bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and functional analysis of the CD33 promoter
Authors: Bodger Michael P. , Hart Derek N. J.
Published: March 2003

CD33 is a leucocyte differentiation antigen restricted to myeloid cells in blood and bone marrow. Two mRNA transcripts encoding CD33 are observed in leukaemic cell lines. The smaller transcript of 1.5 kb is comparable in size to the isolated CD33 cDNA but the origin of the larger 1.8 kb transcript is unknown. To study the regulation of human CD33 expression, a 5′ genomic clone from the CD33 gene was isolated and studied for promoter activity. The clone, although lacking a TATAA box, exhibits other sequences characteristic of a promoter. Two transcriptional start sites were identified, 414 and 527 nucleotides 5′ of the ATG initiation codon, suggesting that these sites are used to generate the 1.8 kb transcript observed in CD33+ cell lines. The CD33 genomic sequence directed high expression of a luciferase reporter gene in myeloid cell lines. Using deletion mutants of the promoter sequence, maximal expression was localized to the first 220 bp 5′ of the ATG initiation codon. Site‐directed mutagenesis of an Sp‐1 and PU.1 binding site within this region showed that the PU.1, but not Sp‐1, was critical for CD33 expression in myeloid lines. Given the restricted expression of CD33 on haemopoietic cells, the identification of the CD33 promoter may be useful for the study of transcription factors that regulate gene expression during early myeloid differentiation.
Leucocyte filterability: comparing diluted with undiluted blood
Authors: Cook Alison M. , Evans Shelley‐Ann , Lane Ian F. , Jones J. Gareth
Published: March 2003

Red blood cells and about 95% of white blood cells have an immediate and constant effect on the flow of undiluted or diluted blood through 5 μm filters. The remaining 5% of all leucocytes exert an increasing influence on flow such that the rate of flow of diluted and undiluted blood through these filters is continually declining over a period of 150 s. Analysis of this declining flow rate enables these cells to be counted and their rheological properties to be deduced. Approximately 50% of these slow leucocytes pass through the filters with a transit time of about 30 s and the remaining cells act as pore blockers for 150 s. The numbers and flow properties of slow leucocytes was approximately the same in blood from young women (25 years) and older men (65 years). However, the number of slow leucocytes was increased in a group of men (65 years) suffering from peripheral arterial occlusive disease. Dilution of the blood with phosphate‐buffered saline increased the numbers of slow leucocytes in both of the older, but not the younger, group of volunteers. This effect was particularly noticeable in the patient group. It is recommended that filtration studies of the rheological profile of leucocytes can, and must, be performed with undiluted blood. The properties after dilution may sometimes, but not invariably, reflect changesex vivoas well as inherent differences in the cells themselves.
Is there a link between African iron overload and the described mutations of the hereditary haemochromatosis gene?
Authors: Mcnamara  , Macphail  , Gordeuk  , Hasstedt  , Rouault
Published: March 2003

Over 80% of Caucasian patients with hereditary haemochromatosis are homozygotes for a C282Y mutation in the HFE gene on chromosome 6. Recent evidence suggests that a genetic factor may also be involved in the pathogenesis of African iron overload, although the locus has not been described. PCR analysis of DNA from 25 southern Africans, identified by segregation analysis as having a high probability of carrying the putative African iron‐loading gene, failed to identify any subjects with the C282Y mutation. The possible genetic defect in African iron overload appears to be different from that described in most cases of hereditary haemochromatosis in Caucasians.
Conservative management of follicular non‐Hodgkin's lymphoma in childhood
Authors: Atra  , Meller  , Stevens  , Hobson  , Grundy  , Carter  , Pinkerton
Published: March 2003

Among 447 children with non‐Hodgkin's lymphoma (NHL) on the childhood U.K. registry, seven children with follicular (NHL) were identified. Four were male and their age ranged from 4.25 to 13.5 years (median 7.5); all had localized disease, Murphy's stage I (n = 4) and II (n = 3). Sites involved at presentation were cervical lymph nodes and tonsils (n = 5), ileum (n = 1) and parotid gland (n = 1). Three had complete surgical excision only and four had complete (n = 1) or incomplete excision (n = 3) followed by a short multi‐agent chemotherapy regimen (UKCCSG 9001 protocol). With a median follow‐up of 1.5 years (range 0.25–5 years) from diagnosis, six are alive in complete remission (CR) including three who had no chemotherapy.

These results confirm previous reports that follicular lymphomas in children are rare (1.5%) and tend to be localized at presentation. Their rarity makes it difficult to produce guidelines about treatment, but in localized cases a period of non‐intervention may be justified.
IL‐4 improves the detection of cytogenetic abnormalities in multiple myeloma and increases the proportion of clonally abnormal metaphases
Authors: HernÁndez  , GutiÉrrez  , Almeida  , GarclÍa  , Sánchez  , Mateo  , Ríos  , San Miguel
Published: March 2003

