bluuebunny/crossref_metadata_2025 huggingface dataset:
The C807T/G873A polymorphism in the platelet glycoprotein Ia gene and the risk of acute coronary syndrome in the Italian population
Authors: Casorelli Ida , De Stefano Valerio , Leone Antonio Maria , Chiusolo Patrizia , Burzotta Francesco , Paciaroni Katia , Rossi Elena , Andreotti Felicita , Leone Giuseppe , Maseri Attilio
Published: March 2003

Membrane glycoprotein (GP) Ia/IIa mediates platelet adhesion to collagen. The linked C807T/G873A polymorphisms in the GP Ia gene are correlated with a variable expression of the platelet surface receptor, the 807 TT/873 AA genotype being associated with a higher receptor density. Our study aimed to evaluate the possible role of the GP Ia C807T/G873A polymorphism as a risk factor for acute coronary syndrome in the Italian population. We investigated 157 patients with acute coronary syndrome (117 with myocardial infarction and 40 with severe unstable angina) as the first manifestation of coronary disease occurring before 65 years of age, compared with 312 healthy controls. All individuals were of Italian ancestry and were genotyped for the GP Ia C807T/G873A polymorphism. Complete linkage between the 807 and 873 sites was found in all samples. The 807 TT genotype was present in 12·7% of cases and in 4·8% of controls; the odds ratio for acute coronary syndrome was 2·9 (95% CI 1·4–5·8) for the 807 TT genotype compared with C‐allele carriers and 0·6 (95% CI 0·4–0·9) for the 807 CC genotype compared with T‐allele carriers. For the TT genotype, compared with CC homozygotes, the increase in risk was 3·4‐fold in patients with at least one risk factor (smoking, hypercholesterolaemia, diabetes, systemic hypertension) and 4·1‐fold in patients with angiographically diagnosed two‐ or three‐vessel disease. We conclude that the GP Ia 807 TT (873 AA) genotype is associated with an increased risk of acute coronary syndrome in the Italian population; conversely, the GP Ia 807 CC (873 GG) genotype seems to represent a protective factor.
Response to vaccination against different types of antigens in patients with chronic lymphocytic leukaemia
Authors: Sinisalo Marjatta , Aittoniemi Janne , Oivanen Petri , Käyhty Helena , Ölander Rose‐Marie , Vilpo Juhani
Published: March 2003

We investigated responses to vaccination against pneumococcal polysaccharide, Haemophilus influenzae b (Hib) conjugate and tetanus toxoid antigens in 31 patients with chronic lymphocytic leukaemia (CLL) and 25 controls. While in the control group all antibody responses against different antigens were highly significant, in the patient group clear evidence for responsiveness was detected only in the case of Hib polysaccharide antigen. Certain CLL patient subgroups showed low reactivity against tetanus toxoid antigen. In conclusion, plain polysaccharide vaccines seem to be ineffective in patients with CLL. Conjugate vaccines, in turn, are immunogenic and may offer protection against infections caused by encapsulated bacteria in these patients. Further studies concerning an optimal vaccination scheme and clinical efficiency are warranted.
A phase II trial of pirfenidone (5‐methyl‐1‐phenyl‐2‐[1H]‐pyridone), a novel anti‐fibrosing agent, in myelofibrosis with myeloid metaplasia
Authors: Mesa Ruben A. , Tefferi Ayalew , Elliott Michelle A. , Hoagland H. Clark , Call Timothy G. , Schroeder Georgene S. , Yoon Soo‐Young , Li Chin‐Yang , Gray Leigh A. , Margolin S. , Hook C. Christopher
Published: March 2003

The anti‐fibrotic and cytokine modulatory properties of pirfenidone suggest its usefulness in the treatment of myelofibrosis with myeloid metaplasia (MMM). In a prospective study, 28 patients with MMM were treated with oral pirfenidone. Twelve patients completed 1 year of therapy; 13 were withdrawn because of disease progression and three because of drug intolerance. Only one patient experienced a clinically relevant benefit with respect to anaemia and splenomegaly. The overall lack of clinical benefit correlated with no significant improvement in the bone marrow morphological features of the disease. We conclude that pirfenidone has no significant clinical or biological activity in MMM.
Autoimmune neutropenia in pregnant women causing neonatal neutropenia
Authors: Kameoka Junichi , Funato Tadao , Miura Toshihiko , Harigae Hideo , Saito Junko , Yokoyama Hisayuki , Takahashi Shinichiro , Yamada Minami , Sasaki Osamu , Imaizumi Masue , Takata Noboru , Meguro Kuniaki , Sasaki Takeshi
Published: March 2003

