bluuebunny/crossref_metadata_2025 huggingface dataset:
Coexistence of normal and clonal haemopoiesis in aplastic anaemia patients treated with immunosuppressive therapy
Authors: Piaggio  , Podestà  , Pitto  , Sessarego  , Figari  , Fugazza  , Benvenuto  , Bruno  , Van Lint  , Truini  , Frassoni  , Bacigalupo
Published: March 2003

Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow‐up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI‐AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG‐AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG‐AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively).

This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow‐up of 4 years.
Generation of anti‐idiotype immune responses following vaccination with idiotype‐protein pulsed dendritic cells in myeloma
Authors: Cull  , Durrant  , Stainer  , Haynes  , Russell
Published: March 2003

Myeloma cells produce immunoglobulin which is unique to the malignant clone and presents antigenic determinants, or idiotypes, which may function as a tumour‐specific antigen. The availability of significant quantities of idiotype protein in the serum makes immunotherapeutic strategies utilizing this protein to generate an anti‐idiotype immune response an attractive prospect. We treated two patients with advanced refractory myeloma with a series of four vaccinations using autologous idiotype‐protein pulsed dendritic cells combined with adjuvant GM‐CSF. The vaccinations were well tolerated with a mild fever post‐vaccination in one patient. An idiotype‐specific T‐cell proliferative response developed in both patients. This T‐cell response was associated with the production of γ‐interferon, indicating a TH‐1‐like response. Furthermore, one patient developed anti‐idiotype IgM antibodies. However, no idiotype‐specific cytotoxic T‐cell response could be demonstrated. Further investigation is warranted to define the optimal conditions for dendritic cell culture and priming to maximize the anti‐tumour immune response.
Clinical significance of urinary albumin excretion in patients with non‐Hodgkin's lymphoma
Authors: Pedersen Lars Møller , Sørensen Peter Grundtvig
Published: March 2003

Slightly increased urinary albumin excretion rates (UAE) have been reported in patients with various types of human cancer. We measured UAE in 24 h urine samples from 48 untreated patients with non‐Hodgkin's lymphoma at diagnosis. In patients with a pretreatment UAE 20 μg/min, post‐treatment value of UAE was determined following completion of the last treatment. The median UAE was 15.0 μg/min and the prevalence of microalbuminuria (UAE 20 μg/min) was 39.6%. Increased UAE was significantly associated with Ann Arbor stage, performance status, serum lactate dehydrogenase (LDH) level, and the International Prognostic Index (IPI). The median posttreatment value of UAE was significantly lower than the pretreatment value (P < 0.0001). Our data suggest a clinical and prognostic significance of UAE in patients with non‐Hodgkin's lymphoma.
Phenotypic and functional characteristics of monocyte‐derived dendritic cells from patients with myelodysplastic syndromes
Authors: Matteo Rigolin Gian , Howard Julie , Buggins Andrea , Sneddon Claire , Castoldi Gianluigi , Hirst William J. R. , Mufti Ghulam J.
Published: March 2003

We have compared the phenotypic and functional characteristics of dendritic cells (DC) generated in vitro from the peripheral blood mononuclear fraction of myelodysplastic syndrome (MDS) patients (four refractory anaemia, four refractory anaemia with excess of blasts) with DCs generated in a similar way from eight healthy donors. After 10 d of culture in the presence of GM‐CSF and IL‐4, reduced numbers and percentages of DCs were obtained in MDS subjects. MDS DCs exhibited significantly lower expression of CD1a, CD54, CD80 and MHC class II molecules. Their ability to stimulate T lymphocytes in an allogeneic mixed leucocyte reaction was reduced in comparison to normal subjects. Furthermore, MDS DCs also showed a reduced receptor‐mediated endocytosis as demonstrated by FITC‐dextran uptake. Simultaneous fluorescence in situ hybridization (FISH) and immunophenotypic analysis demonstrated that MDS DCs have the same cytogenetic abnormality of the malignant clone. Taken together these findings indicate that, in MDS, DCs are part of the malignant clone and exhibit a deficient antigen uptake and presentation.
Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins
Authors: Bendz BjOslash;rn , Hansen John‐Bjarne , Andersen Trine O. , Oslash;stergaard Per , Sandset Per Morten
Published: March 2003

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross‐over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti‐factor Xa activity and anti‐factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre‐injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.
The prothrombin Denver patient has two different prothrombin point mutations resulting in Glu‐300→Lys and Glu‐309→Lys substitutions
Authors: Lefkowitz Jerry B. , Haver Tara , Clarke Susan , Jacobson Linda , Weller Ann , Nuss Rachelle , Manco‐Johnson Marilyn , Hathaway William E.
Published: March 2003

