bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin and insulin‐like growth factor I support the proliferation of erythroid progenitor cells in bone marrow through the sharing of receptors
Authors: Miyagawa Shin‐ichiro , Kobayashi Masao , Konishi Nakao , Sato Takashi , Ueda Kazuhiro
Published: March 2003

The effects of insulin and insulin‐like growth factor I (IGF‐I) on the proliferation of erythroid progenitor cells in bone marrow were studied in serum‐deprived culture. Primitive human bone marrow cells were purified by cell sorting on the basis of the expression of CD34 and the Kit receptor. Insulin and IGF‐I with erythropoietin (EPO) dose dependently supported the formation of erythroid colonies of CD34+/Kit+ cells in bone marrow. The direct effect of insulin and IGF‐I on the stimulation of primitive erythroid progenitor cells was confirmed by single‐cell proliferation studies in serum‐deprived liquid suspension culture. The addition of insulin and/or IGF‐I to stem cell factor (SCF) resulted in an additive increase in the number of erythroid colonies. The erythroid colonies formed by insulin and IGF‐I with EPO were different in size from those formed by SCF with EPO. These findings imply that erythroid progenitor cells responding to insulin and IGF‐I might be at a different developmental stage of erythropoiesis from those responding to SCF in CD34+/Kit+ cells. Similarly, insulin and IGF‐I with EPO supported the proliferation of the mature erythroid progenitor cells in light‐density bone marrow mononuclear cells (LDBMCs). The addition of the anti‐receptor antibody to IGF‐I receptor or insulin receptor partially suppressed erythroid colony formation supported with insulin or IGF‐I in both CD34+/Kit+ cells and LDBMCs. The simultaneous addition of both receptor antibodies completely abrogated the erythroid colony formation. These results suggest that insulin and IGF‐I directly stimulate the proliferation of the late stage of primitive erythroid progenitor cells and mature erythroid progenitor cells through the sharing of receptors.
Heritability of elevated factor VIII antigen levels in factor V Leiden families with thrombophilia
Authors: Kamphuisen P. W. , Lensen R. , Houwing‐Duistermaat J. J. , Eikenboom J. C. J. , Harvey M. , Bertina R. M. , Rosendaal F. R.
Published: March 2003

Factor VIII activity (factor VIII:C) and factor VIII antigen (factor VIII:Ag) levels above150 IU/dl are associated with a five‐ to sixfold increased risk of venous thrombosis compared with levels < 100 IU/dl. These high levels are present in 25% of patients with a first episode of deep‐vein thrombosis and in 11% of healthy controls. von Willebrand factor (VWF) and blood group are important determinants of the factor VIII level in plasma and therefore contribute to thrombotic risk, while factor VIII appears to be the final effector. Previously, we found familial clustering of factor VIII:C levels in women, which remained after adjustment for VWF and blood group. In the present study, we analysed the familial influence on factor VIII:Ag levels exceeding 150 IU/dl in 12 large families with thrombophilia in which high factor VIII:Ag levels contribute to thrombotic risk. As expected, blood group was a main determinant of the plasma factor VIII level: 58 relatives (32%) had factor VIII levels above 150 IU/dl and 50 (86%) of these had blood group non‐O. After adjustment for blood group and age, we found an association between factor VIII:Ag levels in sister pairs (0·35, P = 0·003), brother pairs (0·35, P = 0·003), brother–sister pairs (0·35, P < 0·001) and in mother–son pairs (0·45, P = 0·02), but not in father–daughter or father–son pairs. The familial aggregation test was strongly positive for factor VIII:Ag levels (P < 0·001) and remained so after adjustment for the influence of blood group. We conclude that high factor VIII:Ag levels are a highly prevalent risk factor for venous thrombosis and contribute to risk in families with thrombophilia, and that these high levels are likely to be genetically determined by factors other than just blood group.
Clonal Th2 lymphocytes in patients with the idiopathic hypereosinophilic syndrome
Authors: Roufosse F. , Schandené L. , Sibille C. , Willard‐Gallo K. , Kennes B. , Efira A. , Goldman M. , Cogan E.
Published: March 2003

Idiopathic hypereosinophilic syndrome (HES) and Gleich's syndrome are related disorders characterized by persistent or recurrent hypereosinophilia of unknown origin. Elevated IgE levels and polyclonal hypergammaglobulinaemia are considered as markers of benign outcome in this setting as they are generally associated with predominant cutaneous manifestations and favourable response to glucocorticoid therapy. In a previous study, we identified a clonal population of CD3−CD4+ Th2‐like lymphocytes secreting interleukin (IL)‐5 and IL‐4 in peripheral blood of a patient fulfilling the diagnostic criteria of HES with associated serum hyper‐IgE. We now extend this observation by describing identical findings in three additional patients, and we compare their clinical and biological parameters with five other patients with HES. Chromosomal abnormalities were detected in purified CD3−CD4+ Th2 cells from three patients, among whom one developed anaplastic null cell lymphoma. We therefore suggest that a careful search for T‐lymphocyte clonality and cytogenetic changes should be included in the work‐up of HES for adequate management.
Haematopoietic stem cell transplantation for amegakaryocytic thrombocytopenia
Authors: Lackner A. , Basu O. , Bierings M. , Lassay L. , Schaefer U. W. , Révész T. , Havers W. , Kremens B.
Published: March 2003