In the present study the incidence of abnormal karyotypes and the number and proportion of abnormal metaphases obtained in multiple myeloma (MM) using three culture conditions were compared: unstimulated culture (72 h), IL‐6/GM‐CSF‐stimulated culture (120 h) and IL‐4‐stimulated culture (120 h). The three types of culture conditions were assessed simultaneously on bone marrow samples from 30 consecutive myeloma patients. In addition DNA content (DNA ploidy and cell cycle) was analysed by flow cytometry. The number of MM samples with clonal karyotypic abnormalities was higher after IL‐4‐stimulated cultures (53%) than it was after IL‐6 + GM‐CSF (37%) and unstimulated (30%) cultures. The benefit of IL‐4 was also observed in cases with low numbers of plasma cells in the bone marrow, in early clinical stages and in untreated patients. In those cases in whom clonal chromosomal abnormalities were detected by the three culture methods, the cytogenetic findings were always identical. According to our results the addition of IL‐4 to the cultures of bone marrow cells in MM increases the number of abnormal metaphases.
Delayed haemolytic transfusion reaction due to anti‐M antibody
Authors: Sancho Juan Manuel , Pujol Misericordia , FernÁndez Francesc , Soler Manuel , Manzano Pedro , Feliu Evarist
Published: March 2003

A female patient with delayed haemolytic transfusion reaction due to anti‐M antibody is described. Diagnosis was based on laboratory evidence of haemolysis and on characteristic serological findings. Anti‐M was detected in the recipient's serum 7 d after the last transfusion episode. This alloantibody had not been present in the pretransfusion serum. In addition, the direct antiglobulin test was positive on post‐transfusion testing and the implicated antibody was eluted from post‐transfused red cells.

Delayed haemolytic transfusion reactions have long been recognized as a potencial hazard of transfusion therapy, but such cases due to anti‐M are extremely rare.
Establishment of an IL‐2‐dependent cell line derived from ‘nasal‐type’ NK/T‐cell lymphoma of CD2+, sCD3−, CD3ɛ+, CD56+ phenotype and associated with the Epstein‐Barr virus
Authors: Kagami Yoshitoyo , Nakamura Shigeo , Suzuki Ritsurou , Iida Shinsuke , Yatabe Yasushi , Okada Yasutaka , Kobayashi Tomoko , Tsurumi Tatsuya , Seto Masao , Ogura Michinori , Taguchi Osamu , Morishima Yasuo
Published: March 2003

A novel interleukin‐2 (IL‐2)‐dependent cell line, HANK1, was established from a patient with CD56+ NK/T‐cell lymphoma arising in the retroperitoneum. Morphologically, HANK1 is a pleomorphic large cell line with irregular nuclei, which contains azurophilic granules in the cytoplasm. Immunophenotypic analysis showed that HANK1 expressed CD2, CD3ɛ, CD56, TIA‐1, granzyme B, and HLA‐DR, but no other T‐lineage markers. These features were the same as seen in the original tumour, and are highly characteristic of nasal and ‘nasal‐type’ NK/T‐cell lymphoma as described in the proposed W.H.O. classification. Genotypically, this cell line also demonstrated the germline configuration of the T‐cell receptor β, γ and the immunoglobulin heavy chain genes and clonal integration of the Epstein‐Barr virus (EBV) together with antigen expression with a type II latency pattern (LMP‐1+ and EBNA2−). Furthermore, Southern blot analysis using the EBV termini as probes confirmed its derivation from the original lymphoma, and revealed that it contained multiple copies of the EBV genome. Dose‐dependent growth on IL‐2 was observed in an in vitro study with a doubling time of 3 d at maximal stimulation. These data indicate that HANK1 seemed to preserve the biological characteristics of the original tumour and therefore may serve as a good model for the further analysis of unusual ‘nasal‐type’ NK/T‐cell lymphoma.
Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two‐step separation procedure
Authors: Pick Marjorie , Nagler Arnon , Grisaru Dan , Eldor Amiram , Deutsch Varda
Published: March 2003

Cord blood (CB) transplantation is primarily performed in children, rather than in adults, due to the low number of haemopoietic progenitor cells obtained from the small volume of a single CB collection. Prolonged thrombocytopenia is a major problem following CB transplantation. Efforts are currently underway to expand the number of CB progenitor cells ex vivo, in order to enable transplantation in adults and to decrease the period of thrombocytopenia. In this study we investigated different techniques for enrichment and expansion of megakaryocyte (Mk) progenitor cells and haemopoietic stem cells from CB. CBs from 20 normal deliveries were depleted of red blood cells (RBC) by dividing each sample and testing cell separation on 3% gelatin, Hespan, Ficoll‐Paque or a two‐step 3% gelatin followed by Ficoll‐Paque separation. The two‐step procedure was found to be superior to the other methods in enrichment of the Mk progenitor cells (CFU‐Mk) (34.3‐fold), while at the same time retaining the number of myeloid and erythroid progenitors, CD34+ and CD41+ cells. In short‐term (14 d) liquid culture of non‐adherent nucleated cells isolated by gelatin and Ficoll‐Paque, a 40‐fold expansion of clonable Mk progenitor cells was obtained in the presence of thrombopoietin (r‐hu‐TPO) and stem cell factor (r‐hu‐SCF). In similar cultures of isolated CD34+ cells, a 100‐fold clonable Mk progenitor was obtained at day 14. Therefore this new technique may facilitate the ex vivo expansion of Mk progenitor cells and be adopted for future use in CB transplantation.
High prevalence of low bone mass in thalassaemia major
Authors: Jensen  , Tuck  , Agnew  , Koneru  , Morris  , Yardumian  , Prescott  , Hoffbrand  , Wonke
Published: March 2003