Dysprothrombinaemia is a rare, congenital cause of bleeding. Fewer than 25 families who express a functional prothrombin (factor II) defect have been reported. The original patient with prothrombin Denver had a severe haemophilia‐like bleeding disorder treated with weekly prophylactic factor replacement. Analysis of factor II activity and antigen in the patient showed a factor II activity of 5 units/dl and factor II antigen of 21 units/dl. Genomic DNA from the patient, mother and brother was obtained from peripheral blood white cells. Oligonucleotides were constructed, and prothrombin exons were amplified via polymerase chain reaction (PCR). The entire sequence of the thrombin portion of the molecule (exons VIII–XIV) and that of exons I–II and IV–VII was determined. This moderately severe dysprothrombinaemia was found to be associated with compound heterozygosity for two different Glu→Lys point mutations, at amino acid positions 300 and 309. Assays of plasma from the prothrombin Denver proband suggested that the functional defect was in the activation of zymogen to enzyme.
Effects of interferon and hydroxyurea on bone marrow fibrosis in chronic myelogenous leukaemia: a comparative retrospective multicentre histological and clinical study
Authors: Thiele Juergen , Kvasnicka Hans Michael , Schmitt‐Graeff Annette , Spohr Marlene , Diehl Volker , Zankovich Rudolf , Niederle Norbert , Leder Lutz‐Dietrich
Published: March 2003

A retrospective multicentre clinicopathological study was performed on sequential bone marrow trephine biopsies in 100 patients with Ph1+‐chronic myelogenous leukaemia (CML) to elucidate the effect of interferon (IFN) α 2b and hydroxyurea (HU) treatment on myelofibrosis and megakaryopoiesis. According to strictly defined therapeutic regimens, 38 patients received IFN as monotherapy, 23 patients a combination of IFN and HU and 39 patients HU only. Using standardized intervals of biopsies and histochemical and morphometric methods, a significant increase in reticulin fibre density and in the number of CD61+ megakaryocytes was detectable in the majority of IFN‐treated patients. To a lesser degree, these changes were also expressed in the cohort with a combined IFN and HU regimen. In contrast to these findings, in the group of patients with HU as single‐agent treatment, a stable state or reversal of myelofibrosis was detectable together with corresponding changes in megakaryopoiesis. Further evaluations revealed that these effects had occurred within the first year, mostly after 6 months of treatment, and were prominently expressed in those patients with a slight to relevant grade of myelofibrosis at presentation. In conclusion, this study provides persuasive evidence that monotherapy by IFN exerts a fibrogenic effect, while HU treatment seems to prevent and even resolves bone marrow fibrosis in CML. Probably, in relation to the complex pathomechanisms responsible for the generation of myelofibrosis, the changing content of reticulin fibres was usually accompanied by corresponding alterations in the number of CD61+ megakaryocytes, including atypical microforms and precursor cells.
A rapid one‐stage whole‐blood HPA‐1a phenotyping assay using a recombinant monoclonal IgG1 anti‐HPA‐1a
Authors: Garner  , Smethurst  , Merieux  , Aeby  , Smith  , Armour  , Scott  , Williamson  , Metcalfe  , Goodall  , Clark  , Rigal  , Schawaller  , Ouwehand
Published: March 2003

Severe neonatal alloimmune thrombocytopenia and patients with HPA‐1a‐specific antibodies require transfusion of HPA‐1a‐negative platelets. Identifying HPA‐1a‐negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)‐conjugated recombinant IgG1 anti‐HPA‐1a (CAMTRAN007) to develop a rapid and reliable enzyme‐linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA‐1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA‐1a genotype demonstrated that HPA‐1a‐negative samples had OD values of < 0.266, whereas HPA‐1a‐positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut‐off values and identified 45 HPA‐1a‐negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA‐1a‐negative sample had an OD value of 0.303. The HPA‐1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence‐specific primers (PCR‐ SSP).
Human osteoblast‐like cells and osteosarcoma cell lines synthesize macrophage inhibitory protein 1α in response to interleukin 1β and tumour necrosis factor α stimulation in vitro
Authors: Taichman  , Reilly  , Matthews
Published: March 2003

Recent investigations have demonstrated that macrophage inhibitory protein 1α (MIP‐1α) plays a critical role in haematopoiesis. In part, MIP‐1α limits the differentiation of early haematopoietic cells, thereby ensuring that sufficient quantities of blood precursors are available to meet haematopoietic demands. MIP‐1α is produced by cells of the marrow microenvironment (marrow stromal cells) in response to a variety of stimuli, including interleukin 1β (IL‐1β) and tumour necrosis factor α (TNF‐α). Our recent investigations demonstrated that normal human osteoblast‐like cells (HOBs) maintain the early phenotype of haematopoietic precursors, like other members of the bone marrow stroma. Although the precise molecular mechanisms for these observations have not been determined, the production of MIP‐1α remains one such possibility. In the present study, we investigated whether cells of the osteoblast lineage under basal, IL‐1β and/or TNF‐α stimulation produce MIP‐1α. We observed that IL‐1β and TNF‐α stimulated HOBs and human osteosarcoma cells to rapidly express MIP‐1α mRNA and to secrete large quantities of the protein. MIP‐1α mRNA and protein was not, however, detected under basal conditions. Perhaps more importantly, enriched human CD34+ bone marrow cells in co‐culture may be capable of stimulating the expression of MIP‐1α mRNA by HOBs in vitro. These findings suggest that human osteoblast‐like cells may produce MIP‐1αin vivo to support haematopoiesis at sites where osteoblasts and haematopoietic cells are closely associated.
Waldenström's macroglobulinaemia: a prospective study comparing daily with intermittent oral chlorambucil
Authors: Kyle Robert A. , Greipp Philip R. , Gertz Morie A. , Witzig Thomas E. , Lust John A. , Lacy Martha Q. , Therneau Terry M.
Published: March 2003