Autoimmune neutropenia (AIN) can occur during pregnancy. However, neonatal neutropenia occurring in an infant born to a mother with AIN has only rarely been documented. Recently, we have experienced two cases of AIN during pregnancy, both of which caused severe yet transient neonatal neutropenia (< 0·3 × 109/l), probably as a result of transplacental maternal anti‐neutrophil autoantibodies. The anti‐neutrophil antibodies seemed to be against antigens other than NA1/NA2 because the autoantibodies did not bind to neutrophils of specific NA types selectively in the granulocyte indirect immunofluorescence test. Although AIN is a relatively uncommon disease, neonatal neutropenia caused by maternal AIN may not be quite as rare.
Cyclosporin A for the treatment of patients with chronic idiopathic thrombocytopenic purpura refractory to corticosteroids or splenectomy
Authors: Kappers‐Klunne M. C. , Van'T Veer M. B.
Published: March 2003

Patients with chronic immune thrombocytopenic purpura (ITP) who are unresponsive to corticosteroids require splenectomy, but if this fails, treatment is difficult. We tried to induce durable remissions in ITP patients refractory to corticosteroids before or after splenectomy by applying strong immunosuppression with the combination of cyclosporin A (CyA 5 mg/kg/d) and prednisone (0·4 mg/kg/d). Patients were assigned to one of two groups. Group 1, 10 patients refractory to prednisone; and group 2, 10 patients refractory to at least prednisone and splenectomy. Overall response rate was 55% (50% in group 1 and 60% in group 2 patients). Nine of the 10 patients in group 1 finally had a splenectomy because of relapse, insufficient response or toxicity of CyA. Thirty percent of the patients discontinued CyA because of side‐effects; hypertension, severe headache and muscle pain being the most frequent encountered. It is concluded that CyA treatment does not avoid, but only postpones, splenectomy in chronic ITP patients who are refractory to corticosteroids. However, CyA can be useful in a subgroup of patients with corticosteroid‐ and splenectomy‐refractory ITP, but treatment toxicity is high.
The effect of leucocyte depletion on the quality of fresh‐frozen plasma
Authors: Cardigan R. , Sutherland J. , Garwood M. , Krailadsiri P. , Seghatchian J. , Beard M. , Beckman N. , Williamson L. M.
Published: March 2003

The aim of this study was to evaluate the quality of leucodepleted (LD) fresh‐frozen plasma (FFP) produced using one of five whole blood filters (Baxter RS2000 & RZ2000, NPBI T2926, Macopharma LST1 and Terumo WBSP) or two plasma filters (Pall LPS1 and Baxter FGR7014). Whole blood or plasma was filtered within 8 h of collection at an ambient temperature. Samples were taken pre‐ and post filtration for analysis of coagulation factors and complement activation (n = 7–12 for each type of filter). All filtered units (209–286 ml) contained < 5 × 106 residual leucocytes and < 30 × 109/l platelets. Statistically significant losses of factors V, VIII, IX, XI and XII and increases in markers of coagulation activation were observed (0–21%), which were dependent on filter type. None of the filters had a significant effect on von Willebrand factor (VWF) multimeric distribution or the activity of VWF and factors II, VII or X. The effect on levels of C3a appeared to be related to the filter surface charge: positively charged filters resulted in C3a generation, whereas negatively charged resulted in C3a removal. None of the observed changes are likely to be clinically significant unless subsequent processing of plasma (such as pathogen inactivation) results in further losses of coagulation factors.
Fluorescent 5′‐exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias
Authors: Kreuzer Karl‐Anton , Saborowski Antje , Lupberger Joachim , Appelt Christine , Na Il‐Kang , Le Coutre Philipp , Schmidt Christian Andreas
Published: March 2003