Cooley's original description of β‐thalassaemia major included marked bone deformities as a characteristic feature. These were thought to be due to expansion of haemopoiesis attempting to compensate for the congenital anaemia. Regular blood transfusions from infancy prevents these skeletal problems. Nevertheless, symptoms due to bone disease frequently occur in adult patients. Osteoporosis has not previously been reported as a cause of severe morbidity in thalassaemia major. The present study shows a high prevalence of low bone mass among thalassaemia major patients and analyses the predisposing causes. Bone density scans were performed in 82 patients with transfusion‐dependent β thalassaemia. Factors known to be associated with low bone mass such as gender, endocrine disorders and lifestyle activities, together with factors specific to the thalassaemia and its management, were included in a series of univariate analyses to ascertain any significant associations. 42 (51%) of the patients had severely low bone mass and a further 37 (45%) had low bone mass. The three factors showing a statistically significant association with severely low bone mass were male sex, 24/38 (63%) males had severely low bone mass, compared with 18/44 (41%) females, the lack of spontaneous puberty, 22/32 (69%) who required therapeutic induction of pubertal development had severely low bone mass, compared with 19/47 (40%) with spontaneous puberty and diabetes, 8/10 (80%) diabetic patients had severely low bone mass, compared with 23/56 (41%) with normal glucose tolerance. There was no association between the bone mineral density measurements and the haematological characteristics or treatment details of these patients. Severely low and low bone mass are common findings in patients with β‐thalassaemia major despite optimal transfusion and iron chelation. The associated features suggest that the severely low bone mass is due to endocrine abnormalities, in contrast to the haematological causes of bone disease characteristically seen in untreated thalassaemics.
Iron overload in patients with sideroblastic anaemia is not related to the presence of the haemochromatosis Cys282Tyr and His63Asp mutations
Authors: Beris P. , Samii K. , Darbellay R. , Zoumbos N. , Tsoplou P. , Kourakli A. , Preud'Homme C. , Fenaux P.
Published: March 2003

Forty Caucasian patients with primary acquired sideroblastic anaemia (SA), were investigated for the presence of the Cys282Tyr and/or His63Asp mutation as possible cofactor(s) for iron overload. One patient was heterozygous for the Cys282Tyr mutation and 13 heterozygotes and one homozygote for the His63Asp mutation were found (no difference compared with controls). SA patients with normal codon 63 had a mean ferritin level of 923 ± 815 μg/l whereas those with codon 63 mutation had 769 ± 577 μg/l (P = 0.64). We conclude that ineffective erythropoiesis with no associated mutation in the HFE gene can lead to iron overload in SA patients.
Pharmacokinetic dose adjustment of Erwinia asparaginase in protocol II of the paediatric ALL/NHL‐BFM treatment protocols
Authors: Vieira Pinheiro J. P. , Ahlke E. , Nowak‐Göttl U. , Hempel G. , Müller H. J. , Lümkemann K. , Schrappe M. , Rath B. , Fleischhack G. , Mann G. , Boos J.
Published: March 2003

Native forms of asparaginase stem from different biological sources. Previously reported data from children treated with ErwinaseTM showed significantly lower trough levels and pharmacokinetic dose intensity than after E. coli‐derived preparations. Hence, schedule optimization was initiated to achieve relevant serum activities. 21 children on reinduction therapy received Erwinase on Mondays, Wednesdays and Fridays for 3 weeks (9 × 20 000 IU/m2 i.v.) instead of 4 × 10 000 IU/m2 of E. coli asparaginase (twice weekly for 2 weeks). Asparaginase trough activities were measured as the primary parameter, targeting 100–200 IU/l after 2 d and >50 IU/l after 3 d. Concurrently, asparagine trough concentrations were monitored. The mean trough activity was 156 ± 99 IU/l, with 2/108 samples showing no detectable activity. Regarding trough levels per individual (three or more measurements/patient), means ranged from 52 ± 29 to 276 ± 114 IU/l (20 patients, 106 samples), with nine, six, and five children inside, below, and above the target range, respectively. The mean 3 d trough activity was 50 ± 39 IU/l (20 patients, 51 samples). In 11 of these samples no activity was measurable. Mean trough activities calculated per individual ranged from < 20–84 ± 30 IU/l (14 patients, 42 samples) with seven children below the target limit of 50 IU/l and asparagine concentrations < 0.2–1.5 μm. We concluded that an increased dose of 9 × 20 000 IU/m2 of Erwinia asparaginase within 3 weeks resulted in a pharmacokinetic dose intensity comparable to former observations made with 4 × 10 000 IU/m2 of the E. coli product CrasnitinTM which is no longer marketed. High interindividual variability and the phenomenon of ‘silent’ inactivation necessitate monitoring wherever possible.
Expression of vascular endothelial growth factor in sera and lymph nodes of the plasma cell type of Castleman's disease
Authors: NISHI JUN‐ICHIRO , ARIMURA KIMIYOSHI , UTSUNOMIYA ATAE , YONEZAWA SUGURU , KAWAKAMI KIYOSHI , MAENO NOBUAKI , IJICHI OSAMU , IKARIMOTO NAOAKI , NAKATA MASANORI , KITAJIMA ISAO , FUKUSHIGE TAKAHIKO , TAKAMATSU HIDEO , MIYATA KOICHIRO , MARUYAMA IKURO
Published: March 2003