This prospective study compared continuous and intermittent chlorambucil therapy of untreated Waldenström's macroglobulinaemia. The diagnosis was established by the presence of an IgM monoclonal (M‐) protein in the serum, an infiltrate of lymphocytes and plasma cells in the bone marrow, anaemia or other laboratory abnormalities, physical findings or constitutional symptoms. Patients were randomized to receive chlorambucil 0.1 mg/kg/d or chlorambucil 0.3 mg/kg/d orally for 7 d, repeated every 6 weeks. Criteria for response included 50% or more reduction of serum M‐protein, increase in haemoglobin level of 2 g/dl without transfusion, ≥ 50% decrease of urine M‐protein, or a reduction of 2 cm in the size of the liver, spleen or lymph nodes. Forty‐six patients were randomized to continuous chlorambucil (n = 24) or to intermittent chlorambucil (n = 22). Nineteen (79%) (95% CI = 58–93) of the 24 patients given continuous therapy had an objective improvement by either reduction of serum M‐protein or increase in haemoglobin. Fifteen (68%) (95% CI = 45–86) of the 22 patients given chlorambucil intermittently had an objective response. The size of the liver decreased by ≥ 2 cm in 55% of patients, and the size of the spleen decreased ≥ 2 cm in 67%. Lymphadenopathy decreased in 71%. Acute leukaemia or refractory anaemia developed in four patients. The median duration of survival was 5.4 years, and there was no difference between the regimens. Chlorambucil is effective for the treatment of Waldenström's macroglobulinaemia. Patients must be treated for at least 6 months before therapy is abandoned because response is slow. Chlorambucil is an effective agent and should be compared with purine analogues or rituxan (Rituximab) in a prospective study.
Interleukin 10 regulates cellular responses in monocyte/endothelial cell co‐cultures
Authors: Noble  , Harkness  , Yong
Published: March 2003

Adhesive interactions between monocytes and vascular endothelial cells increase the expression of the inflammatory genes, tissue factor (TF) and E‐selectin, thus contributing to the inflammatory process. In this study, we have shown that these responses could be regulated by the immunomodulatory cytokine interleukin 10 (IL‐10). IL‐10 reduced TF generation in monocyte/endothelium co‐cultures (64.3 ± 3.3% reduction, P < 0.01, n = 4) by acting directly on monocytes, whereas IL‐4 inhibited TF expression in both monocytes and endothelium. Similarly, IL‐10 reduced the induction of endothelial E‐selectin by monocytes (100% reduction at 21 h), but had no effect on cytokine‐induced E‐selectin expression. IL‐10 itself was not able to induce E‐selectin protein or mRNA in endothelial cells. IL‐10 mRNA was detected in monocytes after 6 h co‐culture with endothelial cells, and was sustained for up to 30 h. Finally, IL‐10 significantly reduced the adhesion of monocytes to endothelium (45% reduction), which may account in part for the inhibitory actions of IL‐10. We conclude that IL‐10 has an anti‐inflammatory effect on monocyte/endothelium interactions, and may itself be produced as a result of such interactions.
Monovalent binding of autoantibodies to β2‐glycoprotein I, detected using surface plasmon resonance at low antigen density
Authors: Regnault Véronique , De Maistre Emmanuel , Wahl Denis , Lecompte Thomas
Published: March 2003

The precise mechanism of interaction between autoantibodies and β2‐glycoprotein I (β2GPI) and the experimental conditions to be used for their detection are still under debate. Until now, these interactions have been studied under static conditions. We have investigated the interactions of purified IgG from 25 lupus anticoagulant‐positive patients with immobilized β2GPI under flow conditions by real‐time analysis based on surface plasmon resonance technology. Sensor chips were coated with purified human β2GPI coupled to dextran via amino groups at low densities (1·4, 1·8 or 2·4 ng β2GPI/mm2). Four patients' IgG displayed efficient binding and had the highest so‐called antiphospholipid IgG levels by enzyme‐linked immunosorbent assay (ELISA) and the highest absorbance values in an anti‐ β2GPI ELISA at a β2GPI density reported to be around 12 ng/mm2. Binding of antibodies to the β2GPI sensor chips proved to be dependent upon the IgG concentration and β2GPI density and was inhibited by a rabbit antibody against β2GPI. Similar association and dissociation profiles were observed for the four efficient binders. The fast rate of dissociation limited the binding of autoantibodies to β2GPI and was highly suggestive of a monovalent association, confirmed by binding of Fab fragments under similar experimental conditions. In conclusion, monovalent binding of low‐affinity antibodies to β2GPI immobilized at a density as low as 1·8 ng/mm2 could be detected using surface plasmon resonance.
Long‐term survival of infants with idiopathic myelofibrosis
Authors: Altura Rachel A. , Head David R. , Wang Winfred C.
Published: March 2003

