bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo platelet activation in atherothrombotic stroke is not determined by polymorphisms of human platelet glycoprotein IIIa or Ib
Authors: Meiklejohn D. J. , Vickers M. A. , Morrison E. R. , Dijkhuisen R. , Moore I. , Urbaniak S. J. , Greaves M.
Published: March 2003

Platelet membrane glycoprotein polymorphisms are candidate risk factors for thrombosis, but epidemiological data are conflicting. Thus, demonstration of a genotype‐dependent alteration in function is desirable to resolve these inconsistencies. We investigated in vivo platelet activation in acute thrombosis and related this to platelet genotype. Frequencies of the 1b and 2b alleles of the HPA 1a/1b and HPA 2a/2b platelet glycoprotein polymorphisms were determined in 150 (52 men/98 women, mean age 58·3 years) patients with atherothrombotic stroke, and the influence of genotype on markers of platelet activation was assessed. Platelet P‐selectin (CD62P) expression and fibrinogen binding was measured using whole blood flow cytometry within 24 h of stroke and 3 months later in 77 patients who provided a repeat blood sample. Results were compared with matched controls. Neither the 1b allele [allele frequency 0·11 vs. 0·13, odds ratio (OR) confidence interval (CI) 0·8 (0·5–1·3)] nor the 2b allele [0·09 vs. 0·07, OR (CI) 1·4 (0·8–2·4)] was significantly over‐represented in patients. Increased numbers of activated platelets were found following stroke (acute mean P‐selectin expression 0·64% vs. control 0·35%, P < 0·001; acute mean fibrinogen binding 1·6% vs. control 0·9%, P < 0·001). Activation persisted in the convalescent phase (P < 0·001 and P = 0·005 vs. controls for P‐selectin and fibrinogen respectively). Expression of P‐selectin and fibrinogen was not influenced by either the HPA 1a/1b genotype (P > 0·95 for each marker, Scheffe's test) or the 2a/2b genotype (P > 0·95 for each). Although persisting platelet activation is seen in atherothrombotic stroke, it is independent of HPA 1a/1b and 2a/2b genotypes. These data suggest an underlying prothrombotic state, but do not support the polymorphisms studied as risk factors for thrombotic stroke in this population.
Biological action of nitric oxide donor compounds on platelets from patients with sickle cell disease
Authors: Mondoro Traci Heath , Ryan Beth Brockner , Hrinczenko Borys W. , Schechter Alan N. , Vostal Jaroslav G. , Alayash Abdu I.
Published: March 2003

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2‐(N, N‐diethylamino)‐diazenolate‐2‐oxide (DEANO), S‐nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP‐induced platelet aggregation, CD62P expression, PAC‐1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC‐1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.
Residual risk of transfusion in Ghana
Authors: Candotti Daniel , Sarkodie Francis , Allain Jean‐Pierre
Published: March 2003

Plasma samples from replacement and volunteer blood donors in Kumasi, Ghana were pooled and tested using a duplex human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA detection method. Individual plasmas constitutive of reactive pools were confirmed using reverse transcription‐polymerase chain reaction. HIV and HCV infections were significantly higher in 1569 replacement donors than in 1169 volunteers; 2·4 and 1·7 versus 0·3 and 0·7% respectively (P < 0·01). Two duplex RNA‐positive plasma pools contained a confirmed/seronegative HIV or HCV RNA individual plasma. The residual post‐transfusion risk of HIV and HCV infection of blood collected from replacement blood donors ranged between 1:260 and 1:16 393 after screening for anti‐HIV, p24 antigen and anti‐HCV. These data indicate that in high‐prevalence HIV and HCV blood donor populations, a substantial residual post‐transfusion risk of infection remains. This risk might be reduced by collecting blood in younger volunteer donors or by genomic screening.
Ex vivo generation of human cytomegalovirus‐specific cytotoxic T cells by peptide‐pulsed dendritic cells
Authors: Kleihauer Annette , Grigoleit Ulrich , Hebart Holger , Moris Arnaud , Brossart Peter , Muhm Alexandra , Stevanovic Stefan , Rammensee Hans Georg , Sinzger Christian , Riegler Susanne , Jahn Gerhard , Kanz Lothar , Einsele Hermann
Published: March 2003

Adoptive transfer of donor‐derived human cytomegalovirus (HCMV)‐specific T‐cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV‐specific T cells using HCMV‐infected fibroblasts as stimulator cells confines this approach to HCMV‐seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV‐specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV‐specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide‐pulsed dendritic cells as antigen‐presenting cells. HCMV‐specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV‐seropositive and 2 out of 11 HCMV‐seronegative donors. The CTLs recognized HCMV‐infected autologous fibroblasts. No lysis was observed with either non‐infected autologous or HLA‐mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide‐specific T cells of up to 28% and > 90% of CD8+ T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 × 109 HCMV‐specific T cells was possible after 6–7 weeks when cultures were initiated with 1–5 × 106 responder cells. Thus, the approach with peptide‐pulsed DCs to generate HCMV‐specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.
Treatment of refractory T‐cell malignancies using gemcitabine
Authors: Sallah Sabah , Wan Jim Y. , Nguyen Nam P.
Published: March 2003