Congenital amegakaryocytic thrombocytopenia (CAT), a rare syndrome with failure of megakaryopoiesis, cannot be cured by immunoglobulins, steroids or cyclosporin, but only by allogeneic bone marrow transplantation (BMT). We report on eight patients with CAT, all of whom were dependent at the time of BMT on platelet transfusion. Sources of haematopoietic progenitor cells were bone marrow (n = 5), peripheral stem cells (n = 2) and cord blood (n = 1). Seven patients engrafted. Both patients with matched unrelated donor transplants died, six patients are well with stable platelet counts 3–27 months after transplantation. BMT represents a curative option for CAT. The benefit of using alternative marrow donors should be carefully evaluated.
Mobilization of CD34+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4
Authors: Lichterfeld Mathias , Martin Simona , Burkly Linda , Haas Rainer , Kronenwett Ralf
Published: March 2003

The β1 integrin very late antigen 4 (VLA‐4) plays a central role in mobilization and homing of CD34+ cells. In this study, we examined the activation state of VLA‐4 on CD34+ cells from bone marrow (BM) and peripheral blood (PB) by flow cytometry using a vascular cell adhesion molecule I–immunoglobulin (VCAM‐I/IgG) fusion protein as soluble ligand. In an intraindividual analysis, we found a significantly reduced affinity and avidity of the VLA‐4 receptor on CD34+ cells from PB during granulocyte colony‐stimulating factor (G‐CSF)‐enhanced marrow recovery in comparison with steady‐state BM. Moreover, the amount of circulating CD34+ cells during marrow recovery was inversely related to the activation state but not to the expression level of VLA‐4, suggesting that a modulation of the functional state of VLA‐4 is involved in the mobilization of CD34+ cells. Moreover, VLA‐4 function on CD34+ cells from BM was associated with the maturation state of CD34+ cells as high‐affinity VLA‐4 receptors were observed on the vast majority of more primitive CD34+ cells. In addition, we found that Mg2+ ions as well as co‐incubation of CD34+ cells with endothelial cells resulted in an activation of the VLA‐4 receptor. In conclusion, modulation of the functional state of VLA‐4 appears to be of relevance for the mobilization and homing of CD34+ haematopoietic stem cells.
In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia
Authors: Verschuur Arnauld C. , Van Gennip Albert H. , Leen Rene , Meinsma Rutger , Voute P. A. , Van Kuilenburg André B. P.
Published: March 2003

Cytidine triphosphate (CTP) synthetase is a key enzyme for the synthesis of cytosine (deoxy)ribonucleotides, catalysing the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in several malignancies. In this preclinical study, the enzyme activity and mRNA expression of the enzyme and (deoxy)ribonucleotide concentrations were analysed in leukaemic cells of 57 children suffering from acute lymphocytic leukaemia (ALL). In addition, in vitro experiments were performed with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC). A significantly higher activity of CTP synthetase (6·5 ± 3·9 nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1·8 ± 0·9 nmol CTP/mg/h, P < 0·001) that was independent of white blood cell (WBC) count, blast percentage, age, gender or type of ALL. The enzyme activity was not correlated with the CTP synthetase mRNA expression. The activity of CTP synthetase in ALL cells compared with non‐malignant CD34+ bone marrow controls (5·6 ± 2·4 nmol CTP/mg/h) was not statistically different. In vitro treatment of ALL cells with CPEC induced a dose‐dependent decrease of the CTP concentration. The lowest concentration of CPEC (0·63 µm) induced a depletion of CTP of 41 ± 20% and a depletion of dCTP of 27 ± 21%. The degree of CTP depletion of ALL cells after treatment with CPEC was positively correlated with the activity of CTP synthetase. The inhibition of CTP synthetase in situ was confirmed by flux studies using radiolabelled uridine. From these results, it can be expected that CPEC has a cytostatic effect on lymphoblasts of children with ALL.
Influence of the −675 4G/5G dimorphism of the plasminogen activator inhibitor 1 promoter on thrombotic risk in patients with factor V Leiden
Authors: Visanji J. M. , Seargent J. , Tahri D. , Croft S. A. , Makris M. , Preston F. E. , Peake I. R. , Daly M. E.
Published: March 2003

Elevated plasminogen activator inhibitor 1 (PAI‐1) levels are associated with venous thromboembolism, although their significance is unclear. PAI‐1 levels are influenced by a PAI‐1 promoter dimorphism (4G/5G), the 4G allele being associated with increased PAI‐1 activity. We investigated whether the 4G allele influenced thrombotic risk by studying 99 symptomatic factor V (FV) Leiden heterozygotes and 99 healthy subjects. The 4G allele was more prevalent among cases than among healthy subjects (χ2 = 8·00, P = 0·005) and the odds ratio (OR) for thrombosis associated with either heterozygosity or homozygosity for the 4G allele was 2·43 (P = 0·011). We conclude that carriership of the 4G allele was more prevalent in patients who already carried factor V Leiden than in control subjects without factor V Leiden.
Functional response of leukaemic blasts to stromal cell‐derived factor‐1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia
Authors: Möhle Robert , Schittenhelm Marcus , Failenschmid Cornelia , Bautz Frank , Kratz‐Albers Karsten , Serve Hubert , Brugger Wolfram , Kanz Lothar
Published: March 2003

