bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary prophylaxis in severe haemophilia should be started at an early age but can be individualized
Authors: Astermark Jan , Petrini Pia , Tengborn Lilian , Schulman Sam , Ljung Rolf , Berntorp Erik
Published: March 2003

The frequency of joint bleeds and orthopaedic joint scores were evaluated in 121 patients with severe haemophilia who had started prophylactic treatment with clotting factor concentrates at least once weekly before the age of 10. 75 of the patients started before the age of 3, 31 at the age of 3–5 and 15 at the age of 6–9. Each subgroup was evaluated separately. In addition, a regimen of one infusion weekly was compared with that of two (haemophilia B) or three (haemophilia A) infusions weekly in each patient.

A significant decrease in the overall number of joint bleeds per year was found after shortening the infusion interval (P < 0.005), but the individual bleeding pattern varied. In survival analysis of the first pathologic joint score event, those who started prophylaxis before the age of 3 had a better outcome overall than those starting at later ages (P = 0.001). However, in subgroup analysis, no significant difference was seen in the annual number of joint bleeds and the development of arthropathy between those starting with, or shifting to, the more intensive regimen before the age of 3 and those that were put on this regimen at the age of 3–5. Age at start of prophylaxis was found to be an independent predictor for the development of arthropathy (P = 0.0002), whereas dose and infusion interval at start were not.

Our data emphasize the importance of starting replacement therapy during the first years of life. However, it seems that when beginning the regimen it can be individualized and adjusted according to the bleeding pattern. In this way, the need for a venous access system may be assessed on an individual basis.
Involvement of the CD27‐CD70 co‐stimulatory pathway in allogeneic T‐cell response to follicular lymphoma cells
Authors: Schmitter Doris , Bolliger Ursula , Hallek Michael , Pichert Gabriella
Published: March 2003

Non‐activated follicular lymphoma (FL) cells do not function as effective antigen‐presenting cells in vivo. CD40 activation of FL cells up‐regulates critical adhesion and co‐stimulatory molecules, thereby inducing lymphoma‐specific cytotoxic T cells in vitro. However, other evidence suggests that CD70 is another important co‐stimulatory molecule involved in antigen dependent T‐cell activation. Here, we showed that freshly isolated FL cells from eight diagnostic biopsies expressed intermediate to high levels of CD27, whereas only insignificant levels of CD70 were detected on their cell surface. Together with the low to intermediate expression of B7, these findings help to explain the poor antigen‐presenting capacity of non‐activated FL cells. Activation of FL cells by CD27 and CD40 induced a significantly higher alloantigen T‐cell response than CD40 alone, whereas CD27 activation induced only a mostly insignificant T‐cell proliferation. Both CD40 and CD27 + CD40 activation resulted in a high up‐regulation of CD70 and B7 molecules, whereas CD27 activation up‐regulated CD70 but not B7 expression on the surface of FL cells. Thus, expression of both CD70 and B7 co‐stimulatory molecules appears to be essential for an efficient T‐cell‐mediated immune response. However, the molecules responsible for the significantly higher alloantigen T‐cell response to FL cells activated through CD27 and CD40 remain to be identified. In summary, we conclude that CD27+CD70 represents another co‐stimulatory pathway involved in T‐cell‐mediated immune responses to FL cells. Our findings suggest that several co‐stimulatory pathways exist and should be taken into consideration to optimize antigen presentation for the generation of lymphoma‐directed cytotoxic T cells for adoptive immunotherapy.
Altered drug sensitivity in response to idarubicin treatment in K562 human leukaemia cells
Authors: Locke Vicki L. , Davey Ross A. , Davey Mary W.
Published: March 2003

Relative to the commonly used anthracyclines, little is known about idarubicin and the development of multidrug resistance. We have previously shown the K562/IDA subline resulting from intermittent treatment of the K562 human leukaemia cell line with 20 ng/ml idarubicin did not develop multidrug resistance but became more sensitive to etoposide. Additional similar treatments of this subline produced the K562/IDA20 subline which partially retained its etoposide sensitivity although these cells expressed P‐glycoprotein and were resistant to paclitaxel. Sensitization to etoposide was associated with increased decatenation activity of topoisomerase II, although there were no changes in topoisomerase IIα expression or formation of etoposide‐dependent cleavable complexes. In comparison, the K562/IDA10 subline produced by intermittent treatment of the K562 cells, firstly with 5 ng/ml then 10 ng/ml idarubicin, showed no detectable expression of P‐glycoprotein, decreased topoisomerase IIα expression and increased resistance to etoposide and amsacrine, but not to idarubicin or genistein. Even though intermittent treatment with idarubicin caused increased drug resistance in both sublines, they remained sensitive to idarubicin. Therefore the potential of idarubicin as a substitute for other anthracyclines in the treatment of cancer warrants further investigation.
Interleukin‐15 blocks apoptosis and induces proliferation of the human myeloma cell line OH‐2 and freshly isolated myeloma cells
Authors: Hjorth‐Hansen Henrik , Waage Anders , Börset Magne
Published: March 2003