To evaluate the possible involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of Castleman's disease, we studied VEGF levels in sera and supernatants of cultured lymph nodes from two patients with the plasma cell type of Castleman's disease, and analysed the expression of VEGF immunohistochemically in the lymph nodes. Clinically, one patient was classified as the localized type and the other as the multicentric type. Histologically, mature plasma cells and hyalinized vessels were prominent in the interfollicular region. The VEGF levels of the sera and the supernatants of cultured lymph nodes of both patients were higher than those of normal controls. VEGF was strongly expressed in plasma cells in the interfollicular region of the lymph nodes of both patients, but rarely in normal lymph nodes. Our results suggest that VEGF may be involved in the marked vascular proliferation in the interfollicular region of the lymph nodes of the plasma cell type of Castleman's disease.
Thromboxane‐dependent platelet activation in vivo precedes arterial thrombosis in thrombocythaemia: a rationale for the use of low‐dose aspirin as an antithrombotic agent
Authors: VAN GENDEREN PERRY J. J. , PRINS FRANSISCO J. , MICHIELS JAN J. , SCHRÖR KARSTEN
Published: March 2003

The clinical course of patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) is frequently complicated by arterial thrombotic events. The pathogenesis is not clearly understood but attributed to abnormalities in platelet function. An increase in platelet thromboxane formation has been described in the majority of asymptomatic patients with thrombocythaemia, probably reflecting spontaneous platelet activation in vivo. In the present study we prospectively investigated whether an increase in platelet thromboxane formation actually precedes arterial microvascular thrombosis. In addition, we studied the effect of selective inhibition of platelet thromboxane formation on clinical outcome by reinstitution of low‐dose aspirin (50 mg/d). Six ET patients and one PV patient participated in this study. Within 10 d after withdrawal of aspirin, three patients developed arterial microvascular thrombosis of extremities (erythromelalgia), which was preceded by a 3–30‐fold increase in urinary thromboxane excretion as compared with patients who remained asymptomatic. The increased urinary thromboxane excretion and clinical signs could be inhibited by a platelet‐specific aspirin regimen of 50 mg/d without affecting vascular cyclooxygenase, indicating that platelets were the main source of the increased thromboxane generation. These data suggest that in symptomatic patients an enhanced formation of thromboxane by platelets, reflecting platelet activation in vivo, precedes the development of arterial microvascular thrombosis. These data provide a rationale for using low‐dose aspirin as an antithrombotic agent in thrombocythaemia.
A rapid method for haemophilia B mutation detection using conformation sensitive gel electrophoresis
Authors: Hinks J. L. , Winship P. R. , Makris M. , Preston F. E. , Peake I. R. , Goodeve A. C.
Published: March 2003

Conformation sensitive gel electrophoresis (CSGE) was confirmed as an effective procedure for screening the factor IX (FIX) gene by detecting 10/10 previously known FIX gene mutations. The FIX genes of a further 11 haemophilia B patients with unknown mutations were then screened and an abnormal CSGE profile was identified in all cases. Subsequent DNA sequencing demonstrated one of these to be a novel mutation (31133insT, Arg338Fs), the remaining 10 having been previously reported on the haemophilia B database. Mutation screening of the FIX gene using CSGE was demonstrated to be a rapid and efficient means of carrier analysis in families with haemophilia B.
Fludarabine‐containing regimens severely impair peripheral blood stem cells mobilization and collection in acute myeloid leukaemia patients
Authors: Visani G. , Lemoli R. M. , Tosi P. , Martinelli G. , Testoni N. , Ricci P. , Piccaluga P. P. , Pastano R. , Leopardi G. , Dizdari A. , Motta M. R. , Rizzi S. , Tura S.
Published: March 2003

We studied the effects of an intensified induction/consolidation treatment containing fludarabine (ICE/FLAN/FLAN) on the mobilization and collection of peripheral blood stem cells (PBSC) in 31 consecutive untreated acute myeloid leukaemia (AML) patients. The complete remission (CR) rate was comparable to classic inductions (68% after ICE; 84% after ICE‐FLAN I). To mobilize PBSC, 19 patients received 10 μg/kg/d of granulocyte‐colony stimulating factor (G‐CSF) starting at day 13 after FLAN, 13 (69%) of whom were found to be nonmobilizers. When a second G‐CSF administration was performed in 10/13 patients mobilization was either not achieved (8/10) or was considered insufficient (<1 × 106 CD34+ cells/kg) (2/10) and all 13 were subsequently submitted to bone marrow harvest. The harvest was considered adequate in 12/13 (92%) patients and autologous BMT (ABMT) has so far been performed in 10/12 cases with a mean of 8.6 × 108/kg nucleated reinfused cells. The median times to neutrophil and platelet recovery after ABMT did not significantly differ from those of two previous series of patients treated with ABMT without fludarabine‐containing regimens. Adequate amounts of PBSC were obtained in 6/19 (31%) patients, who were then reinfused. Median times for platelet recovery were significantly longer than in a previous series of 26 AML cases reinfused with PBSC after treatment with the ICE‐NOVIA induction/consolidation regimen (125 v 20 d to 20 × 109 plt/l, P < 0.02; 218 v 37 d to 50 × 109 plt/l, P < 0.02). In addition, times for platelet recovery after ICE/FLAN/FLAN were not significantly different from those in a previous group treated with ABMT performed after ICE/NOVIA,without fludarabine. We conclude that fludarabine‐containing regimens severely impair mobilization and collection of PBSC in AML patients and seem unsuitable when PBSC autotransplantation is programmed.
Serum G‐CSF levels are not increased in patients with antibody‐induced neutropenia unless they are suffering from infectious diseases
Authors: Bux Juergen , Hofmann Christine , Welte Karl
Published: March 2003