Idiopathic myelofibrosis can develop in children as well as adults. However, the disease appears to be much more aggressive in adults, being characterized by poor survival rates and a high frequency of malignant transformation. Here, we describe three cases of idiopathic myelofibrosis in infants, two of whom were followed for 16 and 22 years after diagnosis. Neither of these patients required more than minimal supportive care, and both have had spontaneous erythropoietic recovery as early as 2–3 years after diagnosis. There have been no indications of malignant transformation or clinical deterioration. Thus, idiopathic myelofibrosis may have a different pathogenesis and clinical course in infants from adults, requiring a more conservative approach to management.
Summary curves for patients transplanted for chronic myeloid leukaemia salvaged by a donor lymphocyte infusion: the current leukaemia‐free survival curve
Authors: Klein John P. , Keiding Niels , Shu Youyi , Szydlo Richard M. , Goldman John M.
Published: March 2003

A significant number of patients who relapse after allogeneic stem cell transplantation (SCT) for chronic myeloid leukaemia (CML) will achieve sustained molecular remissions after treatment with donor lymphocyte infusions (DLI) from the original stem cell donor. Leukaemia‐free survival, defined as survival without evidence of relapse at any time after transplant does not account for patients who are successfully treated with DLI. To summarize adequately the response to treatment, a new summary probability, called the current leukaemia‐free survival (CLFS), is proposed. This quantity is defined as the probability that a patient is alive and in remission at a given time after transplant. We discuss two statistical methods for estimating CLFS. The first is based on a multistate modelling approach. The second is based on an estimate constructed by looking at appropriate differences between Kaplan–Meier estimates. We compare these estimates using data on 189 consecutive patients who underwent SCT over a 7‐year period.
Association of extracellular matrix proteins fibulin‐1 and fibulin‐2 with fibronectin in bone marrow stroma
Authors: Gu Yu‐Chen , Nilsson Kenneth , Eng Hubert , Ekblom Marja
Published: March 2003

Extracellular matrix (ECM) molecules, together with growth factors and stromal cells, regulate haematopoietic cell development in bone marrow (BM). We report here expression of ECM proteins fibulin‐1 and fibulin‐2 in mouse BM. In other tissues, fibulin‐1 and fibulin‐2 associate with fibronectin and other ECM proteins. Fibulin‐2 has also been found to adhere to cells via β3 integrins. We studied the association of fibulins with fibronectin in BM stroma. By confocal microscopy, fibulin‐1 and fibulin‐2 immunostainings were co‐localized with fibronectin in the adherent layer of long‐term BM cultures. In cell adhesion assays using recombinant proteins, mouse fibulin‐2 adhered to human erythroid‐megakaryocytic leukaemia cell line HEL. This adhesion was mediated by β3 integrins. However, HEL cells did not adhere to human fibulin‐2. We therefore studied a possible species‐specific cell‐adhesive activity of mouse fibulin‐2 by using mouse megakaryocytes, obtained by culture of BM cells in the presence of thrombopoietin. These megakaryocytes did not adhere to mouse fibulin‐2. Our findings suggested that the functional role of fibulin‐1 and fibulin‐2 in BM stroma is related to binding to the major cell adhesion protein fibronectin, whereas adhesion of mouse fibulin‐2 to human cells containing the integrin β3 chain is not related to an apparent physiological function of the protein.
Affinity purification of heparin‐dependent antibodies to platelet factor 4 developed in heparin‐induced thrombocytopenia: biological characteristics and effects on platelet activation
Authors: Amiral J. , Pouplard C. , Vissac A. M. , Walenga J. M. , Jeske W. , Gruel Y.
Published: March 2003

Antibodies to heparin platelet factor 4 (H‐PF4) complexes were purified from the plasma of three patients with heparin‐induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 m NaCl at pH 7·5 (peak 1) and the remainder was obtained using 0·1 m glycine/0·5 m NaCl at pH 2·5 (peak 2). In an enzyme‐linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H‐PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H‐PF4 complexes. Finally, IgG in patient 3 only bound to H‐PF4 and was unreactive with PF4 alone. Using [14C]‐serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H‐PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.
Prevalence of factor V Leiden and prothrombin G20210A mutations in unselected patients with venous thromboembolism
Authors: De Moerloose P. , Reber G. , Perrier A. , Perneger T. , Bounameaux H.
Published: March 2003