The growth factor‐dependent myeloma cell line OH‐2, which has previously been shown to be responsive to interleukin (IL)‐6, tumour necrosis factor (TNF)‐α and lymphotoxin, was examined for response to other growth factors. Enhanced proliferation was found in the presence of IL‐10, IL‐15, IL‐2 and insulin growth factor (IGF)‐1. Proliferation was strongest in response to IL‐6, intermediate and roughly equipotent in response to IL‐15, IL‐10 and TNF‐α, and modest in response to IL‐2 and IGF‐1. IL‐15 was synergistic with TNF‐α, whereas combinations of IL‐15 and the other cytokines were merely additive. IL‐15‐induced proliferation could not be blocked by neutralizing antibody against gp 130, the common transducer chain of IL‐6 and related cytokines. IL‐15 and IL‐6 prevented apoptosis equally well, both better than TNF‐α, IL‐10, and IGF‐1. In four out of six samples of purified primary cells, IL‐15 and IL‐6 induced proliferation. Furthermore, IL‐15 mRNA was detected by RT‐PCR in most myeloma cell lines and freshly isolated purified patient samples. IL‐15 protein was detectable only in one out of about 20 tested cell supernatants from patients and myeloma cell lines. The OH‐2 cell line is multi‐responsive to cytokines and is a good system for the study of integration of cytokine signal transduction and growth control in myeloma. IL‐15 represents a novel modality of growth regulation in myeloma.
Gain of chromosome 7, which marks the progression from indolent to aggressive follicle centre lymphomas, is restricted to the B‐lymphoid cell lineage: a study by FISH in combination with morphology and immunocytochemistry
Authors: Bernell P. , Arvidsson I. , Jacobsson B.
Published: March 2003

In a previous fluorescent in situ hybridization (FISH) study of patients with high‐grade follicle centre lymphomas (FLCs), we often found additional copies of chromosome 7 in bone marrow (BM) cell nuclei even though obvious malignant tumour cells could not always be morphologically identified in the corresponding cell smears. This raised the question whether the gains of chromosome 7 are really confined to B‐lymphoid tumour cells or whether other cell lineages are also of clonal origin. In the present investigation we employed FISH in combination with immunomarkers and morphological studies on BM smears and lymph node imprints from seven patients with high‐grade FCLs and diffuse large B‐cell lymphomas (DLBCLs). Three out of seven BM samples were found to contain clonal CD20‐positive B‐lymphoid cells (range 0.4–96% of the cells) and no extra copies of chromosome 7 were detected in the myelomonocytoid or erythroid cells or in the CD3‐positive T lymphocytes. All seven patients showed additional copies of chromosome 7 in the lymph nodes and, again, this cytogenetic abnormality was also restricted to the CD20‐positive cells (range 0.7–80% of the cells). Thus the present findings confirm that high‐grade B‐cell lymphomas with or without BM engagement involve the CD20‐positive B‐lymphoid cells exclusively and not the T lymphocytes, erythroid or myelomonocytoid cell lineages. These findings may indicate that anti‐CD20 immunotherapy could be of value in high‐grade B‐cell lymphomas.
Skeletal survey in advanced multiple myeloma: radiographic versus MR imaging survey
Authors: Lecouvet Frédéric E. , Malghem Jacques , Michaux Lucienne , Maldague Baudouin , Ferrant Augustin , Michaux Jean‐Louis , Vande Berg Bruno C.
Published: March 2003

In an attempt to compare the sensitivity of bone radiographs and bone marrow magnetic resonance (MR) imaging for bone lesion detection in patients with stage III multiple myeloma (MM) and to evaluate the possible consequences of the replacement of the conventional radiographic skeletal survey (RSS) by an MR survey of the spinal and pelvic bone marrow in these patients, we obtained MR studies of the thoracic and lumbar spine, pelvis and proximal femurs in addition to the conventional RSS (including radiographs of the skull, entire spine, pelvis, ribs, humerus and femurs) in 80 consecutive patients with newly diagnosed stage III MM according to the Durie and Salmon staging system (based on blood tests and on the RSS). The performance of MR and radiographic studies to detect bone lesions in given anatomic areas and in given patients were compared. The consequences on MM staging following the substitution of the RSS by the MR survey were assessed.

MR imaging was superior to radiographs for lesion detection in the spine (76% v 42% of patients) and pelvis (75% v 46% of patients). The RSS was superior to the limited MR imaging survey for the detection of bone involvement in the patient population (87.5% v 79% of patients). If the RSS had been replaced by the MR imaging survey for patient staging, 7/80 patients would have been categorized as stage I and one as stage II MM on the basis of normal MR findings and biological findings consistent with these stages.