By the use of a G‐CSF‐specific ELISA we determined the serum granulocyte‐colony stimulating factor (G‐CSF) levels in 63 patients with antibody‐induced neutropenia including neonatal immune neutropenia, autoimmune neutropenia, and drug‐induced immune neutropenia. In the sera of 20 patients, elevated G‐CSF levels of 60–1006 pg/ml (normal <39 pg/ml) were observed. These patients suffered from infectious diseases at the time of blood collection. G‐CSF levels normalized after successful antibiotic treatment, indicating that increased G‐CSF production in patients with immune neutropenia may be primarily the result of infection and not of neutropenia.
Enhanced circulatory parameters of human platelets cryopreserved with second‐messenger effectors: an in vivo study of 16 volunteer platelet donors
Authors: Currie Laura M. , Lichtiger Benjamin , Livesey Stephen A. , Tansey Wayne , Yang David J. , Connor Jerome
Published: March 2003

Platelet transfusion represents an important component of the therapy for thrombocytopenic patients. Prolonged storage capabilities for platelets would alleviate many problems associated with blood banking. Unfortunately, current cryopreservation methods are complex to implement and result in loss of cell number and functional activity. Previous in vitro studies have shown that the use of ThromboSolTM, a platelet‐stabilizing formulation, in the cryopreservation of platelets results in significant retention of cell number and in vitro functional activities in addition to reducing the DMSO requirement to only 2%. We evaluated the in vivo circulatory parameters of platelets cryopreserved with ThromboSol. Single donor platelet units were obtained from healthy volunteers (n = 16); the units were then split and cryopreserved with either ThromboSol and 2% DMSO or 6% DMSO alone. Following storage at −80°C for 7–10 d the samples were thawed, washed and radiolabelled with either 51Cr or 111In. The paired samples were then mixed and reinfused into the autologous volunteer. At various time intervals following transfusion a blood sample was drawn and the quantity of circulating labelled platelets was determined. The percent recovery and survival time was determined by multiple‐hit analysis. The ThromboSol‐treated platelets, as compared to the 6% DMSO‐treated platelets, displayed statistically higher percent recovery (40.2% v 28.8%) and survival time (166.3 h v 152.1 h). These results demonstrated that platelets cryopreserved with ThromboSol displayed superior in vitro and in vivo characteristics as compared to the standard 6% DMSO method. The use of ThromboSol allowed for a 3‐fold reduction in the DMSO concentration in conjunction with a 40% increase in circulating cell number and normal survival times.
Syngeneic stem cell transplantation for HIV‐related lymphoma
Authors: Campbell P. , Iland H. , Gibson J. , Joshua D.
Published: March 2003

The treatment of human immunodeficiency virus (HIV)‐related lymphoma is beset by a number of therapeutic limitations. High‐dose chemotherapy followed by peripheral blood stem cell transplantation (PBSCT) for relapsed disease is one option, but may be compromised by unacceptable treatment‐related morbidity and mortality. We describe an HIV‐positive male with relapsed immunoblastic non‐Hodgkin's lymphoma (NHL) who successfully received salvage chemotherapy followed by a syngeneic PBSCT from his HIV‐negative (hepatitis C positive) brother. At 15 months post‐transplant he remains in complete remission with low‐level HIV viral load, an improved CD4 lymphocyte count and absent anti‐hepatitis C antibodies. We believe selected patients with relapsed HIV‐related NHL should be considered for high‐dose therapy.
Human dendritic cells express the thrombopoietin receptor, c‐Mpl
Authors: Kumamoto Tadashi , Azuma Eiichi , Tanaka Minoru , Qi Jiang , Hirayama Masahiro , Zhang Shao‐Li , Kobayashi Michihiro , Iwamoto Shotaro , Komada Yoshihiro , Yamamoto Hatsumi , Nakashima Kunio , Sakurai Minoru
Published: March 2003

Human thrombopoietin (TPO) is a haemopoietic growth factor that is essential for the growth and development of megakaryocytes. However, c‐Mpl, the TPO receptor, has been detected in human leukaemic cell lines with a myelomonocytic phenotype. These results raise the possibility that dendritic cells (DC), a putative myeloid lineage cell, may also express c‐Mpl and respond to TPO. In haemopoietic stem cell transplantation, DC could induce graft‐versus‐host disease by its strong antigen‐presenting capacity. In this study we have examined the effect of TPO on differentiation and the antigen‐presenting capacity of DC. To differentiate DC, cord blood CD34+ cells were cultured in the presence of a cytokine cocktail either in serum‐free medium or RPMI1640 containing 10% fetal bovine serum. Flow cytometric analysis and immunocytochemical staining demonstrated that c‐Mpl was expressed on DC. Furthermore, the expression of c‐Mpl mRNA was detected in DC by RNase protection assay. However, when TPO was added to the culture system there were no significant changes in the differentiation and mixed leucocyte‐stimulating capacity of DC. These findings suggest that TPO may be administered following cord blood transplantation without significant augmentation of antigen presentation mediated by DC.
Two British families with variants of the ‘cryohydrocytosis’ form of hereditary stomatocytosis
Authors: Coles Suzanne E. , Chetty Margaret C. , Ho Mei M. , Nicolaou Anna , Kearney J. W. , Wright Stephen D. , Stewart Gordon W.
Published: March 2003