We have treated 10 patients with relapsed or refractory T‐cell malignancies using gemcitabine. The drug was administered intravenously over 30 min at a dose of 1200 mg/m2 on d 1, 8 and 15 of each 28‐d cycle. The mean age of the patients was 62 years and the mean number of administered cycles was three. Of the 10 patients, two achieved a complete response and four a partial response, for an overall response rate of 60% (95% confidence interval 20–35%). The toxicity of chemotherapy was mild and manageable in all patients. These encouraging results warrant further investigation of gemcitabine either as a single agent or in combination regimens as early salvage treatment for patients with refractory T‐cell haematological neoplasms.
16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype
Authors: Westhoff C. M. , Silberstein L. E. , Wylie D. E. , Skavdahl M. , Reid M. E.
Published: March 2003

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti‐e but did not react with some monoclonal anti‐e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G→C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti‐e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R0 (Dce) haplotype.
Chronic disseminated intravascular coagulation after surgery for abdominal aortic aneurysm: clinical and haemostatic response to dalteparin
Authors: Cummins David , Segal Helen , Hunt Beverley J. , Awad Reda , Maddox Anthony
Published: March 2003

A 77‐year‐old man developed chronic disseminated intravascular coagulation (DIC) after surgical repair of a large infrarenal aortic aneurysm. Self‐administered subcutaneous dalteparin therapy (5000 units o.d.) led to rapid relief of symptoms and sustained improvements in his platelet count and fibrinogen level; activation of coagulation and fibrinolysis appeared to be relatively unaffected. Long‐term treatment with low‐molecular‐weight heparin can provide good symptomatic control of chronic DIC associated with abdominal aortic aneurysm.
Accumulation of γ‐globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine
Authors: Bianchi Nicoletta , Chiarabelli Cristiano , Borgatti Monica , Mischiati Carlo , Fibach Eitan , Gambari Roberto
Published: March 2003

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5‐azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo‐fetal globin genes, such as the ζ‐, ε‐ and γ‐globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating γ‐globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal β‐globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of γ‐globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine‐mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of γ‐globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.
In vitro induction of apoptosis for nasal angiocentric natural killer cell lymphoma‐derived cell line, NK‐YS, by etoposide and cyclosporine A
Authors: Uno Masatoshi , Tsuchiyama Junjiroh , Moriwaki Akiyoshi , Noguchi Toshio , Mizoguchi Kazuhiro , Ogino Tetsuya , Yoshino Tadashi , Okada Shigeru , Harada Mine
Published: March 2003

Epstein‐Barr virus (EBV)‐associated nasal T/natural killer (NK) cell lymphoma has often been reported in Asian countries and has been recently confirmed as a novel clinicopathological entity. The prognosis of advanced stage disease is quite poor and an effective chemotherapeutic modality is strongly advocated. We have established the novel cell line NK‐YS, which preserves the original characteristics of EBV‐associated nasal angiocentric T/NK cell lymphoma. Using this cell line, we investigated the induction of apoptosis by apoptosis‐inducing agents, and expression of P‐glycoprotein (P‐gp), p53 and bcl‐2 proteins. NK‐YS showed resistance towards apoptosis‐inducing agents and expressed bcl‐2 and P‐gp but not p53. To overcome this drug resistance, we added cyclosporine A (CsA) and these agents to culture media as a P‐gp antagonist. The combination of CsA and etoposide or CsA and doxorubicin induced apoptotic cell death. These results indicated that P‐gp‐mediated drug resistance is an essential mechanism of drug resistance of the NK‐YS cell line. Combined therapy of conventional anti‐cancer agents with CsA may have an important place in the establishment of a curative therapy for disseminated nasal angiocentric NK cell lymphoma.
Flavopiridol circumvents Bcl‐2 family mediated inhibition of apoptosis and drug resistance in B‐cell chronic lymphocytic leukaemia
Authors: Pepper Chris , Thomas Alun , Hoy Terry , Fegan Chris , Bentley Paul
Published: March 2003

Flavopiridol, a synthetic flavone, is currently under clinical investigation for the treatment of B‐cell chronic lymphocytic leukaemia (B‐CLL). In this study, we examined the in vitro effects of flavopiridol and fludarabine on B‐CLL cells from 64 patients (36 treated and 28 untreated) in terms of apoptosis induction and Bcl‐2 family expression. Both flavopiridol and fludarabine induced apoptosis in all the samples tested with mean LD50 values (± SD) of 59·7 nmol/l (± 36·5) and 6·2 μmol/l (± 7·5) respectively. Mean flavopiridol LD50 values were not significantly different between the treated and untreated patient groups (P = 0·35), whereas the fludarabine LD50 values were significantly higher in the previously treated patient group (P = 0·01). Bcl‐2 and Mcl‐1 expression were downregulated in both flavopiridol and fludarabine‐induced apoptotic cells, but the increase in Bax expression that accompanied fludarabine‐induced apoptosis was not evident in flavopiridol‐treated cells. In addition, Bcl‐2:Bax ratios were not predictive of flavopiridol cytotoxicity (P = 0·82), whereas they were highly predictive of in vitro responsiveness to fludarabine (P = 0·001). Overall, these findings suggest that flavopiridol exerts its cytotoxic effect through a novel cell‐death pathway that is not subject to the Bcl‐2 family mediated resistance mechanisms that reduce the efficacy of many conventional chemotherapeutic drugs.
Analysis of chimaerism in thalassaemic children undergoing stem cell transplantation
Authors: Amrolia P. J. , Vulliamy T. , Vassiliou G . , Lawson S. , Bryon J. , Kaeda J. , Dokal I. , Johnston R. , Veys P. , Darbyshire P. , Roberts I. A. G.
Published: March 2003