We determined the prevalence of factor V Leiden and of prothrombin G20210A mutations in a cohort of unselected outpatients (n = 748) referred for suspected deep vein thrombosis (DVT) and/or pulmonary embolism (PE) and a pooled analysis of similar studies was also performed. Based on the clinical presentation, the prevalence of factor V Leiden was 15·7% in the 83 patients with DVT and 14·1% in the 99 patients with PE compared with 5·3% in patients without DVT and/or PE (control group). The prevalence of the prothrombin G20210A mutation did not differ among the three groups (3·9% for controls, 4·8% for DVT and 3·9% for PE patients). We then divided the 99 patients with PE by separately analysing those with PE but without DVT (n = 57) and those with PE and DVT (n = 42). Compared with the control group, the prevalence of factor V Leiden was 10·5%, odds ratio (OR) 2·10 [95% confidence interval (95% CI) 0·68–5·45] in patients with primary PE and 19·1%, OR 4·20 (95% CI 1·54–10·30) in patients with DVT and PE. For the prothrombin G20210A mutation, no statistically significant differences were found between the control group and the three other groups. In conclusion, our data and the pooled analysis indicate that patients with primary PE are less often affected by the factor V Leiden mutation. No statistically significant differences were observed between patients and controls for the prothrombin G20210A mutation.
Induction of apoptosis by vitamin D3 analogue EB1089 in NCI‐H929 myeloma cells via activation of caspase 3 and p38 MAP kinase
Authors: Park Woo Hyun , Seol Jae Goo , Kim Eun Shil , Hyun Jung Mi , Jung Chul Won , Lee Chung Choo , Binderup Lise , Koeffler H. Phillip , Kim Byoung Kook , Lee Young Yiul
Published: March 2003

EB1089, a novel 1,25‐dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI‐H929 human myeloma cells. EB1089 inhibited cell growth of NCI‐H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose‐dependent manner. We could also detect apoptosis in NCI‐H929 cells exposed to EB1089 (1 × 10−7 m for 72 h) using the sub‐G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down‐regulation of the Bcl‐2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089‐treated NCI‐H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose‐ and time‐dependent manner. In addition, EB1089 caused the down‐regulation of p44 extracellular signal‐related kinase (ERK) activity and up‐regulation of the p38 kinase activity during apoptosis of NCI‐H929 cells. These results suggest that EB1089 inhibits growth of NCI‐H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.
Successful modulation of high‐dose cytosine arabinoside metabolism in acute myeloid leukaemia by haematopoietic growth factors: no effect of ribonucleotide reductase inhibitors fludarabine and gemcitabine
Authors: Braess Jan , Wegendt Christine , Jahns‐Streubel Gerlinde , Kern Wolfgang , Keye Susan , Unterhalt Michael , Schleyer Eberhardt , Hiddemann Wolfgang
Published: March 2003

High‐dose cytosine arabinoside (AraC)‐containing regimens have shown the highest antileukaemic efficacy of all currently used regimens in the treatment of acute myeloid leukaemia (AML). This study aimed at increasing the antileukaemic potential of high‐dose AraC by raising intracellular levels of AraC triphosphate (AraCTP), which is the mediator of cytotoxicity, via biochemical modulation by inhibitors of ribonucleotide reductase (RR) or haematopoietic growth factors (HGFs). Blasts from patients with de novo AML were analysed for their formation of AraCTP under high‐dose AraC conditions (20 µm over 3 h) without prior modulation (n = 47) after a 2‐h pre‐exposure with fludarabine (50 µg/ml) (n = 40) or gemcitabine (30 ng/ml) (n = 40) and after a 48‐h pre‐exposure to granulocyte colony‐stimulating‐factor (G‐CSF; 100 ng/ml) (n = 27) or granulocyte–macrophage colony‐stimulating‐factor (GM‐CSF; 100 U/ml) (n = 28). Unmodulated formation of AraCTP (median 239·8 ng/107 cells) could not be increased via modulation by gemcitabine (232·4 ng/107 cells) or fludarabine (247·8 ng/107 cells). The lack of effect of RR inhibitors was also observed for all other known metabolites of AraC [Ara‐cytosine monophosphate (CMP), Ara‐cytosine diphosphate (CDP), AraCDP‐choline, Ara‐uridine monophosphate (UMP), Ara‐uridine diphosphate (UDP) and Ara‐uridine triphosphate (UTP)]. In contrast, pre‐exposure to HGFs led to significant increases in AraCTP formation (G‐CSF 556·0 ng/107 cells, 2·31‐fold increase, P < 0·001; GM‐CSF 447·9 ng/107 cells, 1·87‐fold increase, P < 0·0001). To establish the mechanism responsible for these effects, the activity of the rate‐limiting enzyme of AraC metabolism, deoxycytidine kinase (dCK), was investigated (n = 33). In vivo exposure to GM‐CSF led to increases in dCK activity from unmodulated values at 0 h (29·8 pmol/min/mg protein) to 34·3 pmol/min/mg protein at 24 h (1·15‐fold increase) and 54·5 pmol/min/mg protein at 48 h (1·83‐fold increase). The raise in dCK activity over 48 h was significant (P < 0·013).
A comparative study of once‐daily versus twice‐daily filgrastim administration for the mobilization and collection of CD34+ peripheral blood progenitor cells in normal donors
Authors: Anderlini Paolo , Donato Michele , Lauppe Mary J. , Huh Yang O. , Martin Thomas G. , Chan Ka‐Wah , Champlin Richard E. , Körbling Martin
Published: March 2003