Substitution of the RSS by a limited spinal and pelvic marrow MR survey would lead to ‘understaging’ of 10% of patients with otherwise stage III MM on the basis of blood tests and the conventional RSS.
Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction
Authors: Fontenay‐Roupie Michaëla , Bouscary Didier , Guesnu Martine , Picard Françoise , Melle Josiane , Lacombe Catherine , Gisselbrecht Sylvie , Mayeux Patrick , Dreyfus François
Published: March 2003

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)‐induced BFU‐E and CFU‐E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo‐R in MDS marrow, we focused on Epo‐induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro‐apoptotic signals rather than the defect of anti‐apoptotic pathways resulting from Epo‐stimulated Jak2‐STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.
Myelodysplastic syndrome in a patient with adult T‐cell leukaemia
Authors: Kawabata Hisashi , Utsunomiya Atae , Hanada Shuichi , Makino Torahiko , Takatsuka Yoshifusa , Takeuchi Shogo , Suzuki Shinsuke , Suzumiya Junji , Ohshima Kouichi , Horiike Shigeo
Published: March 2003

A 53‐year‐old female who developed myelodysplastic syndrome (MDS) after chemotherapy for adult T‐cell leukaemia (ATL) is described. The latent period of therapy‐related MDS (t‐MDS) from the time of diagnosis of ATL was approximately 35 months. Cytogenetic analysis of the bone marrow cells at the time of diagnosis of t‐MDS revealed a clonal abnormality; 46,XX,add(7)(p13), der(17)t(3;17)(p11;p13). Although monoclonal integration of human T lymphotropic virus type I (HTLV‐I) proviral DNA was detected in the peripheral blood lymphocytes at ATL diagnosis, bone marrow cells at t‐MDS diagnosis did not show monoclonal integration of HTLV‐I. To our knowledge, this is the first report of t‐MDS associated with ATL.
A family with hereditary factor X deficiency with a point mutation Gla32 to Gln in the Gla domain (factor X Tokyo)
Authors: Zama Takeru , Murata Mitsuru , Watanabe Reiko , Yokoyama Kenji , Moriki Takanori , Ambo Hironobu , Murakami Hiroshi , Kikuchi Masao , Ikeda Yasuo
Published: March 2003

We report a new family with hereditary factor X deficiency. The propositus had a markedly prolonged prothrombin time, a mild prolongation of activated partial thromboplastin time and a clotting time activated by Russell's viper venom. Factor X activity in plasma was 3 u/dl (normal range 56–138 u/dl). Factor X antigen level was 61 u/dl. Molecular analysis revealed a homozygous mutation, Glu (GAG) to Gln (CAG) at residue 32 which normally undergoes γ‐carboxylation within the γ‐carboxyglutamic acid rich domain. The genotypes of family members completely correlated with their factor X activities. It is suggested that the Glu32 to Gln mutation is the molecular basis for the abnormal factor X in this family.
A long‐term study of patients with chronic natural killer cell lymphocytosis
Authors: Rabbani Gita R. , Phyliky Robert L. , Tefferi Ayalew
Published: March 2003

Chronic natural killer cell lymphocytosis is a persistent state of natural killer (NK) cell (CD3−CD16/CD56+) excess in the peripheral blood that is not associated with clinical lymphoma. In 16 consecutive patients (median age 60.5 years, range 7–77), males were overrepresented (M:F 7:1) and the median absolute NK cell count was 4.09 × 109/l (range 1.2–16.6). Bone marrow examination was performed in 14 patients and showed atypical granulomata in two; chromosome studies in seven patients were normal. Clonal T‐cell receptor gene rearrangement was not found in any of 12 patients evaluated. At presentation, seven patients (44%) had no clinical symptoms or signs and the others had vasculitic skin lesions (three patients), non‐neutropenic fever (three patients), recurrent neutropenic infection (two patients), musculoskeletal symptoms (two patients), peripheral neuropathy (two patients), aphthous ulcers (one patient), and splenomegaly (one patient). Five patients had anaemia, five had neutropenia, and two had thrombocytopenia. After a median follow‐up of 5.1 years (range 0–10.2) from immunophenotypic diagnosis or 5.7 years (range 0.1–14.1) from documentation of absolute lymphocytosis, vasculitic glomerulonephritis developed in one patient, accelerated splenomegaly developed in a patient receiving myeloid growth factor treatment, and severe aplastic anaemia developed in one patient. Treatment with nonsteroidal anti‐inflammatory drugs or immunosuppressive agents was variably successful.
Role of FcγRI (CD64) in erythrocyte elimination and its up‐regulation in thalassaemia
Authors: Wiener  , Allen  , Porter  , Wickramasinghe  , Porter  , Chinprasertsuk  , Siripanyaphinyo  , Pattanapanyasat  , Fucharoen  , Wanachiwanawin
Published: March 2003

To examine any role of the high affinity Fcγ class I receptor (FcγRI) (CD64) in erythrocyte elimination by mononuclear phagocytes (MP) in thalassaemia (thal), we investigated the in vitro interaction of β‐thalassaemic erythrocytes with monocytes (Mo) whose FcγR expression had been modulated by cytokines.

Treatment of Mo with interferon (IFN)‐γ or interleukin (IL)‐10 which up‐regulate FcγRI, caused a dose‐dependent increase in binding of β‐thalassaemic erythrocytes, whereas stimulation with IL‐4 which down‐regulates the receptor, reduced this interaction, in a dose‐dependent manner, to that of normal erythrocytes. Binding of thalassaemic erythrocytes by IFN‐γ or IL‐10‐treated Mo was inhibited by FcγRI‐specific reagents. In addition, Mo expression of FcγRI and HLA class II DR was determined by flow cytometry in Thai patients with HbH disease (α1/α2 or α1/Hb Constant Spring) (n = 15) or β°‐thal/HbE (n = 16). In both groups of patients FcγRI expression was increased as compared to normal controls (n = 14): mean fluorescence intensity (±SD) 124.79 ± 38.77 in HbH disease and 121.86 ± 18.23 in β°‐thal/HbE versus 91.94 ± 17.36 in normal controls (P < 0.01 and P < 0.001, respectively). In contrast, HLA class II DR expression was similar in patients and controls.