The chemokine stromal cell‐derived factor‐1 (SDF‐1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF‐1‐induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B‐lineage ALL), determined the effect of BM stromal cell‐conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF‐1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF‐1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5‐fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B‐lineage ALL. We conclude that, in AML, SDF‐1 is preferentially active in myelomonocytic blasts as a result of differentiation‐related expression of CXCR4. Functional activity of SDF‐1 and high expression of CXCR4 in B‐lineage ALL is in accordance with the previously described activity of SDF‐1 in early B cells. SDF‐1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM‐derived blasts compared with circulating cells.
Severe congenital dyserythropoietic anaemia type I: prenatal management, transfusion support and alpha‐interferon therapy
Authors: Parez N. , Dommergues M. , Zupan V. , Chambost H. , Fieschi J. B. , Delaunay J. , Miélot F. , Cramer E. M. , Dommergues J.‐P. , Wickramasinghe S. N. , Tchernia G.
Published: March 2003

We report a case of congenital dyserythropoietic anaemia, type I, with severe pre‐ and postnatal manifestations. Exchange transfusions were required for fetal anaemia (3·5 g/dl) at 28 and 30 weeks of gestation. Transfusions were administered at birth (Caesarean section at week 35) and at regular intervals thereafter. At 14 months, α‐interferon therapy was initiated (106 units three times a week). This resulted in stabilization of the haemoglobin at or above 11 g/dl and a reduction in the percentage of erythroblasts with ultrastructurally abnormal heterochromatin. After 9 months, the dose of α‐interferon was decreased to 106 units twice a week. No relapse of anaemia was noted during an additional 4 months of follow‐up.
Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia
Authors: Van Den Oudenrijn Sonja , Bruin Marrie , Folman Claudia C. , Peters Marjolein , Faulkner Lawrence B. , De Haas Masja , Von Dem Borne Albert E. G. KR.
Published: March 2003

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow‐derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c‐mpl gene, encoding the Tpo receptor, exists. Sequencing of c‐mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c‐mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c‐mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti‐apoptotic effect on stem cells. Therefore, mutations in c‐mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.
Bleeding symptoms and coagulation abnormalities in 337 patients with AL‐amyloidosis
Authors: Mumford Andrew D. , O'donnell James , Gillmore Julian D. , Manning Richard A. , Hawkins Philip N. , Laffan Michael
Published: March 2003

Haemorrhage is a frequent manifestation of amyloidosis. We performed a retrospective clinical analysis of 337 patients with systemic immunoglobulin light‐chain (AL)‐amyloidosis, in whom whole‐body serum amyloid P component (SAP) scintigraphy and a clotting screen had been performed. Abnormal bleeding was noted in 94 cases (28%), and the coagulation screen was abnormal in 172 cases (51%). The most common abnormalities were prolongation of the thrombin time (TT; 108 cases, 32%) and the prothrombin time (PT; 82 cases, 24%). In multivariate analysis, a prolonged PT was the only coagulation abnormality associated with abnormal bleeding (P = 0·0012), but this was independent of the whole‐body amyloid load. Prolongation of the TT was associated with hepatic amyloid infiltration (P < 0·00001), with proteinuria (P < 0·001) and low serum albumin (P < 0·00001). In 154 patients who were studied further, subnormal factor X activity (FX:C) was found in 22 cases (14%). In cases with subnormal FX:C, the corresponding factor X antigen (FX:Ag) measurements were consistently higher (median FX:Ag/FX:C 2·5, range 0·81–9·25, n = 16) than cases with normal FX:C (median FX:Ag/FX:C 0·96, range 0·65–1·29, n = 28, P < 0·0001). No evidence was found of an FX inhibitor. Of the 48/154 (31%) cases with a prolonged TT, the reptilase time was also prolonged in 38/48 cases (79%). These data show that haemorrhage and abnormal coagulation are common in AL‐amyloidosis and are multifactorial in origin. We provide evidence suggesting that hepatic amyloid infiltration and nephrotic syndrome are determinants of the TT. In most patients, prolongation of the PT was explained by reduction in FX:C, but this was not wholly explained by a reduction in FX:Ag.
Intermittent injections vs. continuous infusion of Factor VIII in haemophilia patients undergoing major surgery
Authors: Batorova Angelika , Martinowitz Uri
Published: March 2003