Eighty‐one first‐time normal donors underwent leukapheresis for peripheral blood progenitor cell (PBPC) collection after mobilization with filgrastim administered either twice‐daily (6 µg/kg every 12 h; n = 40) or once‐daily (12 µg/kg; n = 41) subcutaneously for 3 d. The groups were similar for age, donor blood volume and target CD34+ cell dose to be collected (≥ 4 × 106 CD34+ cells/kg recipient). There was no statistically significant difference in the apheresis yield of CD34+ PBPCs (× 106) per kg recipient weight (5·6 ± 3·3 vs. 5·6 ± 4·3; P = 0·94) and per litre of blood processed (30 ± 17·2 vs. 30·4 ± 19·5; P = 0·92).
b3a2 BCR–ABL fusion peptides as targets for cytotoxic T cells in chronic myeloid leukaemia
Authors: Norbury Louise C. , Clark Richard E. , Christmas Stephen E.
Published: March 2003

Peptide sequences spanning the BCR–ABL protein junction potentially constitute novel leukaemia‐specific antigens. 9‐mer b3a2 fusion peptides have been reported to bind with high affinity to HLA‐A3, ‐A11 and ‐B8. We have studied the effect of b3a2 BCR–ABL junctional peptides on the cytotoxic T‐cell (CTL) response against normal and chronic myeloid leukaemia (CML) cells. Antigen‐presenting cells (APCs) were prepared from HLA‐A3‐ or ‐B8‐positive peripheral blood mononuclear cells (PBMCs) by incubation with phytohaemagglutinin (PHA) and interleukin (IL)‐2 for 7 d. These APCs were pulsed with the respective b3a2 junctional peptide in the presence of β2‐microglobulin and were then used to challenge autologous PBMCs at 7‐d intervals in the presence of IL‐2, IL‐6, IL‐7 and IL‐12. On subsequent exposure to target cells (either further pulsed normal APCs or unpulsed CML cells), specific HLA‐restricted CTL responses were observed against all HLA‐A3/‐B8 matched normal target cells tested, but not to targets that were HLA mismatched. Cytotoxicity was also induced against HLA‐A3/‐B8 unpulsed CML cells, but not against unmatched CML cells. These data indicate (i) that endogenous BCR–ABL junctional peptides may be presented by CML cells and (ii) that exogenous peptides are potential stimulators of autologous antileukaemic CTLs.
Incidence of mixed chimaerism and clinical outcome in 101 patients after myeloablative conditioning regimens and allogeneic stem cell transplantation
Authors: Wäsch Ralph , Bertz Hartmut , Kunzmann Regina , Finke Jürgen
Published: March 2003

In the light of reduced intensity conditioning regimens for allogeneic transplantation, monitoring of donor cell engraftment acquires new relevance. We analysed the clinical significance of haematopoietic chimaerism as a parameter of patient outcome and detection of relapse for early intervention by donor lymphocyte infusion (DLI) after allogeneic transplantation. Between July 1994 and March 1999, 101 adult patients with malignant disease were evaluated. Median follow‐up was 15 months (range 0·7–56·5) after transplantation. Patients received busulphan‐containing (n = 82) or total body irradiation (TBI)‐containing (n = 19) regimens. Fifteen out of 98 (15%) patients with predictive chimaerism analyses relapsed, 5 out of 20 (25%) with mixed chimaerism (MC) and 10 out of 78 (13%) with complete donor chimaerism (CC) before apparent relapse. Seven patients received donor lymphocyte infusions (DLI) as relapse therapy with conversion from MC to CC in all three patients with successful DLI. Our data stress the importance of chimaerism monitoring after allogeneic transplantation and demonstrate the more frequent occurrence of disease relapse in patients showing MC, rather than CC, after transplantation. Moreover, the assessment of chimaerism has been shown to be a valuable tool in monitoring the efficiency of donor lymphocyte infusions for relapse.
Reduced intensity thiotepa–cyclophosphamide conditioning for allogeneic haemopoietic stem cell transplants (HSCT) in patients up to 60 years of age
Authors: Raiola A. M. , Van Lint M. T. , Lamparelli T. , Gualandi F. , Mordini N. , Berisso G. , Bregante S. , Frassoni F. , Sessarego M. , Fugazza G. , Di Stefano F. , Pitto A. , Bacigalupo A.
Published: March 2003