The results suggest that, in thalassaemia, up‐regulated FcγRI on mononuclear phagocytes plays a role in their interaction with erythrocytes and that this process can be modified by cytokines.
An in vitro system for expression analysis of mutations of the β‐globin gene: validation and application to two mutations in the 5′ UTR
Authors: Ho P. Joy , Sloane‐Stanley Jackie , Athanassiadou Aglaia , Wood William G. , Thein Swee Lay
Published: March 2003

We describe the setting up of an in vitro expression system for the analysis of mutations of the β‐globin gene. The system is based on the stable transfection of a normal or mutated β‐globin gene into mouse erythroleukaemia (MEL) cells. The expression construct contains an Aγ gene as an internal control and both globin genes are under the control of the HS2 element of the β LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non‐mutant genes, high‐level expression of both β and Aγ genes is seen and both mRNAs are stable.

The system was validated by comparing the expression of the β654 thalassaemia splicing mutation in MEL cells with its well‐characterized expression in vivo. The level of the initial transcript, the proportion of abnormally spliced mRNA and its instability during erythroid cell maturation were all faithfully reproduced. The system was used to examine the mechanism by which two mutations in the β‐globin 5′ untranslated region (5′ UTR) result in β thalassaemia. Surprisingly, the mechanism appeared to differ in the two cases, with the C‐G substitution at position +33 affecting transcription, whereas the −T deletion at position +10 resulted in a translational defect. The stably transfected MEL cells, with an internal control and an endogenous enhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should find wide application in the analysis of the effects and mechanisms of gene inactivation in mutations affecting the β‐globin as well as other genes.
Evidence that anti‐HBc but not HBV DNA testing may prevent some HBV transmission by transfusion
Authors: Allain Jean‐Pierre , Hewitt Patricia E. , Tedder Richard S. , Williamson Lorna M. ,   for the anti‐HBc screening study group
Published: March 2003

Blood donor screening for antibody to hepatitis B core antigen (anti‐HBc) implemented in some countries as a surrogate marker for non‐A, non‐B hepatitis has been superseded by anti‐HCV screening. To assess the value of anti‐HBc screening for the detection of hepatitis B surface antigen‐negative blood donations that might contain infectious HBV, HBV genomic detection and recipient testing were used. Blood donations were screened and confirmed by multiple anti‐HBc assays. Donations containing isolated anti‐HBc and those with anti‐hepatitis B surface antigen (anti‐HBs) level < 0.1 IU/ml were tested for the presence of HBV DNA. Recipients of previous donations from the corresponding donors during the previous 5 years were traced and tested for markers of HBV infection. Of 103 869 donations screened, 586 (0.56%) were anti‐HBc positive, two of which contained HBsAg, and 413 (0.4%) had protective ( 0.1 IU/ml) levels of anti‐HBs. Anti‐HBs < 0.1 IU/ml was found in 102 of these donations (0.1%) and isolated anti‐HBc in 69 (0.07%). No donations with isolated anti‐HBc were HBV DNA confirmed positive. Of 278 recipients of previous donations from 171 donors at risk of HBV carriage, 12 had markers of HBV infection. Six recipients had other identified risk factors. An association with blood transfusion was considered probable in two and possible in four recipients. None of the six corresponding donors had detectable HBV DNA 6–40 months after the implicated donation. The frequency of HBV transmission by chronic carriers negative for hepatitis B surface antigen was estimated in this study to be 1 in 52 000 donations (CI 0.3–7.8/100 000) from HBsAg‐negative donors. Such HBV infectious donations may not be detected by DNA amplification.
Allogeneic bone marrow transplantation in aggressive non‐Hodgkin's lymphoma (excluding Burkitt and lymphoblastic lymphoma): a series of 73 patients from the SFGM database
Authors: Dhedin  , Giraudier  , Gaulard  , Esperou  , Ifrah  , Michallet  , Milpied  , Rio  , Cahn  , Molina  , Laporte  , Guilhot  , Kuentz  ,   for the Société Française de Greffe de Moelle
Published: March 2003

The place of allogeneic bone marrow transplantation (BMT) in the treatment of aggressive non‐Hodgkin's lymphoma (NHL) remains controversial. We conducted a retrospective study of French experience in allografting NHL between 1984 and 1994. To improve the homogeneity of the study population, cases of low‐grade, Burkitt and lymphoblastic NHL were excluded. 73 patients were included in the analysis. Median age at transplantation was 35 years (range 9–61 years); 64 patients were in stage IV and 45 had bone marrow involvement at diagnosis. At the time of transplantation, 46 patients had sensitive disease (25 in complete remission; CR).