Continuous infusion (CI) of factor VIII (FVIII) has been proved to be a safe alternative to intermittent bolus injections (BI) in haemophilia A. Most reports on CI suggest a considerable saving in FVIII compared with historical controls treated with BI, but some recent reports failed to demonstrate such an effect. The present study prospectively compared safety, efficacy and factor requirements in 43 major surgical procedures performed in severe haemophilia A patients who were treated with either BI (18 operations) or CI (25 operations). The aim was to maintain factor VIII levels above the same minimum levels. Improved safety of CI over BI was observed, despite a bias in favour of the BI group (all underwent unilateral operations, compared with 24% of the CI group who underwent bilateral operations). Higher nadir levels were found in the CI group (0·44 ± 0·06 vs. 0·31 ± 0·09 IU/ml; P < 0·01) with a lower incidence of dangerous drops below 0·3 IU/ml (8% vs. 44% of patients respectively; P < 0·01), and a lower drop in haemoglobin (Hb) (1·56 ± 1·21 vs. 3·01 ± 2·13 g/dl; P < 0·05) and blood transfusion requirements (12% vs. 39%; P < 0·01). Major bleeding complications developed in three out of 18 patients (17%) in the BI group and none of the CI group (P = 0·06). The FVIII dosage was lower by 36% in the CI group (467 ± 104 vs. 733 ± 126 IU/kg; P < 0·01). Had the trough factor levels been maintained at the target levels, a greater difference of 72% would probably have been observed.
Evaluation of apoptosis as a prognostic factor in myelodysplastic syndromes
Authors: Shimazaki K. , Ohshima K. , Suzumiya J. , Kawasaki C. , Kikuchi M.
Published: March 2003

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo‐ or hypercellular bone marrow. This is thought to be as a result of the apoptosis of haematopoietic bone marrow cells resulting in ineffective haematopoiesis. To clarify the relationship between prognosis and apoptosis and/or cell proliferation in the bone marrow, we studied 51 cases with MDS. Bone marrow biopsies were stained immunohistochemically for MIB‐1 (marker for proliferating cells) and CD34 (marker for stem cells). Apoptosis was visualized by detection of DNA fragmentation using TdT incorporation of nucleotides on 3′ ends of DNA (TUNEL technique) and expressed as the apoptotic rate. MDS patients included 32 with refractory anaemia (RA), one RA with ringed sideroblasts (RARS) patient, seven RA with excess of blasts (RAEB) patients, eight patients with RAEB in transformation (RAEB‐t) and three patients with chronic myelomonocytic leukaemia (CMMoL). In addition, we also studied six cases with acute myeloid leukaemia (AML) arising from MDS (AML‐MDS) and ten control subjects. Fatal pancytopenia was the cause of death in 19 out of 51 patients. The apoptotic rate was higher in MDS patients (5·5%) than in control subjects (0·6%) and AML‐MDS patients (0·4%). The percentage of MIB‐1 positive cells was higher in MDS and AML‐MDS than in control. The percentage of CD34‐positive cells was higher in AML‐MDS, RAEB, RAEB‐t and CMMoL patients than control subjects and RA patients. Our findings indicate the activation of both the proliferative and apoptotic rates in MDS. Poor prognosis correlated significantly with higher apoptotic rates, but not with percentages of MIB‐1 and CD34‐positive cells. Our results suggest that apoptosis might be a useful prognostic factor and inhibition of apoptotic mechanisms may induce leukaemic transformation in MDS.
Two‐step immunoablative treatment with autologous peripheral blood CD34+ cell transplantation in an 8‐year‐old boy with autoimmune haemolytic anaemia
Authors: Paillard C. , Kanold J. , Halle P. , Yakouben K. , Boiret N. , Rapatel C. , Berger M. , Malpuech G. , Deméocq F.
Published: March 2003

Life‐threatening haemolysis in children with autoimmune haemolytic anaemia (AIHA) occurs rarely. Many cases of severe autoimmune disease are currently treated with immunosuppressive high‐dose chemotherapy and autograft. We report here a case of a child with severe AIHA who did not respond to conventional treatments, but was cured with an autologous peripheral blood CD34+ cell transplantation. After d 16 post autograft, no further red cell transfusions were required. At 20 months post autograft, haematological complete remission persists.
Sensitization by 5‐aza‐2′‐deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all‐trans retinoic acid and 1α,25‐dihydroxyvitamin D3
Authors: Niitsu Nozomi , Hayashi Yasuhide , Sugita Kanji , Honma Yoshio
Published: March 2003

Most chromosomal abnormalities associated with breakage at 11q23 in acute leukaemia involve the MLL gene, and the presence of this breakage strongly predicts a poor clinical outcome. We assessed the possibility of differentiation‐inducing therapy for acute leukaemias with chromosomal translocations involving 11q23. Among the cell lines with MLL translocations that we examined, KOCL48 and KOPN‐1 cells were induced to differentiate into granulocytes by all‐trans retinoic acid (ATRA) or into monocytes by 1α,25‐dihydroxyvitamin D3 (VD3). These cells expressed p16 mRNA before treatment with 5‐aza‐2′‐deoxycytidine (5‐AZA), an inhibitor of DNA methylation. On the other hand, differentiation was not induced in SN‐1, KOCL33, KOCL51 or KOCL44 cells by ATRA or VD3, and these cells did not express mRNA of this gene. However, these cells were effectively induced to differentiate by ATRA or VD3 in the presence of 5‐AZA, and concomitantly exhibited p16 gene expression, suggesting an association between DNA demethylation and restoration of sensitivity to differentiation‐inducing activity of ATRA or VD3 in leukaemia cells with MLL abnormalities. Based on these findings, combined treatment with ATRA or VD3 plus 5‐AZA may be clinically useful in therapy for acute leukaemia with MLL abnormalities.
Engagement of the CrkL adaptor in interferon α signalling in BCR–ABL‐expressing cells
Authors: Grumbach Isabella M. , Mayer Ingrid A. , Uddin Shahab , Lekmine Fatima , Majchrzak Beata , Yamauchi Hayato , Fujita Shigeru , Druker Brian J. , Fish Eleanor N. , Platanias Leonidas C.
Published: March 2003