Transplant‐related mortality (TRM) remains a major problem in older patients undergoing allogeneic haemopoietic stem cell transplants (HSCTs). We have therefore explored a less intensive conditioning in 33 patients with a median age of 52 years (range 43–60) transplanted from human leucocyte antigen (HLA)‐identical siblings. The underlying disease was chronic myeloid leukaemia (n = 15), acute myeloid leukaemia (n = 6), myelodysplasia (n = 7) or a chronic lymphoproliferative disorder (n = 5); 15 patients (45%) had advanced disease. The regimen consisted of thiotepa (THIO; 10 mg/kg) on day −5 and cyclophosphamide (CY; 50 mg/kg) on days −3 and −2 (total dose 100 mg/kg). The source was bone marrow (BM) (n = 17) or granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood (PB) (n = 16), which were infused without manipulation. Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and a short course of methotrexate. Mean time to achieve a neutrophil count of 0·5 × 109/l was 17 d (range 11–23) and full donor chimaerism was detected in 79% of patients by day 100. Acute GVHD grade III or IV occurred in 3% of patients. Chronic GVHD was seen in 45% of patients, with a significant difference for PB (69%) compared with BM transplants (23%) (P = 0·009). For BM grafts, the actuarial 2‐year TRM was 6%, the relapse 56% and survival 87%; for PB grafts, these figures were, respectively, 27%, 33% and 68%. Twenty‐five patients are alive at a median follow‐up of 762 d (range 216–1615) and 20 patients (60%) remain free of disease. Thirteen patients (39%) received donor lymphocyte infusion (DLI) either for persisting or relapsing disease and six patients had complete remission. In conclusion: (i) patients up to the age of 60 years can be allografted with reduced intensity conditioning; (ii) the procedure was associated with a low transplant‐related mortality, particularly for bone marrow grafts, because of a lower risk of chronic GVHD; and (iii) DLI were required after transplant in half the patients for persisting disease or relapse.
The pharmacodynamic basis for the increased antileukaemic efficacy of cytosine arabinoside‐based treatment regimens in acute myeloid leukaemia with a high proliferative activity
Authors: Braess J. , Voß S. , Jahns‐Streubel G. , Schoch C. , Haferlach T. , Kern W. , Keye S. , Schleyer E. , Hiddemann W.
Published: March 2003

The current study was initiated to explore the mechanisms underlying the previously demonstrated association between the proliferative activity of leukaemic blasts and the response to cytosine arabinoside (AraC)‐based therapy in de novo acute myeloid leukaemia (AML). The activity of key enzymes of AraC metabolism–deoxycytidine kinase (DCK), cytidine deaminase (DCD) and polymerase α (PolyA) were determined in blast cells from 33 patients. In addition, formation and retention of intracellular levels of AraC triphosphate (AraCTP) and DNA incorporation of AraC were measured, as was the proliferative activity of leukaemic blasts by [3H]‐TdR incorporation before and after stimulation with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) or granulocyte CSF (G‐CSF) for 48 h. AraC incorporation into the DNA (median 0·60 pmol/105 cells) was significantly related to the proliferative activity of AML blasts (r = 0·74, P < 0·001). Similarly, priming with GM‐CSF or G‐CSF increased both the proliferative activity of AML blasts by a median of 1·84‐ and 1·64‐fold, respectively, and the incorporation of AraC into the DNA (1·29‐ and 1·40‐fold respectively). In contrast, no relationship was found between the endogenous proliferative activity (EPA) and enzyme activities regulating AraC activation (DCK; median 4·70 pmol/min/mg protein), inactivation (DCD; median 2·92 pmol/min/mg protein) or inhibitory effects (PolyA; median 1·50 pmol/min/mg protein), nor the formation or retention of AraCTP (median 306·1 ng/107 cell and 1·6 h respectively). When samples were grouped according to EPA (more than or less than the median), slowly proliferating specimens had a higher response to cytokine priming for proliferative activity and incorporation of AraC into DNA. Clinical data of 15 patients were available. Although all eight patients with a high endogenous proliferative activity reached complete remission, only four out of seven patients with a low proliferative activity responded, whereas the other three patients were non‐responders (P = 0·077).
Significance of lung resistance‐related protein in the clinical outcome of acute leukaemic patients with reference to P‐glycoprotein
Authors: Tsuji Kazue , Motoji Toshiko , Sugawara Isamu , Shiozaki Hiroko , Wang Yan‐Hua , Motomura Sayuri , Okada Michiko , Yasunami Takeshi , Sano Fumiaki , Takahashi Masatomo , Kawada Hiroshi , Maseki Nobuo , Hoshino Shigeru , Ishida Masashi , Mizoguchi Hideaki
Published: March 2003

Lung resistance‐related protein (LRP) overexpression in leukaemic blast cells from acute leukaemia patients and the effect of LRP or P‐glycoprotein (P‐gp) on the clinical outcome of acute leukaemia were investigated individually by dividing patients into four groups. The complete remission rate of group I (LRP and P‐gp both negative) was 81·7%, group II (only LRP positive) 87·5%, group III (only P‐gp positive) 87·1% and group IV (LRP and P‐gp both positive) 40·0%. There were no statistical differences between group I and groups II or III, but a significant difference was observed between groups I, II or III and group IV. Median overall survival in group IV was significantly shorter (4·6 months) than in groups I, II or III, although no significant differences were observed between group I and groups II or III (18·9, 20·5 and 31·8 months). There was a tendency for disease‐free survival in group III to be longer than that in groups I, II or IV. The reasons for these findings are discussed. Our present results indicate that the co‐existence of LRP and P‐gp strongly influenced the effectiveness of induction chemotherapy and long‐term prognosis, whereas the isolated presence of LRP or P‐gp did not.
Central venous access devices in children with congenital coagulation disorders: complications and long‐term outcome
Authors: McMahon C. , Smith J. , Khair K. , Liesner R. , Hann I. M. , Smith O. P.
Published: March 2003