The overall survival (OS) and progression‐free survival (PFS) rates were 41% and 40% respectively at 5 years (median follow‐up of survivors 90 months). The probability of disease progression was 30% at 5 years, and only one relapse occurred after 15 months. 32 patients died of transplantation‐related complications. In multivariate analysis, pretransplant complete remission was the main factor associated with longer survival (OS at 60 months of 76% among the 25 patients in CR at transplant and of 23% among the 48 patients not in CR at transplant). Neither acute nor chronic graft‐versus‐host disease (GvHD) influenced the relapse rate.

In conclusion, in this high‐risk population the overall results of allogeneic BMT were encouraging, despite a high transplant‐related mortality rate. We believe this procedure should be studied further in prospective controlled trials.
Bleeding and thrombosis in 55 patients with inherited afibrinogenaemia
Authors: Lak  , Keihani  , Elahi  , Peyvandi  , Mannucci
Published: March 2003

Knowledge of the spectrum of symptoms in patients with inherited afibrinogenaemia is limited by the rarity of this coagulation defect. We compared a large series of 55 afibrinogenaemic patients from Iran with 100 patients with severe factor VIII deficiency. In afibrinogenaemia there was a higher frequency of mucosal‐type bleeding symptoms but joint and muscle bleeding was less frequent and severe than in haemophilia. Umbilical cord bleeding was relatively frequent only in afibrinogenaemic patients. Two young patients developed spontaneous thrombotic episodes and three women had recurrent abortions. Overall, in afibrinogenaemia bleeding symptoms are qualitatively different and less severe than in haemophilia. Afibrinogenaemia can also be accompanied by thrombotic manifestations.
Time‐course expression of polypeptides carrying blood group antigens during human erythroid differentiation
Authors: Bony Viviane , Gane Pierre , Bailly Pascal , Cartron Jean‐Pierre
Published: March 2003

The time course expression of blood group antigens was examined by flow cytometry using a two‐phase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71++) and glycophorin A (GPA+). Four main categories of blood group markers were identified: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epo‐independent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wrb (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epo‐dependent phase 2 and (iv) those like Jk3 and Lub which were expressed in late phase 2.

Regarding blood group molecules exhibiting adhesive properties (LW/ICAM‐4, Oka and Lu) the most significant event was a sharp decrease of Oka (neurothelin) expression with the concomitant loss of ICAMs expression during the later stage of differentiation. These studies suggest that Oka, ICAMs and LW might contribute to the adhesive interactions involved in the formation of erythroblastic islands and attachment to stroma cells and the extracellular matrix. We also noted an asynchronous expression of the proteins that compose the core of the Rh complex, since Rh50 glycoprotein was expressed earlier than Rh(D, CE) proteins.
CD82 (KAI1), a member of the tetraspan family, is expressed on early haemopoietic progenitor cells and up‐regulated in distinct human leukaemias
Authors: Burchert Andreas , Notter Michael , Menssen Hans Dietrich , Schwartz Stefan , Knauf Wolfgang , Neubauer Andreas , Thiel Eckhard
Published: March 2003

CD82 (KAI1) is a member of the tetraspan transmembrane protein family which has been cloned from lymphoblastoid variant cell lines. However, a role for CD82 in early normal and malignant haemopoiesis has not yet been characterized. We studied the CD82 expression in 33 normal donor samples and 98 leukaemias by fluorescence activated cell sorting (FACS) and reverse transcriptase polymerase chain reaction (RT‐PCR). We demonstrated that CD82 was moderately expressed in the vast majority of normal granulocytes and monocytes. In contrast, only about one third of the peripheral blood lymphocytes were weakly CD82 positive (CD82+). Interestingly, judgement of the CD82 transcription and expression in various leukaemias revealed that CD82 was overexpressed in chronic myeloid leukaemia (CML) patients in accelerated or blastic phase (CML‐AP/BP) as well as in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) patients. Analysis of AML patients with CD34+/CD82+ blasts prompted us to expand our studies on haemopoietic CD34+ progenitor cells. Intriguingly, 84–95% of the CD34+ cells isolated from healthy bone marrow, cord blood or peripheral blood were highly CD82+. CD82 was abundantly expressed on primitive as well as on committed haemopoietic progenitor cells. After in vitro induction of myeloid differentiation in CD34+ peripheral blood progenitor cells (PBPC), the expression of CD82 decreased to levels similar to those found on peripheral blood granulocytes. These observations suggest for the first time a role for CD82 in normal and malignant haemopoiesis.
Pgp‐positive leukaemic cells have increased mtDNA but no increased rate of proliferation
Authors: Jia Li , Liu Kan‐Zhi , Newland Adrian C. , Mantsch Henry H. , Kelsey Stephen M.
Published: March 2003