Interferon α (IFNα) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR–ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR–ABL‐expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNα‐signalling in the CML‐derived KT‐1 cell line, which expresses BCR–ABL and is sensitive to the growth inhibitory effects of IFNα. CrkL is constitutively associated with BCR–ABL in these cells and treatment with IFNα had no effect on the BCR–ABL/CrkL interaction. After IFNα stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNα‐dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N‐terminus SH3 domains of CrkL. The resulting CrkL–Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the β‐casein promoter or the promoter of the PML gene, an IFNα‐inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT‐1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR–ABL in CML‐derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti‐proliferative effects of IFNα in CML.
Mutations in ras proto‐oncogenes are associated with lower mdr1 gene expression in adult acute myeloid leukaemia
Authors: Schaich M. , Ritter M. , Illmer T. , Lisske P. , Thiede C. , Schäkel U. , Mohr B. , Ehninger G. , Neubauer A.
Published: March 2003

Mutations in ras genes have been found to be the most frequent genetic aberrations in adult myeloid leukaemia (AML). Some reports have shown an improved outcome of ras‐mutated AML. In order to understand the biology of ras mutation in AML, we studied a cohort of patients treated in a prospective multicentre trial for ras mutational status and resistance gene expression. Blast samples from 162 adult patients with de novo or secondary AML were examined for resistance gene expression (mdr1, mrp1 and lrp) and ras mutations using reverse transcription‐polymerase chain reaction and protein nucleic acid‐competitive polymerase chain reaction strategies respectively. Ras mutations were confirmed using DNA sequencing. Ras mutations leading to an exchange of amino acids were found in 40 (25%) patients. Thirty AML patients had N‐ras mutations and nine patients had K‐ras mutations. One patient showed both N‐ras and K‐ras mutations. Resistance gene expression was positive for mdr1 in 30%, for mrp1 in 43% and for lrp in 62% of patients. There was a strong inverse correlation between the presence of ras mutation and mdr1 expression (P = 0·005). However, no significant difference was seen between patients with or without ras mutations and mrp1 or lrp expression. Whereas mdr1 expression was associated with a lower complete remission rate (P < 0·04), ras mutations had no significant influence on remission status. Neither ras mutation nor mdr1 expression had a significant impact on overall or disease‐free survival to date. For the first time, there is evidence that activated ras genes are associated with lower mdr1 expression in AML.
The superoxide dismutase content in erythrocytes predicts short‐term toxicity of high‐dose cyclophosphamide
Authors: Dumontet Charles , Drai Jocelyne , Thieblemont Catherine , Hequet Olivier , Espinouse Daniel , Bouafia Fadela , Salles Gilles , Coiffier Bertrand
Published: March 2003

Patients receiving high‐dose cyclophosphamide as a conditioning regimen for peripheral stem cell collection are subjected over a short period of time to significant exposure to reactive oxygen species (ROS). All these patients undergo profound leucopenia. Various other short‐term toxicities are observed in a fraction of the patients, including febrile aplasia requiring hospitalization, thrombocytopenia and mucositis. Although stem cell collection is feasible in the majority of patients stimulated with haematopoietic growth factors, in some instances, graft collection cannot be performed because of insufficient concentrations of stem cells in peripheral blood. There is currently no predictive assay to determine which patients treated with high‐dose cyclophosphamide have a high risk of febrile aplasia or will successfully undergo cytaphereses for stem cell collection. In order to identify such predictive factors, we analysed the level of expression before treatment of various ROS detoxification mechanisms in the peripheral blood of 37 patients receiving high‐dose cyclophosphamide for lymphoproliferative diseases. Various parameters involved in the metabolism of ROS were measured in plasma and/or erythrocytes, including superoxide dismutase, glutathione, glutathione peroxidase, glutathione reductase and malondialdehyde. High levels of erythrocyte superoxide dismutase before cyclophosphamide therapy were correlated with an increased risk of hospitalization for febrile aplasia (65% vs. 29%, P = 0·013). High superoxide dismutase and low erythrocyte glutathione reductase were associated with lower CD34 yields. These data suggest that components of the ROS detoxification system modulate the degree of short‐term toxicity of cyclophosphamide and could be used as predictive markers in individual patients.
In vivo suppression of Bcl‐XL expression facilitates chemotherapy‐induced leukaemia cell death in a SCID/NOD‐Hu model
Authors: Fennell Dean A. , Corbo Margherita V. , Dean Nicholas M. , Monia Brett P. , Cotter Finbarr E.
Published: March 2003