We have prospectively assessed the relative contribution of host and donor to haemopoiesis following stem cell transplantation (SCT) in children with β‐thalassaemia major (n = 35), using karyotype analysis or Southern blot/polymerase chain reaction analysis of variable number tandem repeats on genomic DNA from peripheral blood. Early haemopoiesis was fully donor in origin in 24 out of 35 cases and remained so throughout the post‐transplant course in all but one patient, who evolved to stable mixed chimaerism. The remaining 11 cases (31%) initially showed mixed chimaerism: four of these rejected, one eventually eradicated host haemopoiesis to become fully donor haemopoietic, and the remaining six had persistent mixed chimaerism, with 5–38% host haemopoiesis. The risk of graft rejection was high when > 15% host haemopoiesis was present at 3 months post transplant: four out of six such patients rejected their grafts; conversely, zero out of 29 patients with < 15% host haemopoiesis at 3 months rejected (P < 0·0001). There was a higher incidence of significant acute and chronic graft‐versus‐host disease in patients with full donor chimaerism. These studies confirm that the mixed chimaeric state is common following SCT for thalassaemia, often persists (with up to 4 years follow‐up) and is compatible with long‐term cure. Analysis of chimaerism in patients undergoing SCT for β‐thalassaemia enables monitoring of engraftment in the early post‐transplant period, provides insight into the biology of engraftment and may be useful in identifying patients at high risk of rejection.
The effect of human platelet alloantigen polymorphisms on the in vitro responsiveness to adrenaline and collagen
Authors: Theodoropoulos Ilias , Christopoulos Costas , Metcalfe Paul , Dimitriadou Ekaterini , Economopoulos Panagiotis , Loucopoulos Dimitris
Published: March 2003

A number of clinical studies have suggested that carriage of the low frequency allele (b) of the human platelet antigen 1 (HPA‐1) system is a risk factor for coronary thrombosis. We have examined the effect of a series of HPA biallelic polymorphisms (systems ‐1, ‐2, ‐3 and ‐5) on the in vitro platelet aggregation in response to adrenaline and collagen in 30 healthy volunteers. There was a significantly higher prevalence (10 out of 18) of carriers of the HPA‐1b polymorphism among subjects showing a > 50% aggregation response to adrenaline (‘responders’) than the prevalence (1/12) in ‘non‐responders’ (P < 0·05). Platelets heterozygous for the HPA‐1b polymorphism showed a significantly higher rate (slope) and greater extent (%) of adrenaline‐induced aggregation than platelets not carrying the HPA‐1b allele (P < 0·05). A greater extent of collagen‐induced aggregation was also demonstrated in HPA‐1ab platelets (P < 0·05). Inhibition of adrenaline‐induced aggregation following incubation with aspirin was greater (P < 0·01) in HPA‐1ab than in HPA‐1aa platelets. Collagen‐induced aggregation was slower in carriers of the HPA‐5b allele than in HPA‐5aa subjects (P < 0·05). Polymorphisms of the HPA‐2 and HPA‐3 systems were not associated with different aggregation responses to either adrenaline or collagen. These results support the clinical observation that polymorphism HPA‐1b may predispose to increased platelet thrombogenicity and suggest that the presence of polymorphism HPA‐5b might render the platelet less reactive to collagen.
Analysis of FGFR3 gene mutations in multiple myeloma patients with t(4;14)
Authors: Intini Daniela , Baldini Luca , Fabris Sonia , Lombardi Luigia , Ciceri Gabriella , Maiolo Anna Teresa , Neri Antonino
Published: March 2003

The t(4;14)(p16.3;q32) in multiple myeloma (MM) leads to an apparent deregulation of the FGFR3 and WHSC1/MMSET genes. FGFR3 mutations, known to be associated with genetic skeletal disorders, have also been identified in a few cases of MM (mainly cell lines) with t(4;14). We investigated FGFR3 mutations in a series of 53 MM cases; 11 cases with t(4;14) and FGFR3 overexpression were analysed using reverse transcription polymerase chain reaction, while the remaining cases were studied at DNA level. The Arg248Cys mutation, which is associated with some lethal forms of skeletal disorders, was found in one case with t(4;14). Our results indicate that FGFR3 mutations occur in only a small fraction of MM cases with t(4;14).
Prevalence of HFE mutations in people from North Africa living in southern France
Authors: Aguilar‐Martinez Patricia , Picot Marie‐Christine , Becker Fabienne , Boulot Pierre , Montoya Françoise , Mares Pierre , Bachelard Bruno , Henry Yanne , Delarbre Jean‐Louis , Sarda Pierre , Schved Jean‐François
Published: March 2003

