. , Marie J. P. , Viguié F.
Published: March 2003

We describe two patients with positive t(15;17) acute promyelocytic leukaemia (APL) that developed into a therapy‐related myelodysplasia 2–2·5 years after complete remission (CR) and then evolved into therapy‐related acute myeloid leukaemia (t‐AML). Both patients received anthracyclines as potential leukaemogenic drugs. In both cases, cytogenetic changes usually occurring after use of alkylating agents were noticed: monosomy 7 associated with monosomy 5 or 5q– chromosome. A review of the literature on t‐AML occurring after successful therapy for APL showed only one report similar to these two cases. These observations suggest that anthracyclines can cause t‐AML similar to that induced by alkylating agents.
Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM−) and G1948D (CRMr)
Authors: David Dezsö , Saenko Evgueni L. , Santos I. M. Ana , Johnson Daniel J. D. , Tuddenham Edward G. D. , McVey John H. , Kemball‐Cook Geoffrey
Published: March 2003

In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site‐directed mutagenesis of B domain‐deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross‐reacting material (CRM)‐negative (FVIII‐C329S) or mild/moderate CRM‐reduced (FVIII‐G1948D) haemophilia A. Wild‐type (FVIIISQ‐WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild‐type and two variant constructs. In contrast to FVIIISQ‐WT, immunofluorescence analysis of both CRM− and CRMr variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ‐WT. Phenotypic analysis of the B domain‐deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.
High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis
Authors: Jilma‐Stohlawetz P. , Homoncik M. , Jilma B. , Folman C. C. , Von Dem Borne A. E. G. KR. , Bernaschek G. , Deutinger J. , Ulm B. , Eppel W. , Panzer S.
Published: March 2003

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20‐fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0·05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.