We describe two British families with similar, dominantly‐inherited, temperature‐related variants of hereditary stomatocytosis, consistent with the original description of ‘cryohydrocytosis’. The cells show a 5–6‐fold increase in passive permeability at 37°C with abnormal intracellular Na and K levels at 15–20 and 60–65 mmol/(l cells) respectively. Marked temperature effects were evident: lysis of red cells on storage in the cold was blatant and when whole heparinized blood was stored at room temperature, K accumulated in the plasma, producing ‘pseudohyperkalaemia’. Studies of the temperature dependence of passive permeability showed that the minimum in the passive permeability, which is seen in normal cells at 8–10°C, was shifted up to 23°C in these abnormal cells, such that the permeability at 0°C exceeded that at 37°C. The abnormal temperature dependence in these genetically abnormal red cells strongly resembles that seen in normal cells when suspended in media in which either Na or Cl has been replaced by an organic cation or anion: it could be said these cells had a genetic mutation that somehow rendered the cell resistant to the stabilizing action of NaCl at low temperatures.
Evidence for a continuous decline in haemopoietic cell function from birth: application to evaluating bone marrow failure in children
Authors: Marley  , Lewis  , Davidson  , Roberts  , Dokal  , Goldman  , Gordon
Published: March 2003

There are considerable differences in haemopoietic activity between young children and adults on the one hand, and between adults and the elderly on the other. A fundamental unanswered question is whether these differences relate to discrete stages or are part of a continuous process. We have sought to define aspects of the haematological ageing process, and have found that results from children with bone marrow failure syndromes differ from age‐matched reference values. Haemopoietic cells were obtained from umbilical cord blood, from blood and bone marrow of healthy individuals and from the blood of young patients with bone marrow failure syndromes. Clonogenic myeloid progenitors (CFU‐GM) were grown in semi‐solid medium to measure their frequency; the proliferative capacity of myeloid progenitors was measured by replating colonies and observing secondary colony formation. We found that the frequency of CFU‐GM in normal marrow increased and their proliferative capacity decreased exponentially with age. The proliferative capacity of CFU‐GM in normal blood also decreased exponentially with age. This relationship extrapolated back to the levels of proliferation measured for cord blood CFU‐GM (age = 0). The proliferative capacities of CFU‐GM from children with bone marrow failure syndromes were severely reduced compared with age‐matched reference values. These results indicate that a decline in haemopoietic progenitor cell function begins at birth and continues throughout life. This decline may occur prematurely in childhood marrow failure syndromes with a predisposition to leukaemia.
Reproductive choices of haemophilia carriers
Authors: Tedgård U. , Ljung R. , McNeil T. F.
Published: March 2003

The actual reproductive choices made by slightly over a quarter of all the carriers of severe or moderate haemophilia in Sweden were investigated and compared with those of a randomly selected age‐matched group of women who were not carriers of haemophilia. In general, the 105 carriers had the same number of children as other women of similar age. However, carriers who did not choose prenatal diagnosis (PD) often abstained from further pregnancies after the birth of a haemophilic child, and they had significantly fewer children than the remainder of the carriers, as well as fewer children than women in the control group. Logistic regression analysis showed choice of PD to be correlated to a positive attitude towards abortion following PD and a family history of haemophilia. Carriers who have experienced the complications of haemophilia or its treatment appear to be more in favour of PD than women whose haemophilic children have received modern treatment without complications.
A prospective study of routine antenatal enzyme antibody screening demonstrates lack of clinical value in predicting haemolytic disease of the newborn
Authors: Clark D. , Greiss M. A. , Urbaniak S. J.
Published: March 2003

A prospective study of 7065 consecutive new pregnancies identified 230 with a positive screen, of which 27% (62/230) were ‘enzyme‐only’ antibodies. 32 of these (52%) were potentially clinically important and were all of Rh specificity: 22 anti‐E, seven anti‐Cw, two anti‐D and one anti‐c. However, only three of these enzyme‐only antibodies (one anti‐D, one anti‐c and one anti‐E) became reactive by the indirect antiglobulin test (IAT) during the course of pregnancy, and all were detected in the routine 34–36‐week maternal sample. No babies were affected, and we reaffirm that routine antibody screening by enzyme techniques is unnecessary.
Telomerase activity in chronic lymphoproliferative disorders of B‐cell lineage
Authors: Trentin Livio , Ballon Gianna , Ometto Lucia , Perin Alessandra , Basso Umberto , Chieco‐Bianchi Luigi , Semenzato Gianpietro , De Rossi Anita
Published: March 2003