The two main mutations of the HFE (haemochromatosis) gene, C282Y and H63D, were found previously to be rare or absent among Africans. Dried blood samples of 1276 newborns from southern France were analysed for both HFE mutations, and the origins of the four grandparents of each newborn were recorded. The allele frequency of C282Y and H63D was 3·0% ± 0·7% and 16·9% ± 1·5% respectively. In a subgroup of 171 newborns with four North African ancestries (mainly from Morocco and Algeria) the allele frequency was 0·9%+2·5%−0·2% for the C282Y and 13·2% ± 3·6% for H63D. HFE mutations are not absent in individuals with North African origins living in southern Europe. This finding has implications for the diagnosis and screening of hereditary haemochromatosis in these populations.
Transient pancytopenia preceding acute lymphoblastic leukaemia (pre‐ALL) precipitated by parvovirus B19
Authors: Heegaard Erik D. , Madsen Hans O. , Schmiegelow Kjeld
Published: March 2003

A preleukaemic phase, typified by pancytopenia and bone marrow (BM) hypoplasia, is an uncommon but well‐documented prelude to acute lymphoblastic leukaemia (pre‐ALL) in children. Parvovirus B19 (B19) exhibits a marked tropism to human BM and replicates only in erythroid progenitor cells acting as a confounding, but treatable agent in immunocompromised patients. We present the first case of B19‐associated pre‐ALL characterized by severe and recurring transient pancytopenia in a child who developed ALL 5 months later. The advent of B19‐specific IgG at the time of infection and the subsequent disappearance 1·5 years later has not previously been described. In this patient the observed cytopenias were probably the result of B19 acting in concert with the failing BM and B19 is possibly one of several factors capable of triggering the onset of pre‐ALL.
The ratio of serum transferrin receptor and serum ferritin in the diagnosis of iron status
Authors: Malope Babatyi I. , MacPhail A. Patrick , Alberts Marianne , Hiss Donavon C.
Published: March 2003

Laboratory tests used in the diagnosis of iron status lack specificity in defining iron deficiency anaemia (IDA) and anaemia of inflammation (AI). The serum transferrin receptor (sTfR) may provide more information in this regard. The iron status of 561 pre‐school children was determined and classified using the conventional measurements. The value of the concentration of sTfR, the ratio of sTfR (µg/ml) to LogSF (µg/l) (TfR‐Index), and the Log of the ratio of sTfR (µg/l) to SF (µg/l) − (LogTfR:Fer ratio), in the classification of the iron status were determined by comparing their distributions across the classification of iron status. Although there were significant differences in sTfR and TfR‐Index across the categories of iron status, there was considerable overlap. All subjects with iron deficiency had LogTfR:Fer ratio > 2·55, whereas in all subjects classified as AI it was < 2·55, thus clearly separating the two. The LogTfR:Fer ratio was not able to exclude IDA in the presence of inflammation. However, in cases of combined IDA and AI the LogTfR:Fer ratio was < 2·55 but increased to > 2·55 after resolution of the inflammation. This novel method of calculating the LogTfR:Fer ratio may provide a more precise classification of the iron status of children.
Mutations in the gene encoding neutrophil elastase (ELA2) are not sufficient to cause the phenotype of congenital neutropenia
Authors: Germeshausen Manuela , Schulze Harald , Ballmaier Matthias , Zeidler Cornelia , Welte Karl
Published: March 2003

Mutations in the ELA2 gene encoding human neutrophil elastase have been reported recently to be involved in the aetiology of both, cyclic (CyN) and congenital neutropenia (CN). We analysed the correlation between the occurrence of ELA2 mutations and the neutropenic phenotype in a family with two children affected with CN. The two children harboured the same heterozygous mutation in the ELA2 gene that was inherited from their unaffected father. We conclude that ELA2 mutations are not the single cause of CN although they might be a necessary prerequisite for the expression of the neutropenic phenotype in a subgroup of CN patients.
Internal jugular vein thrombosis after assisted conception therapy
Authors: Arya Roopen , Shehata Hassan A. , Patel Raj. K. , Sahu Satyajit , Rajasingam Daghni , Harrington Kevin F. , Nelson‐Piercy Catherine , Parsons John H.
Published: March 2003

Superovulation therapy during assisted conception may result in a hypercoagulable state. Five cases of upper extremity venous thrombosis were identified in women who conceived after ovarian stimulation for in vitro fertilization (IVF). They presented between 7 and 10 weeks' gestation with neck pain and swelling. Three had been treated for ovarian hyperstimulation syndrome and two had evidence of inherited thrombophilia. Four patients received thromboprophylaxis before presentation. Although thrombosis is an uncommon complication of IVF, patients should be counselled before treatment. Thrombophilia screening may be considered for ‘high‐risk’ patients, although current regimes for thromboprophylaxis remain suboptimal.
Increased circulating platelet–leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis
Authors: Joseph Joanne E. , Harrison Paul , Mackie Ian J. , Isenberg David A. , Machin Samuel J.
Published: March 2003