The Wilms' tumour gene (WT1) has been suggested as a powerful parameter for molecular monitoring of minimal residual disease (MRD) in leukaemias. However, molecular monitoring via WT1 RNA levels is far from being routinely performed, which is possibly owing to the complex and inaccurate quantitative reverse transcription polymerase chain reaction (RT‐PCR) procedures. Using a newly‐developed quantitative real time RT‐PCR, we measured WT1 transcripts in peripheral blood leucocytes of patients with acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid leukaemia (CML). While healthy blood donors did not show measurable amounts of WT1 transcripts, WT1 RNA levels were detectable in all types of leukaemia. Furthermore, intraindividual WT1 transcript kinetics were exclusively dependent on disease progression, treatment and subsequent disease outcome. Using this approach, we could distinguish between treatment response and failure within the first days of therapeutic intervention. Moreover, gradually rising WT1 levels over a period of weeks and months paralleled long‐term disease progression and appeared to be a prognostic indicator for subsequent clinical relapse. A linear correlation between quantities of WT1 and bcr/abl fusion transcripts could be seen in CML. We conclude that quantitative assessment of WT1 transcripts using real‐time PCR is an appropriate method for molecular monitoring of AML, ALL and CML, and can be used independently for both short‐ and long‐term monitoring of leukaemia patients.
Safety and efficacy of thalidomide in patients with myelofibrosis with myeloid metaplasia
Authors: Barosi Giovanni , Grossi Alberto , Comotti Benedetto , Musto Pellegrino , Gamba Gabriella , Marchetti Monia
Published: March 2003

We administered the anti‐angiogenic drug thalidomide to 21 patients (12 men) with myelofibrosis with myeloid metaplasia (MMM), who were not responsive to standard treatment. Patients received thalidomide at an escalating dose from 100 to 400 mg/d. Administration of the drug was discontinued before the planned 6 months of treatment in 19 patients (90·5%), mainly because of somnolence and/or fatigue, neurological symptoms or neutropenia. Of the 13 evaluable patients (who received more than 30 d of therapy), anaemia improved in three out of seven (43%) who were treated because of anaemia; thrombocytopenia improved in two out of three (66·6%) who were treated because of thrombocytopenia; splenomegaly was reduced in four (30·8%). Undesired increases in white blood cell and platelet counts were observed in three (23·1%) and five (38·5%) patients respectively. A severity score, indexed on haematological and clinical parameters, improved in two patients (15·4%), but worsened in five (38·5%). In conclusion, standard‐dose thalidomide in MMM patients is burdened with a high rate of side‐effects, which prevent prolonged treatment. Because the drug is effective in improving anaemia and thrombocytopenia and in reducing splenomegaly, low‐dose therapy warrants evaluation. The unexpected observation of leucocytosis and thrombocytosis suggests biological studies and better criteria for selection of patients for treatment.
A highly polymorphic microsatellite in the factor V gene is an informative tool for the study of factor V‐related disorders
Authors: Castoldi Elisabetta , Lunghi Barbara , Mingozzi Federico , Simioni Paolo , Girolami Antonio , Bernardi Francesco
Published: March 2003

The role of factor V (FV) mutations in activated protein C (APC) resistance and FV deficiency is well established. We report on the identification of a highly polymorphic (AT)n microsatellite marker in the FV gene, which represents an informative tool for the investigation of the origin and evolution of pathologically relevant FV genetic components. A high number of different microsatellite alleles were found to be associated with FV R506Q and FV H1299R, two single‐origin mutations. An example of the use of the microsatellite marker in family studies of thrombophilia and FV deficiency is also provided.
Simultaneous signalling through c‐mpl, c‐kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3‐K, PKC and MAPK pathways
Authors: Minamiguchi Hitoshi , Kimura Takafumi , Urata Yoji , Miyazaki Hiroshi , Bamba Tadao , Abe Tatsuo , Sonoda Yoshiaki
Published: March 2003

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU‐Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood‐derived CD34+IL‐6R− cells as a target. TPO alone induced a significant number of CFU‐Meg colonies. Although stromal cell‐derived factor‐1 (SDF‐1) or SCF alone did not support CFU‐Meg colony formation, these factors had a synergistic effect on CFU‐Meg colony formation in the presence of TPO. The combination of SDF‐1, SCF and TPO induced twice as many CFU‐Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU‐Meg colony formation. LY294002 and GF109203X (inhibitors of PI3‐K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU‐Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF‐1, and TPO + SCF + SDF‐1, whereas it abolished the effect of SDF‐1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c‐mpl, c‐kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU‐Meg, and SDF‐1 is a potentiator of human megakaryocytopoiesis.
Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells
Authors: Quirici Nadia , Soligo Davide , Caneva Lorenza , Servida Federica , Bossolasco Patrizia , Deliliers Giorgio Lambertenghi
Published: March 2003