Bcl‐XL, a member of the Bcl‐2‐related anti‐apoptosis protein family, antagonizes a diverse range of apoptosis‐inducing stimuli by preventing mitochondrial permeability transition, release of apoptogenic factors including cytochrome C, and caspase activation. We have tested the hypothesis that the susceptibility of Bcl‐XL‐expressing leukaemic cells to apoptosis induced by VP16 (etoposide) can be enhanced by pharmacological downregulation of Bcl‐XLin vivo. Two subcutaneous xenograft models of B‐cell leukaemia‐employing SEMK‐2 and BV173 cell lines were established in severe combined immunodeficient/non‐obese diabetic mice followed by 14 d of continuous subcutaneous administration of Bcl‐XL‐specific second generation oligonucleotides ISIS 16009 or ISIS 15999. Tumours were disaggregated, enabling investigation of Bcl‐XL expression and apoptosis susceptibility at single‐cell resolution using cytofluorimetry. Marked sequence‐specific reduction of Bcl‐XL was associated with sequence‐specific enhancement of VP16‐induced mitochondrial permeability transition, caspase‐3 activation and loss of membrane asymmetry. A negative correlation between Bcl‐XL expression and apoptosis susceptibility was observed, together with a positive correlation with respect to a reduced redox state. Bcl‐XL downregulation reduces the threshold for VP16‐induced apoptosis by potentiating mitochondrial dysfunction and its sequelae, and therefore presents a novel therapeutic strategy for reversing chemoresistance.
The clinical features, risk factors and outcome of thrombotic thrombocytopenic purpura occurring after bone marrow transplantation
Authors: Fuge Rhian , Bird Jennifer M. , Fraser Anne , Hart Dan , Hunt Linda , Cornish Jacqueline M. , Goulden Nicholas , Oakhill Anthony , Pamphilon Derwood H. , Steward Colin G. , Marks David I.
Published: March 2003

In this study, we retrospectively analysed the clinical features, risk factors and outcome of 22 patients with thrombotic thrombocytopenic purpura (TTP) occurring after allogeneic stem cell transplantation. All but two of these patients received stem cells from unrelated donors (UDs), two‐thirds were female, three‐quarters were adults and leukaemia was the major reason for transplant. The incidence of TTP was 20 out of 332 patients (6%) with UD transplants and two out of 104 recipients (2%) of matched sibling allografts (P = 0·16). In order to ascertain basic demographic risk factors for the development of TTP, we compared the 22 patients with 434 patients who did not develop TTP. Compared with patients who did not develop TTP, patients with TTP were nearly three times older (P < 0·001) and were more than twice as likely to be female (P = 0·001). Because > 90% of patients were recipients of UD marrow, we then compared the 20 UD‐bone marrow transplantation (BMT) patients with 60 randomly selected UD‐BMT patients who did not develop TTP. On univariate analysis, age and female gender were again significant risk factors, as was grade II–IV acute graft‐versus‐host disease (GvHD) (P = 0·002), and there was a trend towards an association with chronic GvHD (P = 0·083). However, after logistic regression analysis, only age and sex remained significant (P < 0·001 and 0·009 respectively). We report an 86% mortality with only three survivors out of 22 patients, and one of these remains thrombocytopenic and red cell transfusion dependent, possibly in part because of graft hypoplasia. Six out of 17 patients responded to plasmapheresis, but the majority of them ultimately succumbed because of TTP, often in association with GvHD or fungal infection.
Activation of coagulation factor XI, without detectable contact activation in dengue haemorrhagic fever
Authors: Van Gorp Eric C. M. , Minnema Monique C. , Suharti Catharina , Mairuhu Albert T. A. , Brandjes Dees P. M. , Ten Cate Hugo , Hack C. Erik , Meijers Joost C. M.
Published: March 2003

A prospective cohort study was performed in 50 patients with dengue haemorrhagic fever (DHF) to determine the potential role of the contact activation system and factor XI activation (intrinsic pathway) in the coagulation disorders in DHF. To establish whether TAFI (thrombin‐activatable fibrinolysis inhibitor) was involved in the severity of the coagulation disorders, the TAFI antigen and activity levels were also determined. Markers of contact activation (kallikrein‐C1‐inhibitor complexes), the intrinsic pathway of coagulation (factor XIa–C1‐inhibitor complexes) and TAFI were measured and correlated to thrombin generation markers (thrombin–anti‐thrombin complexes (TAT), prothrombin fragment 1+2 (F1+2)) and a marker for fibrinolysis [plasmin–α2–anti‐plasmin complexes (PAP)]. Activation of the intrinsic pathway of coagulation was clearly demonstrated by elevated levels of factor XIa–C1‐inhibitor complexes, without evidence of contact activation, reflected by undetectable kallikrein–C1‐inhibitor complexes. Both TAFI antigen and activity levels were decreased in all patients, which may contribute to the severity of bleeding complications in DHF because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis. These findings in a human viral infection model are in accordance with earlier findings in bacterial sepsis.
Screening for Aspergillus spp. using polymerase chain reaction of whole blood samples from patients with haematological malignancies
Authors: Lass‐Flörl Cornelia , Aigner Johannes , Gunsilius Eberhard , Petzer Andreas , Nachbaur David , Gastl Günther , Einsele Hermann , Löffler Jürgen , Dierich Manfred P. , Würzner Reinhard
Published: March 2003