Cells of solid tumours tend to rely on glycolysis for energy. On the other hand, increased glycolysis in solid tumour cells expressing the multidrug resistance protein MDR‐1 has been associated with increased malignancy in tumours. We have previously shown that cells of the MDR‐1‐positive CEM/VLB100 leukaemic cell line have increased mitochondrial electron transport chain (mtETC) activity compared with parental CEM cells. In the present study we used infrared (IR) spectroscopy to demonstrate that the mitochondrial DNA (mtDNA) content in the CEM/VLB100 cell line was significantly increased compared to that in the parental CEM cells. The increase in mtDNA was not accompanied by an increase in mitochondrial protein as both lipid and protein levels were decreased in CEM/VLB100 mitochondria. The ATP content was similar in these two cell lines. However, the ATP‐dependent membrane efflux pump function in CEM/VLB100 cells was significantly reduced when mitochondrial ATP synthesis was inhibited by oligomycin, a specific inhibitor of mitochondrial F0F1‐ATPase. Proliferation of CEM/VLB100 cells was significantly decreased compared to parental CEM cells, and was independent of p53 expression. Thus, we conclude that: (1) IR spectroscopy is a potential powerful technique for detecting mtDNA, protein and lipid contents simultaneously; (2) leukaemic cells mainly rely on mtDNA for energy; (3) increased expression of an ATP‐dependent membrane efflux pump such as Pgp may up‐regulate ATP generation and mtDNA content. These metabolic perturbations may exist merely to serve the efflux pump and do not result in an increase in leukaemic cell proliferation. In addition, the associated reduction in mitochondrial lipid and protein may contribute to sensitize the cells to cytochrome c release.
Clinical and laboratory evaluation of all‐trans retinoic acid modulation of chemotherapy in patients with acute myelogenous leukaemia
Authors: Seiter Karen , Feldman Eric J. , Dorota Halicka  , Deptala Andrzej , Traganos Frank , Burke Harry B. , Hoang Albert , Goff Heather , Pozzuoli Monica , Kancherla Ramamohana , Darzynkiewicz Zbigniew , Ahmed Tauseef
Published: March 2003

All‐trans retinoic acid (ATRA) is synergistic with chemotherapy in leukaemia cell lines. We treated 53 patients with newly diagnosed acute myelogenous leukaemia (AML) with high‐dose cytarabine‐based chemotherapy followed by ATRA. Peripheral blood and bone marrow samples were obtained to study the effect of in vitro exposure to ATRA and to measure apoptosis and bcl‐2. The response rate was 72% for patients under age 60 years and 46% for patients aged 60 years or above. There was no difference in the percentage of responding patients, time to recurrence or overall survival for patients receiving chemotherapy with ATRA vs. historical controls receiving chemotherapy without ATRA. After in vitro exposure of day 3 bone marrow samples to ATRA, there was an increase in apoptotic cells in 25% of patient samples compared with samples not exposed to ATRA. Later date of peak apoptosis in peripheral blood and higher percentage of apoptotic cells in bone marrow on day 3 of treatment were associated with lack of clinical response to treatment. Increased bcl‐2 in patient samples was associated with shorter time to recurrence and poor cytogenetic risk. The addition of ATRA to chemotherapy did not improve patient outcome. However, evidence of in vitro response to ATRA in 25% of patients suggests that retinoid pathways should be studied further in patients with AML.
Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow‐derived macrophages and human haemopoietic cell lines
Authors: Hodges Vivien M. , Molloy Gael Y. , Wickramasinghe
Published: March 2003

The expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1 and 3A4) was studied in human bone marrow, bone‐marrow‐derived macrophages and blood monocyte‐derived macrophages. Reverse transcriptase polymerase chain reaction (RT‐PCR) detected expression of all five CYPs in each of these cell populations. All five CYPs were also expressed in the haemopoietic cell lines HL‐60 and HEL and in Epstein–Barr virus (EBV)‐transformed B‐lymphoblastoid cell lines. The data suggest that bone marrow macrophages and probably other bone marrow cell types are capable of metabolizing xenobiotics. This metabolic potential may play a role in the bone marrow damage induced by some drugs and chemicals.
A novel polymorphism in intron 1a of the human factor VII gene (G73A): study of a healthy Italian population and of 190 young survivors of myocardial infarction
Authors: Peyvandi  , Mannucci  , Bucciarelli  , Zeinali  , Akhavan  , Sacchi  , Merlini  , Perry
Published: March 2003

We have identified a novel polymorphism located in intron 1a of the human factor VII gene, caused by the nucleotide change G to A at position + 73. In a population of 128 healthy individuals from northern Italy, the variant A73 allele had a frequency of 0.21, whereas the frequency of the previously reported 10 bp insertion allele located at −323 in the promoter region was 0.17 and that of the Q353 allele in the catalytic region of the factor VII gene was 0.20. In 75% of the healthy individuals, the A73 allele was present together with the 10 bp insertion and the Q353 alleles, indicating a strong linkage disequilibrium. The concomitant presence of A73 with both the 10 bp and the Q353 alleles was associated with the lowest factor VII levels, measured as coagulant activity, activated factor VII and factor VII antigen. The G73A polymorphism was also evaluated in 190 survivors of myocardial infarction who had experienced the event before the age of 45 years and in 179 individuals with a negative exercise test matched with patients for sex, age and geographical origin. Patients carrying the A73 allele associated with lower factor VII levels tended to have a lower risk of myocardial infarction (adjusted odds ratio 0.54; 95% confidence intervals 0.29–0.99). In conclusion, we found a novel variant allele in intron 1a of the human factor VII gene that is often associated in healthy individuals with the 10 bp and Q353 alleles in the promoter and catalytic region of the same gene. This intronic mutation, alone or in association with other factor VII gene polymorphisms, might confer protection against myocardial infarction in the young.
The spectrum of Fusarium infection in immunocompromised patients with haematological malignancies and in non‐immunocompromised patients: a single institution experience over 10 years
Authors: Musa  , Al Eisa  , Halim  , Sahovic  , Gyger  , Chaudhri  , Al Mohareb  , Seth  , Aslam  , Aljurf
Published: March 2003