The progressive shortening of telomeres at each cell division is a key mechanism in controlling cell proliferative capacity. The activation of telomerase, a reverse transcriptase that extends telomere length, potentially leads to unlimited cell proliferation, and is believed to play a critical role in the neoplastic process. High levels of telomerase activity have been demonstrated in almost all solid tumours; however, little data is available concerning its expression in chronic B‐cell neoplasms. By using a quantitative polymerase chain reaction‐based method we quantified telomerase activity in normal B lymphocytes, and in various B‐cell malignancies, including chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL) and hairy cell leukaemia (HCL). Compared to normal B cells, which expressed very low levels of telomerase activity, malignant cells from most of the patients showed a significant increase in telomerase activity, with highest values observed in HCL samples. Moreover, among the CLL and HCL cases, significantly higher levels of telomerase activity were found in patients with progressive disease at 1 year follow‐up versus patients with stable disease. These data suggest that telomerase activity might correlate with disease progression.
Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene
Authors: Ghilardi Nico , Wiestner Adrian , Kikuchi Masahiro , Ohsaka Akimichi , Skoda Radek C.
Published: March 2003

Hereditary thrombocythaemia (HT) with clinical features very similar to essential thrombocythaemia (ET) has been found to be transmitted as an autosomal dominant trait in several families. Here we studied the pathogenesis of HT in a previously described Japanese kindred. We found markedly elevated thrombopoietin (TPO) serum levels in all affected individuals and identified a novel point mutation in the TPO gene, a G → T transversion at position 516 of the TPO mRNA (G516T) that co‐segregated with the HT phenotype in all affected family members. This mutation is located in the 5′‐untranslated region (5′‐UTR) of the TPO mRNA and when assayed in reticulocyte lysates, improved translational efficiency of in vitro transcribed TPO mRNA. Cell lines transfected with the mutant TPO cDNA secreted up to 8‐fold more TPO protein than cells transfected with the normal cDNA. We provide a molecular model of how the mutation partially disables the physiologic repression of TPO translation and thereby causes thrombocytosis. This is the third family in which HT has been caused by the loss of translational inhibition of TPO mRNA.
Propagation and senescence of human marrow stromal cells in culture: a simple colony‐forming assay identifies samples with the greatest potential to propagate and differentiate
Authors: DiGirolamo Carla M. , Stokes David , Colter David , Phinney Donald G. , Class Reiner , Prockop Darwin J.
Published: March 2003

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony‐forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony‐forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.
Further characterization of antibody and antigen in heparin‐induced thrombocytopenia
Authors: Newman Peter M. , Chong Beng H.
Published: March 2003

Patients with immune heparin‐induced thrombocytopenia (HIT) possess antibodies that bind to a complex of platelet factor 4 (PF4) and heparin. We observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity‐purify anti‐PF4‐heparin HIT IgG, we are able to provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4‐heparin (Kd = 7–30 nm) and polystyrene adsorbed PF4 alone (Kd = 20–70 nm). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti‐PF4‐heparin antibodies. We conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4‐heparin and therefore most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (suboptimal) promotes recognition of this epitope.
Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B‐cell precursor acute lymphoblastic leukaemia
Authors: Baersch  , Baumann  , Ritter  , Jürgens  , Vormoor
Published: March 2003

To identify residual candidate normal progenitor/stem cell populations in childhood B‐cell precursor acute lymphoblastic leukaemia (ALL), expression of AC133 and CD117 was analysed on the leukaemic cell clone and on immature B‐lineage‐negative CD34+CD19− bone marrow cells. 10/25 patients (40%) had no detectable expression of AC133 within the leukaemic cell clone. 24/26 patients (92%) lacked expression of CD117 on the leukaemic blast cell population. In contrast, a distinct AC133‐positive cell population was found in 8/8 children with AC133‐negative ALL and a CD117‐positive cell population could be identified in 12/12 children with CD117‐negative ALL, within the CD34+CD19− progenitor/stem cell compartment. These observations provide further evidence that in B‐cell precursor ALL, unlike in acute myelogenous leukaemia, it may be possible to distinguish residual normal progenitor/stem cells from the leukaemic cell clone.
Molecular heterogeneity of the NUP98/HOXA9 fusion transcript in myelodysplastic syndromes associated with t(7;11)(p15;p15)
Authors: Hatano Yoshiaki , Miura Ikuo , Nakamura Takuro , Yamazaki Yukari , Takahashi Naoto , Miura Akira B.
Published: March 2003

The reciprocal translocation t(7;11)(p15;p15) has been reported as occurring mainly in acute myelogenous leukaemia (AML) and the acute phase of chronic myelogenous leukaemia (CML). This translocation in AML involves both the nucleoporin gene NUP98 on 11p15 and the homeobox gene HOXA9 on 7p15. The invariant chimaeric NUP98/HOXA9 transcripts are a result of the fact that each breakpoint of the NUP98 and the corresponding breakpoint of the HOXA9 gene cluster occur within the same intron. Only one patient with myelodysplastic syndromes (MDS) carrying this chromosome aberration has been reported, but this study did not involve molecular analysis. We describe two patients with MDS associated with t(7;11): patient 1 was a Japanese man diagnosed with chronic myelomonocytic leukaemia; patient 2 was a Japanese woman with refractory anaemia with excess of blasts in transformation. Within a year both patients developed AML and showed multidrug resistance to chemotherapy. Southern blot analysis showed rearrangements of the NUP98 gene of the two patients and the HOXA9 gene of patient 2. Patient 1 had two types of the novel NUP98/HOXA9 fusion transcripts. Each of them lacked the common 141 bp NUP98 exon which was contained in the NUP98/HOXA9 fusion transcripts detected in patient 2 and the reported AML cases. These data indicated that t(7;11) could determine the development of various myeloid leukaemias and that the resultant chimaeric transcripts are heterogenous.
Three months supplementation of hyperhomocysteinaemic patients with folic acid and vitamin B6 improves biological markers of endothelial dysfunction
Authors: Constans Joël , Blann Andrew D. , Resplandy François , Parrot Françoise , Renard Martine , Seigneur Martine , Guérin Viviane , Boisseau Michel , Conri Claude
Published: March 2003