Sensitive screening for Aspergillus spp. using polymerase chain reaction (PCR) of whole blood samples in patients with haematological disorders has not been performed to date. In a 2‐year study, 121 patients admitted to the University Hospital of Innsbruck for cancer chemotherapy without clinical signs of fungal infection were prospectively screened for Aspergillus spp. In 28 out of 121 (23%) patients, Aspergillus DNAaemia was detected. Of these patients, 16 (57%) were positive only once for Aspergillus DNA, but positivity was never associated with invasive aspergillosis. PCR positive episodes were short and resolved without antifungal treatment. Five patients (18%) had intermittent PCR positive results. Seven (25%) patients presented at least two consecutive positive PCR results; one of these patients developed invasive aspergillosis and another two were strongly suspected as having aspergillosis. Based on the criteria of the European Organization for Research and Treatment of Cancer case definitions, sensitivity and specificity of serial PCR monitoring were 75% and 96%. Positive PCR results became negative shortly after commencement of antifungal treatment, but the changes did not correlate with clinical responsiveness to treatment in three patients. Our results indicate the potential usefulness of PCR for screening for Aspergillus spp. in patients at risk, but without antifungal treatment.
Flow cytometric analysis of the association between blood group‐related proteins and the detergent‐insoluble material of K562 cells and erythroid precursors
Authors: Gane Pierre , Le Van Kim Caroline , Bony Viviane , Nemer Wassim El , Mouro Isabelle , Nicolas Virginie , Colin Yves , Cartron Jean‐Pierre
Published: March 2003

The linkage between blood group‐related cell surface proteins and the detergent‐insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody‐binding capacity of intact and Triton‐X100‐treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton‐insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton‐insoluble. Glycophorin A (GPA), however, behaved as Triton‐soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton‐insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM‐associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM‐associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.
Two consecutive immunophenotypic switches in a child with immunogenotypically stable acute leukaemia
Authors: Bierings M. , Szczepański T. , Van Wering E. R. , Willemse M. J. , Langerak A. W. , Révész T. , Van Dongen J. J. M.
Published: March 2003

A 12‐year‐old girl presented with a CD33+ precursor B‐acute lymphoblastic leukaemia (ALL) and seemed to respond well to ALL treatment. However, 2 weeks after diagnosis her leucocyte count rose rapidly with a predominance of myeloid blasts with M5b morphology and CD19+ myeloid immunophenotype. Acute myeloid leukaemia (AML) treatment was started and remission was achieved after one course of chemotherapy; the AML treatment was continued for 6 months. Two months after cessation of chemotherapy, the patient developed a bone marrow relapse, this time with an undifferentiated blast morphology and a precursor B immunophenotype. Molecular analysis of the immunoglobulin and T‐cell receptor genes showed several clonal gene rearrangements at diagnosis: two IGH, two IGK and two TCRD gene rearrangements. All rearrangements were also detected during the AML phase of the disease, suggesting a phenotypic shift of the same leukaemia. At relapse, 8 months later, all rearrangements were preserved except for one TCRD (Vδ2–Dδ3) rearrangement. The first phenotypic shift in the genotypically stable leukaemia was remarkably fast. The most probable explanation for our observations is an oncogenic event in an undifferentiated haematopoetic progenitor clone, with a highly versatile phenotype.
Gemcitabine as a single agent in the treatment of relapsed or refractory low‐grade non‐Hodgkin's lymphoma
Authors: Dumontet C. , Morschhauser F. , Solal‐Celigny P. , Bouafia F. , Bourgeois Emmanuelle , Thieblemont C. , Leleu Xavier , Hequet O. , Salles G. , Coiffier B.
Published: March 2003

A multicentre phase II trial was conducted to evaluate the efficacy and toxicity of gemcitabine in patients with refractory or relapsed indolent non‐Hodgkin's lymphoma. Thirty‐six patients were enrolled onto the study, including 11 cases of mantle cell lymphoma (MCL), 10 cases of chronic lymphocytic leukaemia (CLL)/lymphocytic lymphoma, nine cases of follicular lymphoma, four cases of lymphoplasmacytic lymphoma and two cases of T‐cell lymphoma. Gemcitabine 1 g/m2 was administered as a 30‐min infusion on d 1, 8 and 15 of a 28‐d schedule, up to a maximum of six cycles. Complete responses were observed in two patients with MCL, and partial responses were observed in seven patients, including three patients with CLL/lymphocytic lymphoma, two patients with T‐cell lymphoma, one patient with MCL and one patient with follicular lymphoma. Minor responses were observed in three patients, including two patients with MCL and one patient with CLL. The median duration of response was 150 d and the overall progression‐free survival was 342 d. Haematological toxicity was observed as grade 3–4 leucopenia in 12 patients (33%) and grade 3–4 thrombocytopenia in 18 patients (50%). Severe non‐haematological toxicity included one case of fatal veno‐occlusive disease, one case of thrombotic microangiopathy leading to terminal renal failure, one case of capillary leak syndrome, one case of myocardial infarction and drug‐induced fever in two patients. These data suggest that gemcitabine displays activity in patients with MCL and CLL/lymphocytic lymphoma. Haematological toxicity was frequent in these heavily treated patients. Severe non‐haematological toxicity was significant and should be taken into account in the design of future trials.
Non‐transferrin‐bound iron during allogeneic stem cell transplantation
Authors: Sahlstedt Leila , Ebeling Freja , Von Bonsdorff Leni , Parkkinen Jaakko , Ruutu Tapani
Published: March 2003