It is possible that platelet activation may play a pathogenic role in the increased risk of thrombosis associated with antiphospholipid antibodies (APA). In this study, levels of in vivo platelet activation were measured in 20 patients with primary antiphospholipid syndrome (PAPS) and 30 systemic lupus erythematosus (SLE) patients (14 of whom had secondary APS) using sensitive flow cytometry. Soluble P‐selectin levels were also assayed. Platelet CD63 expression was significantly higher in PAPS than normal controls (P = 0·007), as well as SLE patients with and without secondary APS (P = 0·03 and P = 0·002 respectively). PAC‐1 binding was significantly higher in PAPS than the control group (P = 0·007) and SLE patients without APS (P = 0·015). Platelet–leucocyte complexes were significantly higher in SLE patients than both PAPS and the control group, and platelet–monocyte complexes were significantly increased in PAPS compared with the control group. (Platelet–leucocyte complexes were also significantly higher than controls in 10 rheumatoid arthritis (RA) patients without APA). Soluble P‐selectin levels were significantly higher in PAPS and SLE patients than the control group. Platelet CD62p expression, annexin V binding and platelet microparticle numbers were not increased in PAPS or SLE patients. We conclude that there is evidence of increased platelet activation in PAPS and SLE, and this is important to note as it may have potential therapeutic implications with respect to use of antiplatelet agents in these patients.
β‐Spectrin Sta Bárbara: a novel frameshift mutation in hereditary spherocytosis associated with detectable levels of mRNA and a germ cell line mosaicism
Authors: Bassères Daniela S. , Duarte Adriana S. S. , Hassoun Hani , Costa Fernando F. , Saad Sara T. O.
Published: March 2003

Hereditary spherocytosis (HS) is a common inherited anaemia characterized by the presence of spherocytic red cells and by a heterogeneous nature in terms of its clinical presentation, molecular basis and inheritance. Defects in several membrane protein genes have been involved in the pathogenesis of HS, including defects in the β‐spectrin gene. We detected a novel frameshift mutation in the β‐spectrin gene, a C deletion at codon 638, in a patient presenting with HS and spectrin deficiency. The mutant protein was not detected in the membrane or in other cellular compartments, but detectable levels of mutant mRNA were found in the patient. Interestingly, this mutation was not present in the patient's parents, suggesting a genetic mosaicism, especially as the patient has an affected brother with the same molecular defect. We analysed DNA from different tissues of the parents and the mutation was absent from all tissues analysed. This mutation seems to be confined to the germ cell lineage of the patient's mother and must present a mosaic pattern in these cells as the patient also has unaffected siblings.
Histone deacetylase inhibitors induce caspase‐dependent apoptosis and downregulation of daxx in acute promyelocytic leukaemia with t(15;17)
Authors: Amin Hesham M. , Saeed Shahnaz , Alkan Serhan
Published: March 2003

Histone deacetylase (HDAC) appears to play an important role in the pathogenesis of acute promyelocytic leukaemia (APL) as it is recruited by both PML–RARα and PLZF/RARα in leukaemic cells with t(15;17) and t(11;17) respectively. Recent studies have demonstrated that HDAC inhibitors can be therapeutically used in various neoplastic disorders including APL. Cell differentiation was considered the major mechanism of the anti‐leukaemic effects of HDAC inhibitors in APL. However, most of these studies either evaluated the effect of HDAC inhibitors in combination with all‐trans retinoic acid (ATRA) or focused on the less common form of APL with t(11;17). To investigate the cellular effects of HDAC inhibitors, including sodium butyrate, trichostatin A, and suberoylanilide hydroxamic acid (SAHA), we used two APL cell lines, NB4 and the ATRA‐resistant derivative NB4.306. Moreover, primary cells from five patients with cytogenetic evidence for t(15;17) were also studied. Our results demonstrated that HDAC inhibitors induce distinct caspase‐dependent apoptosis in APL, which showed both concentration‐and time‐dependence. In addition, changes in the apoptosis‐regulatory proteins, daxx, bcl‐2 and bax were analysed. HDAC inhibitors induced downregulation of daxx, but no significant changes were detected in bcl‐2 or bax. In conclusion, apoptosis induced by HDAC inhibitors in APL could provide an effective strategy for treatment of patients with t(15;17).
Prolonged immune deficiency following allogeneic stem cell transplantation: risk factors and complications in adult patients
Authors: Maury Sébastien , Mary Jean‐Yves , Rabian Claire , Schwarzinger Michael , Toubert Antoine , Scieux Catherine , Carmagnat Maryvonnick , Esperou Hélène , Ribaud Patricia , Devergie Agnès , Guardiola Philippe , Vexiau Patrick , Charron Dominique , Gluckman Eliane , Socié Gérard
Published: March 2003

To evaluate the long‐term immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), we prospectively screened standard immune parameters in a series of 105 patients, at a median time of 15 months after SCT. Analysing lymphoid phenotypes, in vitro immune functions and immunoglobulin levels, we found that, more than 1 year post SCT, cellular and humoral immunity was still altered in a significant number of patients. CD4+ T cells were < 200/µl in one third of patients, and the CD4/CD8 ratio was still reversed in 78% of patients. Almost all patients showed positive T‐cell responses against mitogens, but antigen‐specific proliferation assays identified 20% to 80% of non‐responders. B‐cell counts were reconstituted in 61% of the patients, but levels of total immunoglobulins were still low in 59%. In multivariate analyses, human leucocyte antigen (HLA) disparity between donor and recipient and chronic graft‐versus‐host disease were the leading causes affecting immune reconstitution. Interestingly, cytomegalovirus (CMV) infections were strongly associated with normal CD8+ T‐cell counts. Studying the impact of impaired immune reconstitution on the rate of infections occurring in the 6 years following screening, we identified three parameters (low B‐cell count, inverted CD4/CD8 ratio, and negative response to tetanus toxin) as significant risk factors for developing such late infections.
Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system
Authors: Albertsen Birgitte Klug , Schrøder Henrik , Ingerslev Jørgen , Jakobsen Preben , Avramis Vassilios I. , Müller Hans‐Joachim , Carlsen Niels T. , Schmiegelow Kjeld
Published: March 2003