Fusarium is a newly emerging fungal pathogen associated with significant morbidity and mortality in the immunocompromised host. We have reviewed our hospital's experience with Fusarium between 1985 and 1995. Fusarium species were isolated from 22 specimens, representing 11 patients. Cases were not clustered by time period. The median age of the patients was 36.5 years (range 17–69 years). The sources of the organism were 12 skin lesions from eight patients, seven blood cultures from two patients and one specimen each from a Hickman catheter tip, nail clippings and a bronchoalveolar lavage. Seven of the patients had chemotherapy‐induced neutropenia when the Fusarium was isolated. Five of them developed invasive fusarosis during acute leukaemia induction treatment. They remained neutropenic, and none survived. The other two patients recovered from neutropenia and were treated successfully for this infection. The remaining four patients were not neutropenic or immunocompromised. Three grew Fusarium from skin or nail clippings and one from bronchial alveolar lavage (BAL). There was no evidence of invasive disease in any of the four. None of them received antifungal therapy, and they were all alive at last follow‐up. We conclude that Fusarium is a newly emerging infection in neutropenic patients. A high index of suspicion, especially for skin lesions, will help in early diagnosis before systemic and visceral dissemination. Excision of the initial focus of infection and antifungal therapy, aided by speedy neutrophil recovery, are likely to protect patients threatened with these fatal infections. Fusarium isolated from non‐neutropenic, non‐immunosuppressed patients is not significant and does not merit systemic antifungal treatment.
High FUS/TLS expression in acute myeloid leukaemia samples
Authors: Mills  , Walsh  , Gilkes  , Sweeney  , Mirza  , Woodgate  , Brown  , Burnett
Published: March 2003

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all‐trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto‐oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA‐resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA‐sensitive and ‐resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.
Megakaryopoiesis in vitro in myelodysplastic syndromor and development es and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors
Authors: Liu Yin J. A. , Adams J. A. , Brereton M. L. , Hann A. , Harrison B. D. , Briggs M.
Published: March 2003

Pegylated recombinant human megakaryocyte growth and development factor (PEG‐rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG‐rHuMGDF combined with granulocyte colony‐stimulating factor (G‐CSF), granulocyte–macrophage CSF (GM‐CSF), interleukin 3 (IL‐3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61‐positive cells in suspension cultures increased with PEG‐rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL‐3 and/or SCF were added to PEG‐rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU‐Mk growth was poor overall, but could be enhanced by PEG‐rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG‐rHuMGDF can be augmented by IL‐3 and SCF in many MDS and AML patients.
Prognostic value of serum markers of bone metabolism in untreated multiple myeloma patients
Authors: Fonseca Rafael , Trendle Michael C. , Leong Traci , Kyle Robert A. , Oken Martin M. , Kay Neil E. , Van Ness Brian , Greipp Philip R.
Published: March 2003

Bone involvement is a central feature of multiple myeloma (MM). We investigated whether serum markers of osteoblastic and osteoclastic activity correlate with the presence of bone disease and survival in 313 MM patients enrolled in a phase III trial (E9486). Five markers were measured, including osteocalcin (OC), carboxy‐terminal propeptide of type I collagen (PICP), bone alkaline phosphatase (BAP), carboxy‐terminal telopeptide of type I collagen (ICTP) and tartrate‐resistant acid phosphatase (TRAP). We analysed the relationship between serum levels of these markers and the presence of bone manifestations, and survival. Serum levels of ICTP and BAP correlated significantly with bone pain, lesions and fractures. Serum level of ICTP was also higher in stage II–III compared with stage I disease. The serum level of ICTP was significantly associated with shortened survival in the univariate analysis. The median survival times were 4·1 and 3·5 years for low and high ICTP respectively (P = 0·02). There was a strong relationship between ICTP and beta‐2‐micrgolobulin (B2M). ICTP stands out as a significant marker of bone disease. Incorporation of these markers into clinical trials assessing the use of bisphosphonates in MM is needed to determine whether they might serve as indicators of effectiveness of these agents.
Coagulation factor content of cryoprecipitate prepared from methylene blue plus light virus‐inactivated plasma
Authors: Hornsey V. S. , Krailadsiri P. , MacDonald S. , Seghatchian J. , Williamson L. M. , Prowse C. V.
Published: March 2003