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133+ bone marrow cells, a subset of CD34+ haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 ± 5%), the CD133+ bone marrow cells were grown on fibronectin‐coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF‐1). The CD133+ fraction contained 95 ± 4% CD34+ cells, 3 ± 2% cells expressing VEGF receptor (VEGFR‐2/KDR), but did not express von Willebrand factor (VWF), VE‐cadherin, P1H12 or TE‐7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 ± 5 times. The cells were further purified using Ulex europaeus agglutinin‐1 (UEA‐1)‐fluorescein isothiocyanate (FITC) and anti‐FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45− and CD14−, and expressed several endothelial markers (UEA‐1, VWF, P1H12, CD105, E‐selectin, VCAM‐1 and VE‐cadherin) and typical Weibel–Palade bodies. They had a high proliferative potential (up to a 2400‐fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co‐cultivated with CD34+ cells, in parallel with a purified fibroblastic cell monolayer. CD34+ cells (10 × 105) gave rise to 17 951 ± 2422 CFU‐GM colonies when grown on endothelial cells, and to 12 928 ± 4415 CFU‐GM colonies on fibroblast monolayers. The ECs also supported erythroid blast‐forming unit (BFU‐E) colonies better. These results suggest that bone marrow CD133+ progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo‐angiogenesis.
Hb H hydrops foetalis syndrome: a case report and review of literature
Authors: Lorey Fred , Charoenkwan Pimlak , Witkowska H. Ewa , Lafferty John , Patterson Margaret , Eng Barry , Waye John S. , Finklestein Jerry Z. , Chui David H. K.
Published: March 2003

Haemoglobin H (Hb H) disease is caused by deletion or inactivation of three α‐globin genes, leaving only one intact and active α‐globin gene. People with Hb H disease usually have moderate anaemia, but are generally thought to be asymptomatic. Some Hb H disease patients require transfusions, and there are reports of fetuses with Hb H disease who have severe anaemia in utero resulting in fatal hydrops foetalis syndrome. We now report a case of Hb H hydrops foetalis syndrome, caused by the inheritance of a hitherto novel α‐globin gene point mutation (codon 35 TCC→CCC or Serine→Proline) and an α‐thalassaemia deletion of the Filipino type removing all ζ‐α‐globin genes on the other chromosome 16. The infant was delivered prematurely because of pericardial effusion and fetal distress, and was found to have severe anaemia and congenital anomalies. A review of the relevant literature on this syndrome is presented, and serves to underscore the phenotypic variations of Hb H disease and the need for surveillance for this condition among newborns and genetic counselling in communities with a high proportion of at‐risk populations.
Endothelial dysfunction in patients with polycythaemia vera
Authors: Neunteufl T. , Heher S. , Stefenelli T. , Pabinger I. , Gisslinger H.
Published: March 2003

Patients with polycythaemia vera (PV) are at increased risk of developing arterial and venous thromboembolic complications. We investigated whether endothelium‐dependent, flow‐mediated vasodilatation (FMD) is impaired in PV patients without clinical evidence of artery disease as observed in patients with conventional cardiovascular risk factors. FMD and endothelium‐independent, nitroglycerine‐induced vasodilatation (NMD) were assessed using high‐resolution ultrasound in the brachial artery of 20 patients with PV and 20 sex‐ and age‐matched control subjects (CTL). FMD was markedly impaired in PV patients compared with CTL (7·6 ± 2·9% versus 11·6 ± 5·7%, P = 0·009) whereas NMD was similar in both study groups. The impairment of FMD was independently related to the presence of PV (r = −0·434, P = 0·009) and vessel size (r = −0·107, P = 0·038) but was not related to haematocrit values and platelet counts. The results demonstrate that PV is associated with endothelial dysfunction in the pre‐clinical phase of arterial disease. However, the precise mechanisms by which PV leads to this altered vascular reactivity remain unclear.
Residual expression of functional MHC class II molecules in twin brothers with MHC class II deficiency is cell type specific
Authors: Wolf Hermann M. , Thon Vojtech , Gulle Heinz , Lechleitner Sonja , Eibl Martha M. , Petzelbauer Peter
Published: March 2003