Asparaginase comes from different biological sources and the various preparations have different pharmacokinetic properties, and their tendency to induce side‐effects is different. Erwinia asparaginase (ASNase) has a shorter half‐life than the Escherichia coli preparations, and it has been reported to be less immunogenic than the E. coli preparations and to induce fewer coagulation disorders. Children with newly diagnosed acute lymphoblastic leukaemia (ALL) were included in this study. Twenty‐seven patients were treated with Erwinia ASNase (induction therapy 30·000 IU/m2/d i.m. for 10 d, and re‐induction therapy 30·000 IU/m2 twice a week for 2 weeks) and 15 were treated with ASNase Medac (induction therapy 1·000 IU/m2/d i.m. for 10 d, and re‐induction therapy 5·000 IU/m2 i.m. twice a week for 2 weeks). Blood samples were drawn to determine enzyme activity, l‐asparagine, anti‐asparaginase antibodies, and coagulation parameters. After i.m. administration, Erwinia ASNase displayed a protracted absorption phase compared to ASNase Medac. The mean bioavailability after i.m. administration was 27% for Erwinia ASNase and 45% for ASNase Medac respectively. Mean trough enzyme activities during induction therapy were Erwinia ASNase 1748 IU/l and ASNase Medac 272 IU/l, and during re‐induction therapy Erwinia ASNase 83 IU/l and ASNase Medac 147 IU/l. We conclude that in this setting, therapy with ASNase Medac resulted in sufficient treatment during both phases of therapy, whereas treatment with Erwinia ASNase resulted in unnecessarily intense therapy during the induction phase and insufficient treatment during the re‐induction phase. There was no significant difference in the incidence of antibody formation, and therapy with Erwinia ASNase resulted in a more pronounced influence on the coagulation parameters than therapy with ASNase Medac.
Allogeneic peripheral blood stem cell transplantation with reduced‐intensity conditioning: results of a prospective multicentre study
Authors: Martino Rodrigo , Caballero María Dolores , Canals Carmen , Simón José Antonio Pérez , Solano Carlos , Urbano‐Ispízua Alvaro , Bargay Joan , Rayón Consolación , Léon Antonio , Sarrá Josep , Odriozola Jesús , Conde Javier García , Sierra Jorge , San Miguel Jesús ,   for the ALLOPBSCT subcommittee of the Spanish Group for Haematopoietic Transplantation (GETH) and the Group GEL‐TAMO
Published: March 2003

Reduced‐intensity conditioning (RIC) regimens for allogeneic haematopoietic stem cell transplantation (SCT) have been shown to lead to engraftment of donor stem cells without the severe extra‐haematological toxicities of traditional myeloablative transplants. Between December 1998 and December 2000, 76 patients underwent a RIC peripheral blood SCT in a prospective multicentre study. The median age was 53 years, and 57 patients were beyond the early phase of their disease. The conditioning regimens consisted of fludarabine (150 mg/m2) plus melphalan (140 mg/m2) or busulphan (10 mg/kg). Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporin A plus short‐course methotrexate. The preparative regimens were well tolerated. All patients experienced severe pancytopenia, but haematological recovery was prompt in all but two cases (early deaths). The 100‐d probability of developing grade II–IV acute GVHD was 32% (10% grade III–IV), and the 1‐year probability of developing chronic extensive GVHD was 43%. Early complete donor chimaerism was observed in 52/68 patients, and 16 evaluable patients were in complete chimaerism 1 year post transplant. With a median follow‐up of 283 d (355 in 48 survivors), the 1‐year probability of transplant‐related mortality was 20%, and the 1‐year overall and progression‐free survivals were 60% and 55% respectively. In conclusion, RIC regimens lead to low early toxicity after allografting, with stable donor haematopoietic engraftment, with an apparent low risk of acute GVHD. Chronic GVHD, however, develops in a significant proportion of patients.
Autoimmune haemolytic anaemia triggered by Bartonella henselae infection: a case report
Authors: Van Audenhove A. , Verhoef G. , Peetermans W. E. , Boogaerts M. , Vandenberghe P.
Published: March 2003

Bartonella henselae is a hitherto unidentified cause of autoimmune haemolytic anaemia. Here we report a case of Coombs‐negative autoimmune haemolytic anaemia. The episode was preceded by exposure to a cat and a non‐specific infectious syndrome. Concomitant serum titres of B. henselae antibodies were indicative of a recent infection. The case report suggests that B. henselae infection can trigger secondary autoimmune haemolytic anaemia.
Can positron emission tomography with [18F]‐fluorodeoxyglucose after first‐line treatment distinguish Hodgkin's disease patients who need additional therapy from others in whom additional therapy would mean avoidable toxicity?
Authors: Spaepen Karoline , Stroobants Sigrid , Dupont Patrick , Thomas José , Vandenberghe Peter , Balzarini Jan , De Wolf‐Peeters Christine , Mortelmans Luc , Verhoef Gregor
Published: March 2003