Levels of factor VIII (FVIII) and fibrinogen were assessed in control cryoprecipitate and cryoprecipitate prepared in two centres from plasma subjected to methylene blue (MB) photochemical virus inactivation. The level of coagulation FVIII activity was reduced in plasma by approximately 30% after MB photoinactivation, with only 44% (centre A) and 31% (centre B) of units meeting the current UK specification of 0·7 iu/ml. A revised specification of 0·5 iu/ml is suggested. Losses of less than 11% were seen for von Willebrand factor (VWF)‐related activities. Cryoprecipitate prepared from group O or group A MB‐treated plasma contained 27–40% less FVIII than control units. This reflected the lower levels in MB‐treated plasma. The concentrating power of the cryoprecipitation process was not reduced for FVIII or fibrinogen in MB‐treated units. MB cryoprecipitate from centre A still met the UK guideline specification for FVIII and fibrinogen content, whereas at centre B only 62·5% of the group O cryoprecipitates contained > 70 iu FVIII/unit. This may reflect the lower product volume and lower FVIII content of group O plasma used at centre B and suggests that maintenance of total coagulation factor recovery in MB‐treated cryoprecipitate will require the higher product volume.
Combination of stem cell factor and granulocyte colony‐stimulating factor mobilizes the highest number of primitive haemopoietic progenitors as shown by pre‐colony‐forming unit (pre‐CFU) assay
Authors: Horsfall M. J. , Hui C. H. , To L. B. , Begley C. G. , Basser R. L. , Simmons P. J.
Published: March 2003

Fifty‐two patients with poor prognosis carcinoma of the breast underwent peripheral blood stem cell (PBSC) mobilization using five different regimens. The yields of primitive haemopoietic progenitors were quantified by a recently described pre‐colony‐forming unit (pre‐CFU) assay using limiting dilution analysis (LDA). Results of days 14 and 35 pre‐CFU were also correlated with conventional CD34+ cell enumeration, CFU‐GM (granulocyte–macrophage) and long‐term culture‐initiating cell (LTCIC) assays. The yield of pre‐CFUs with the combination of granulocyte colony‐stimulating factor (G‐CSF) and stem cell factor (SCF) was significantly higher than with G‐CSF alone, cyclophosphamide (Cyclo) and granulocyte–monocyte colony‐stimulating factor (GM‐CSF), interleukin (IL)‐3 and GM‐CSF, or Cyclo alone. No significant correlation between neutrophil engraftment and pre‐CFU could be demonstrated. Furthermore, CFU‐GM was shown to bear a stronger correlation with pre‐CFU and LTCIC than CD34+ cell measurement; thus, CFU‐GM remains a useful biological tool for haemopoietic stem cell assay. We conclude that the combination of G‐CSF and SCF mobilizes the highest number of pre‐CFUs as measured by functional pre‐CFU assay, which provides an alternative measurement of primitive haemopoietic progenitors to the LTCIC assay.
Prevalence of methylenetetrahydrofolate reductase C677T and its association with arterial and venous thrombosis in the Chinese population
Authors: Zheng Yan‐Zhen , Tong Jian , Do Xiao‐Ping , Pu Xiao‐Qun , Zhou Bo‐Tong
Published: March 2003

Moderate hyperhomocysteinaemia (MHH) is associated with arterial and venous thrombosis. A main genetic defect related to MHH is a C to T substitution at nucleotide 677 of the 5,10‐methylenetetrahydrofolate reductase (MTHFR) gene. A prothrombin 20210A mutation was recently identified as a risk factor for arterial and venous thrombosis. However, studies on the prevalence of mutant MTHFR C677T and prothrombin G20210A and their association with thrombosis were controversial and seldom reported in the Chinese population. We investigated the prevalence of MTHFR C677T and prothrombin G20210A genotypes by polymerase chain reaction (PCR) followed by restriction enzyme digestion in 420 Chinese subjects: 53 with deep venous thrombosis (DVT); 145 with cerebrovascular disease [115 cerebral infarction, 30 cerebral haemorrhage (CH)]; 100 with coronary artery disease (CAD); and 122 control subjects. The prevalence of the mutated MTHFR 677TT genotype and the 677T allele in normal controls was 12·3% and 30·7% respectively, similar to that in Caucasians and Japanese. The mutant 677T homozygotes and alleles were more frequent in patients with DVT than in controls (18·9% vs. 12·3%, 0·01 < P < 0·025; 48·1% vs. 30·7%, P < 0·005). The relative risk of DVT among the carriers of 677TT and 677T were significantly increased [odds ratios: 3·4, 95% confidence interval (CI) 1·3–9·5, and 3·6, 95% CI 1·7–7·7, respectively). The mutant MTHFR heterozygous 677C/T carriers were increased in patients with cerebral infarction compared with controls (53·9% vs. 36·9%, 0·01 < P < 0·025). Relative risk of cerebral infarction was 0·96 (95% CI 0·4–2·3) for 677TT homozygotes and 1·99 (95% CI 1·2–3·4) for 677C/T heterozygotes. However, the distribution of the MTHFR TT genotype was less frequent in patients with CAD with coronary artery stenosis of > 50% than in controls (2·8% vs. 12·3%, 0·025 < P < 0·05). Relative risk of CAD was not increased among the carriers of 677TT and 677T (odds ratios: 0·2, 95% CI 0–1·1, and 0·97, 95% CI 0·5–1·8, respectively). There were no differences in the distribution of the MTHFR genotypes among CH, CAD with coronary artery stenosis of < 50% and controls. The prothrombin 20210A mutation was not found in any patients or controls. These results demonstrated that MTHFR 677T was associated with DVT and cerebral infarction but was less associated with CAD in the Chinese population.