Hydroxyl radical formation catalysed by non‐transferrin‐bound iron (NTBI) might contribute to transplantation‐related complications. The occurrence of NTBI in 10 adult allogeneic stem cell transplantation (SCT) patients was followed for 20 d. The transferrin saturation reached 99% on d −4 and remained > 80% thereafter. NTBI, measured as bleomycin‐detectable iron, was detected for 6–18 d in all patients with a peak on d −4. High transferrin saturation levels were associated with the appearance of NTBI with a threshold at 80% saturation. Prevention of the potential deleterious effects of NTBI might reduce transplantation‐related morbidity.
The risk of recurrent venous thromboembolism among heterozygous carriers of the G20210A prothrombin gene mutation
Authors: De Stefano Valerio , Martinelli Ida , Mannucci Pier Mannuccio , Paciaroni Katia , Rossi Elena , Chiusolo Patrizia , Casorelli Ida , Leone Giuseppe
Published: March 2003

The G20210A mutation in the prothrombin gene is associated with an increased risk of a first venous thromboembolic episode; few data are available about the long‐term risk for recurrent venous thromboembolism and it is not known whether or not carriers of the mutation should be recommended lifelong anticoagulant treatment after the first thrombosis. We investigated 624 patients, referred for previous objectively documented deep venous thrombosis of the legs or pulmonary embolism, to determine the risk of recurrent thromboembolism in heterozygous carriers of the G20210A mutation in the prothrombin gene after the first episode of venous thromboembolism. After exclusion of other inherited (anti‐thrombin, protein C, protein S deficiency and factor V Leiden) or acquired (anti‐phospholipid antibody syndrome) causes of thrombophilia, 52 heterozygous carriers of the prothrombin mutation were compared with 283 patients with normal genotype. The relative risk for recurrent venous thromboembolism was calculated between groups using a Cox's proportional hazard model. The patients with the prothrombin mutation had a risk for spontaneous recurrent venous thromboembolism similar to that of patients with normal genotype (hazard ratio 1·3; 95% CI, 0·7–2·3). The circumstances of the first event (spontaneous or secondary) did not produce any substantial variation in the risk for recurrence. In conclusion, the carriers of the prothrombin mutation should be treated with oral anticoagulants after a first deep venous thrombosis for a similar length of time as patients with a normal genotype.
Incidence of factor VIII inhibitors in severe haemophilia: the importance of patient age
Authors: Lorenzo José Ignacio , López Aurelio , Altisent Carmen , Aznar José Antonio
Published: March 2003

The potential effect of age at the start of replacement therapy on the development of factor VIII (FVIII) inhibitors was assessed in 62 severe (FVIII < 2 IU/dl) haemophilia A patients who started FVIII therapy at one of two haemophilia centres. Inhibitors were tested on an annual basis. Persistent or high‐titre inhibitors were detected in 15 patients (24%). Kaplan–Meier cumulative incidence at 3 years from first FVIII exposure was 41% (95% CI 22–67%) in patients starting therapy before the age of 6 months, 29% (95% CI 13–57%) in patients starting therapy between 6 and 12 months of age, and 12% (95% CI 4–34%) in those starting therapy beyond 1 year of age (P = 0·03). By multivariate analysis, the influence of age was shown to be independent of other variables, including calendar year at the onset of therapy and baseline FVIII plasma levels. In conclusion, patient age at initial treatment appears to influence inhibitor formation. If confirmed, this finding would have a major impact on the management of haemophilia.
Interaction of cytokines and growth factor in the regulation of verotoxin‐induced apoptosis in cultured human endothelial cells
Authors: Molostvov G. , Morris A. , Rose P. , Basu S.
Published: March 2003

The role of CD77, inflammatory cytokines and endothelial cell growth factor (ECGF) in verotoxin (VT)‐induced apoptosis in human umbilical vein endothelial cells (HUVECs) was studied. Apoptosis was detected using annexin V and propidium iodide staining. The expression of CD77 antigen was measured on a FACStar flow cytometer using specific monoclonal antibodies. Our experiments showed that HUVECs had very low initial levels of CD77 and were resistant to VT. Treatment with tumour necrosis factor alpha (TNF‐α) resulted in a significant upregulation of CD77 expression and sensitized endothelial cells to VT‐mediated apoptosis. HUVECs incubated with a combination of cytokines [TNF‐α and interferon gamma (IFN‐γ), both 500 U/ml] showed more pronounced upregulation of CD77 expression (> sixfold at 48 h) and underwent apoptosis, suggesting that TNF‐α and IFN‐γ have a synergistic effect on CD77 expression in HUVECs and can induce apoptosis without VT. Cells pretreated with TNF‐α and IFN‐γ and incubated with VT showed the most pronounced (14‐fold) increase in CD77 expression. ECGF had a partial protective effect against cytokine‐ and VT‐induced apoptosis. Taken together, our data suggest that CD77 antigen is involved in the regulation of endothelial cell apoptosis.