To assess the ability of restaging positron emission tomography (PET) scanning to predict clinical outcome after first‐line treatment in patients with Hodgkin's disease, we included 60 patients with histologically proven HD, who underwent whole‐body [18F]‐fluorodeoxygenase ([18F]‐FDG)‐PET studies after first‐line treatment and with a follow‐up of at least 1 year. Persistence or absence of residual disease on PET was related to progression‐free survival (PFS) using Kaplan–Meier survival analysis. After treatment, 55 patients showed a normal [18F]‐FDG‐PET scan; 50 of 55 remained in complete remission (CR), with a median follow‐up of 955 d. Only five patients relapsed (median PFS, 296 d). During follow‐up in all five patients, [18F]‐FDG‐PET was the first tool that became positive for relapse. Persistent abnormal [18F]‐FDG uptake was seen in only five patients; all of them relapsed (median PFS, 296 d). In four of five patients, only PET predicted persistent disease. All relapses were proven histologically. Two‐year actuarial PFS rate for negative patients was 91% compared with 0% for positive patients. We concluded that [18F]‐FDG‐PET has an important prognostic role in the post‐treatment evaluation of HD patients.
Association of the HLA A2‐B46‐DR9 haplotype with autoimmune thyroid dysfunction after bone marrow transplantation in Chinese patients
Authors: Au W. Y. , Hawkins B. R. , Chan E. Y. T. , Lie A. K. W. , Kung A. W. C. , Liang R. , Cheng N. , Kwong Y. L.
Published: March 2003

Autoimmune thyroid disease (AITD) may occur in patients after bone marrow transplantation (BMT). At a median follow‐up of 4 years, among 194 allografts and 28 autografts, four patients (three allografts, one autograft) developed AITD. All carried the human leucocyte antigen (HLA) A2‐B46‐DR9 haplotype, strongly associated with AITD in the Chinese population. No significant thyroid disorder was detected in 190 patients without this haplotype. The frequency of AITD in BMT patients with the HLA A2‐B46‐DR9 haplotype was 12·5%, with a relative risk of 7·8 times that of non‐carriers (P < 0·001). The risk of AITD should be recognized in recipients with high‐risk HLA haplotypes, and regular screening might be warranted.
Homozygosity for the C→T polymorphism at nucleotide 46 in the 5′ untranslated region of the factor XII gene protects from development of acute coronary syndrome
Authors: Endler Georg , Mannhalter Christine , Sunder‐Plassmann Heike , Lalouschek Wolfgang , Kapiotis Sonja , Exner Markus , Jordanova Nelli , Meier Susanne , Kunze Freja , Wagner Oswald , Huber And Kurt
Published: March 2003

Recently, a C→T polymorphism at nucleotide 46 in the 5′‐untranslated region of the factor XII (FXII) gene was shown to be associated with lower levels of FXII. To study the impact of this polymorphism on the development of an acute coronary syndrome (ACS), we compared 303 patients with ACS and 227 patients with stable coronary artery disease (CAD). In the latter group, 54·2% of individuals carried wild‐type FXII:46C, 37·9% were heterozygous FXII:C46T and 7·9% were homozygous for FXII:46T. In contrast, in the ACS group (n = 303), 54·1% were wild‐type FXII:46C, 42·6% were heterozygous FXII:C46T and only 3·3% carried the homozygous FXII:46T genotype. The 2·5‐fold lower prevalence of the FXII:46T genotype in patients with ACS could indicate a protective effect on the development of ACS (odds ratio = 0·4, 95% CI 0·1–0·9) in patients with pre‐existing CAD.
Predicting long‐term (≥ 5 years) event‐free survival in multiple myeloma patients following planned tandem autotransplants
Authors: Tricot Guido , Spencer Trey , Sawyer Jeffrey , Spoon Dan , Desikan Raman , Fassas Athanasios , Badros Ashraf , Zangari Maurizio , Munshi Nikhil , Anaissie Elias , Toor Amir , Barlogie Bart
Published: March 2003

Summary. Although outcome in multiple myeloma (MM) patients has improved significantly with the introduction of autotransplants (AT), the curability of this approach remained to be demonstrated. Therefore, we analysed outcome and prognostic factors using a logistic regression model in 515 consecutive newly diagnosed and previously treated patients intended to receive melphalan‐based tandem transplants with follow up of ≥ 5 years. One quarter ofpatients had event‐free survivals (EFS) ≥ 5 years with no further relapses seen after 7 years (46 patients on plateau). On multivariate analysis, factors associated with EFS ≥ 5 years were absence of chromosome 11 and 13 abnormalities (odds ratio: 6·1), ≤ 12 months of preceding standard‐dose therapy (SDT) (OR: 2·6) and β‐2 microglobulin (B2M) level ≤ 2·5 mg/l at time of first AT (OR: 1·7). Patients with only favourable variables (25%) had a 7‐year EFS in excess of 35%, compared with 15% and 10%, respectively, with one (43%) or two unfavourable variables (27%), and 0% for 5% of patients with three unfavourable variables (P < 0·0001). Using a 1‐year landmark analysis to allow for guaranteed time and thereby excluding early treatment failures, attaining a complete remission (CR) had no significant effect on long‐term survival. Our data are consistent with cure in MM patients with a CR duration ≥ 7 years and re‐establishment of a monoclonal gammopathy of undetermined significance (MGUS) phase in those with persistent evidence of disease post transplantation, but without disease progression ≥ 7 years.
Invasive fungal infections after allogeneic peripheral blood stem cell transplantation: incidence and risk factors in 395 patients
Authors: Martino Rodrigo , Subirá Maricel , Rovira Montserrat , Solano Carlos , Vázquez Lourdes , Sanz Guillermo F. , Urbano‐Ispizua Alvaro , Brunet Salut , De la Cámara Rafael ,   for the alloPBSCT Infectious/Non‐infectious Complications Subcommittees of the Grupo Español de Trasplante Hematopoyético (GETH)
Published: March 2003