Reliable venous access is essential to facilitate the administration of prophylactic factor concentrate or blood products in children with congenital coagulation disorders and immune tolerance therapy (ITT) regimens in those who develop high responding inhibitors. Poor venous access is even more problematic in very young children, the vast majority of whom will require the insertion of central venous access devices (CVADs). Previous studies have suggested that infection rates are low and that there are few long‐term complications associated with CVAD usage. We have reviewed 86 CVADs that have been inserted, since 1988, in 58 children with congenital bleeding disorders, aged 6 d to 16·5 years, attending Great Ormond Street Hospital, London, and the National Children's Hospital, Dublin. The devices have remained in situ for 2 weeks to 92 months (median 22·5 months). Early (0–2 weeks) complications of CVAD insertion included nine bleeding episodes, one extravasation of factor concentrate, three allergic reactions to factor concentrate and five catheter infections. Overall, CVAD infection was the commonest problem encountered, with 52 devices (60%) becoming infected. Twenty‐seven CVADs (31%) required removal. Infection rates in children without inhibitors (29/68) were 1/20 patient–months or 1·6 infections/1000 patient–days, but infection rates for those with inhibitors were 1/8·5 patient–months or 4·3/1000 patient–days. Staphylococcus epidermidis was the predominant organism (25/52) isolated. Blockage of CVAD (four) and catheter disconnection (four) were the most frequently occurring non‐infectious long‐term complications. Skin erosion of the port was also seen in three children, in one child at 20 months, in one at 29 months and in one at 34 months after insertion. This study demonstrates a high CVAD infection rate and highlights the long‐term complications of CVAD usage.
The central role of the P2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity
Authors: Storey Robert F. , Sanderson Heather M. , White Ann E. , May Jane A. , Cameron Kathryn E. , Heptinstall Stan
Published: March 2003

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X1, P2Y1 and P2T receptors. Sustained ADP‐induced aggregation requires co‐activation of P2Y1 and P2T receptors. AR‐C69931MX, a selective P2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P2T receptor. The roles of the P2Y1 receptor and thromboxane A2 were assessed using the selective P2Y1 antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single‐platelet counting and platelet‐rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using [14C]‐5‐hydroxytryptamine (HT)‐labelled platelets. Ca2+ mobilization, P‐selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P2T receptor activation amplified ADP‐induced aggregation initiated by the P2Y1 receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor‐activating peptide (TRAP) and collagen, independent of thromboxane A2 synthesis, which played a more peripheral role. P2T receptor activation sustained elevated cytosolic Ca2+ induced by other pathways. These studies indicate that the P2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P2T receptor antagonists.
Fatal atypical T‐cell proliferation associated with Epstein–Barr virus infection
Authors: Hauptmann Steffen , Meru Nadine , Schewe Christiane , Jung Andreas , Hiepe Falk , Burmester Gerd Rüdiger , Niedobitek Gerald , Buttgereit Frank
Published: March 2003

We report the case of a young Caucasian man who presented with polyneuropathy and severe, ultimately fatal, congestive heart failure in the context of a chronic active Epstein–Barr virus (EBV) infection. Post‐mortem examination revealed both monoclonal and polyclonal proliferation of EBV‐positive atypical T lymphocytes within different organs. Predominant infiltration of the nervous system and heart with extensive myocardial scarring accounted for the clinical symptoms. The remarkable features of this case are (i) the occurrence in a Caucasian patient, (ii) the absence of detectable immunodeficiency, and (iii) the myocardial destruction by EBV‐infected monoclonal T cells.
Haemopoietic stem cell transplantation for advanced polycythaemia vera or essential thrombocythaemia
Authors: Jurado M. , Deeg H. J. , Gooley T. , Anasetti C. , Chauncey T. , Flowers M. E. D. , Myerson D. , Storb R. , Appelbaum F. R.
Published: March 2003

Ten patients with polycythaemia vera (PV) and nine with essential thrombocythaemia (ET) received a haemopoietic stem cell transplant (HSCT) at the Fred Hutchinson Cancer Research Center between May 1988 and March 2000. HSCT was performed because of progression to the spent phase of the disease with myelofibrosis and splenomegaly in 10 patients and evolution into a myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML) in nine patients. Patients were 18–59 years old (median 43). The interval from diagnosis to HSCT was 77–300 months (median 170). Seven patients were splenectomized before transplantation, and all but five had been treated with cytotoxic agents. Eleven patients received a transplant from a related, and eight from an unrelated, donor following conditioning with chemotherapy only or chemotherapy plus total body irradiation regimens. All evaluable patients achieved sustained engraftment. Twelve patients are surviving 5–116 months (median 41) after transplant, 10 in continued complete remission, one in haematological remission with residual marrow fibrosis and one with mixed haemopoietic chimaerism currently receiving therapy with interferon. Seven patients (six with AML/MDS and one with myelofibrosis) died of transplant‐related complications. These data show that HSCT can provide curative therapy for patients with PV and ET with advanced disease.