Hyperhomocysteinaemia is a risk factor for premature atherosclerosis and venous thromboembolic disease. Supplementation with folic acid and vitamin B6 has been shown to decrease plasma homocysteine but data fail to assess an effect on the progression of vascular disease. We measured plasma homocysteine and two markers of endothelial injury (plasma soluble thrombomodulin and von Willebrand factor) at baseline and after 3 months of treatment with folic acid and vitamin B6. After this treatment there was a significant decrease in fasting soluble thrombomodulin (−15 ng/ml, 95%CI 5–22.2). Von Willebrand factor was significantly raised after methionine load at baseline but did not significantly rise after supplementation.
Diagnosis of invasive aspergillosis in bone marrow transplant recipients by polymerase chain reaction
Authors: Williamson Emma C. M. , Leeming John P. , Palmer Helen M. , Steward Colin G. , Warnock David , Marks David I. , Millar Michael R.
Published: March 2003

A nested polymerase chain reaction (PCR) test targeting Aspergillus spp. large ribosomal subunit genes was evaluated retrospectively on 175 serum samples from 37 bone marrow transplant recipients, 70% of whom received grafts from unrelated donors. Six patients had proven infection, seven had probable infection, and three had possible infection, using the revised EORTC case definitions. These 16 patients were all PCR positive (57 out of 93 samples tested). Two additional patients who did not fulfil current diagnostic criteria, but in whom invasive aspergillosis (IA) was thought clinically probable, were also PCR positive (five out of nine samples). Invasive aspergillosis was unlikely in the remaining 19 patients, four of whom were PCR positive on a single occasion (four out of 70 samples). Three samples were inhibitory to PCR. Sensitivity of PCR in diagnosing patients with IA was 100%, specificity was 79% and positive predictive value was 80%, using the criterion of a single positive result. If two positive results were required, these values were 81%, 100% and 100% respectively. The median duration of infection documented by PCR was 36 days (range 3–248 days) in 17 out of 18 patients (94%) who did not survive. Positive PCR results predated the institution of antifungal therapy in two‐thirds of patients. Four patients became PCR positive during pretransplant conditioning therapy.
Heightened proteolysis of the von Willebrand factor subunit in patients with von Willebrand disease hemizygous or homozygous for the C2362F mutation
Authors: Castaman Giancarlo , Eikenboom Jeroen C. J. , Lattuada Antonella , Mannucci Pier Mannuccio , Rodeghiero Francesco
Published: March 2003

We studied the proteolytic pattern of the mutant von Willebrand factor (VWF) in four patients with von Willebrand disease (VWD) who were either homozygous or hemizygous for the mutation C2362F. A significant decrease in the native fragment of 225 kDa was evident in all the patients, together with a marked increase in the 176 and 140 kDa fragments, a pattern usually observed in type 2A VWD. The proteolytic pattern measured in four heterozygotes for C2362F was within the normal range, suggesting that the mutant VWF C2362F present in the plasma of these patients may be protected from proteolysis by normal VWF.
Type 1 von Willebrand disease — a clinical retrospective study of the diagnosis, the influence of the ABO blood group and the role of the bleeding history
Authors: Nitu‐Whalley  , Lee  , Griffioen  , Jenkins  , Pasi
Published: March 2003

This clinical retrospective study investigated the difficulties in diagnosing type 1 von Willebrand disease (VWD). A total of 246 patients previously diagnosed with type 1 VWD were reclassified into ‘possible’ type 1 VWD (patients with low levels of VWF adjusted for the blood group and either a significant bleeding history or family history) and ‘definite’ type 1 VWD, requiring low levels of von Willebrand factor (VWF), a bleeding history and inheritance. On reclassification, only 144/246 (59%) patients had low VWF levels adjusted for blood group, 88/246 (36%) patients met all the criteria for ‘definite’ type 1 VWD and 51/246 (21%) patients were ‘possible’ type 1 VWD. A significant proportion of patients, 102/246 (42%), remained an indeterminate group with blood type O, VWF levels between 35 and 50 U/dl and personal and/or family bleeding history. This subgroup might require reclassification as ‘not VWD’. However, a similar bleeding tendency was found in two matched groups of patients of blood groups O and non‐O and VWF levels between 35 and 50 U/dl. These results suggest that the use of ABO adjusted ranges for VWF levels might not be essential for diagnosis, because bleeding symptoms may depend on the VWF level regardless of the ABO type. Of the diagnostic criteria, the bleeding history was of prime importance in the clinical decision to diagnose and treat type 1 VWD. These observations could help in the reconsideration of how the criteria for diagnosing type 1 VWD could be adjusted in order to maximize their clinical relevance.