Summary. We have analysed the incidence and risk factors for the occurrence of invasive fungal infections (IFI) among 395 recipients of an allogeneic peripheral blood stem cell transplantation (PBSCT) from a human leucocyte antigen (HLA)‐identical sibling. IFI (n = 50) occurred in 46 patients, giving an overall probability of 14%. There were 12 cases of invasive candidiasis (3%), with only one death. Non‐Candida IFI occurred in 37 patients (12% probability), mostly invasive aspergillosis (n = 32). In multivariate analysis the only two significant variables associated with a higher risk of developing a non‐Candida IFI were the development of moderate‐to‐severe graft‐versus‐host disease (GvHD, P < 0·0001; OR 4·6) and having received steroid prophylaxis for GvHD (P = 0·04; OR 2·1). In multivariate analysis the variables associated with a lower overall survival after PBSCT were development of a non‐Candida IFI (P < 0·0001; OR 5·6), non‐early disease phase (P = 0·0001; OR 1·9), steroid prophylaxis (P = 0·02; OR 1·4), moderate‐to‐severe GvHD (P = 0·01; OR 1·6) and cytomegalovirus infection post transplant (P = 0·001; OR 1·8). Our results show that non‐Candida IFI (in particular aspergillosis) was an important cause of infectious morbidity and mortality after an HLA‐identical sibling PBSCT, while invasive candidiasis was rare. Use of steroid prophylaxis and, in particular, the development of moderate‐to‐severe GvHD post transplant were risk factors for non‐Candida IFI. Prophylactic strategies for these infections should thus take into account these risk factors.
Very low frequencies of human normal CD34+ haematopoietic progenitor cells express the Wilms' tumour gene WT1 at levels similar to those in leukaemia cells
Authors: Hosen Naoki , Sonoda Yoshiaki , Oji Yusuke , Kimura Takafumi , Minamiguchi Hitoshi , Tamaki Hiroya , Kawakami Manabu , Asada Momotaro , Kanato Keisuke , Motomura Mari , Murakami Masaki , Fujioka Tatsuya , Masuda Tomoki , Kim Eui Ho , Tsuboi Akihiro , Oka Yoshihiro , Soma Toshihiro , Ogawa Hiroyasu , Sugiyama Haruo
Published: March 2003

Summary. The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1‐expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single‐cell reverse transcriptase polymerase chain reaction (RT–PCR) methods. Using the semiquantitative single‐cell polyA‐PCR + sequence‐specific (SS)‐PCR method, WT1 expression was detected in four (1·3%) out of 319 CD34+ BM single cells. To confirm the above results, a single‐cell nested sequence‐specific (NSS)‐RT–PCR method that was less quantitative but more sensitive than the polyA‐PCR + SS‐PCR method was also performed, and WT1 expression was detected in 15 (1·1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1·2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1‐expressing cells were found between CD34+CD38– and CD34+CD38+ BM single cells. Furthermore, WT1‐expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage‐specific and cell cycle gene expression in WT1‐expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38– and lineage‐committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1‐expressing CD34+ BM cells were normal counterparts of leukaemia cells.
p53‐mediated downregulation of Chk1 abrogates the DNA damage‐induced G2M checkpoint in K562 cells, resulting in increased apoptosis
Authors: Cummings Michele , Siitonen Timo , Higginbottom Karen , Newland Adrian C. , Allen Paul D.
Published: March 2003

Summary. BCR–ABL confers apoptotic resistance to a range of genotoxic agents, and this protection is mediated in part by prolonging the G2 checkpoint. The p53 tumour suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. To investigate the effect of p53 on the BCR–ABL‐mediated G2M checkpoint response, we transiently transfected the BCR–ABL‐positive, p53‐negative cell line K562 with wild‐type human p53. The p53‐transfected cells showed a decreased ability to arrest in G2 and an increase in apoptosis in response to etoposide treatment, relative to the control mock‐transfected cells. p53‐transfected and control cells were treated with etoposide and trapped at mitosis with nocodazole. The mitotic index of p53‐transfected cells was higher than that of the control cells, which suggests that p53 abrogates the G2 checkpoint response to etoposide treatment in K562 cells. We found that the expression of the cell cycle checkpoint protein Chk1 was reduced in the etoposide‐treated p53‐transfected cells by 24 h, and this correlated with a reduction in the extent of etoposide‐induced phosphorylation of CDK1 at tyrosine 15 (Y15). We conclude, therefore, that p53 overrides the strong G2 checkpoint response to etoposide in K562 cells, by directly or indirectly downregulating Chk1 expression, which, in turn, contributes to the proapoptotic effect of p53.