We examined major histocompatibility complex (MHC) class II expression in B cells, peripheral blood monocytes, activated T cells, epidermal Langerhans cells, monocyte‐derived dendritic cells, dermal microvascular endothelial cells (DMEC) and fibroblasts of twin brothers with MHC class II deficiency. Although residual human leucocyte antigen (HLA)‐DR expression was found on a subpopulation of epidermal Langerhans cells and a subset of peripheral blood monocyte‐derived dendritic cells, the patients' B cells, monocytes and activated T cells were HLA‐DR negative. After treatment with interferon‐γ (IFN‐γ), the patients' DMEC expressed HLA‐DR but not ‐DP and ‐DQ at the protein and mRNA level, whereas IFN‐γ failed to induce HLA‐DR expression on dermal fibroblasts. The patients' monocyte‐derived dendritic cells were capable of processing and presenting tetanus toxoid to autologous T cells, and patient‐derived DMEC induced the proliferation of allogeneic CD4+ T cells in an MHC class II‐restricted fashion, indicating that the observed residual MHC class II surface expression was functional. The findings reported show that the defect encountered in these patients is not necessarily expressed to the same extent in different cell lineages, which is relevant for the understanding of the patients' phenotype and also illustrates that only small amounts of MHC class II are needed to mount a functional cellular immune response in vivo.
Clinical, biochemical and molecular findings in a series of families with hereditary hyperferritinaemia–cataract syndrome
Authors: Girelli Domenico , Bozzini Claudia , Zecchina Gabriella , Tinazzi Elisa , Bosio Sandra , Piperno Alberto , Ramenghi Ugo , Peters Jutta , Levi Sonia , Camaschella Clara , Corrocher Roberto
Published: March 2003

Hereditary hyperferritinaemia–cataract syndrome (HHCS) is an autosomal dominant disease caused by mutations in the iron responsive element (IRE) of the l‐ferritin gene. Despite the elucidation of the genetic basis, the overall clinical spectrum of HHCS has been less well studied as, to date, only individual case reports have been described. Therefore, we studied a total of 62 patients in 14 unrelated families, with nine different mutations. No relevant symptoms other than visual impairment were found to be associated with the syndrome. A marked phenotypic variability was observed, particularly with regard to ocular involvement (i.e. age range at which cataract was diagnosed in 16 subjects with the C39T: 6–40 years). Similarly, serum ferritin levels varied substantially also within subjects sharing the same mutation (i.e. range for the A40G: 700–2412 µg/l). We followed an HHCS newborn in whom well‐defined lens opacities were not detectable either at birth or at 1 year. The lens ferritin content was analysed in two subjects who underwent cataract surgery at different ages, with different cataract morphology. Values were similar and about 1500‐fold higher than in controls. These observations suggest that: (i) in HHCS the cataract is not necessarily congenital; (ii) in addition to the IRE genotype, other genetic or environmental factors may modulate the phenotype, especially the severity of the cataract.
Association of clonal T‐cell large granular lymphocyte disease and paroxysmal nocturnal haemoglobinuria (PNH): further evidence for a pathogenetic link between T cells, aplastic anaemia and PNH
Authors: Karadimitris Anastasios , Li Ke , Notaro Rosario , Araten David J. , Nafa Khedoudja , Thertulien Raymond , Ladanyi Marc , Stevens Ann E. , Rosenfeld Calvin S. , Roberts Irene A. G. , Luzzatto Lucio
Published: March 2003

There is mounting evidence to suggest that T‐cell‐mediated suppression of haemopoiesis is a pathogenetic mechanism in three bone marrow failure syndromes: aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplasia (MDS). T‐cell microclones can be detected by sensitive polymerase chain reaction (PCR)‐based methods in all three disorders. Recently, larger clonal populations of T‐cell large granular lymphocytes (T‐LGLs) have been observed in some patients with AA and MDS. Here, we report the development of a large clonal T‐LGL population in a patient with bona fide PNH. In this patient, we defined part of the sequence of the T‐cell receptor (TCR) β‐chain gene, and we have shown that the large T‐LGL population emerged from a background of multiple smaller T‐cell clones. Thus, T‐LGL clones in AA, MDS and PNH probably expand as a result of antigenic stimulation. It is postulated that the antigen driving clonal T‐cell proliferations in these disorders exists on haemopoietic stem cells.
Relationship between bone marrow failure syndromes and the presence of glycophosphatidyl inositol‐anchored protein‐deficient clones
Authors: Maciejewski Jaroslaw P. , Rivera Candido , Kook Hoon , Dunn Daniel , Young Neal S.
Published: March 2003

Because of the insensitivity of the Ham test, paroxysmal nocturnal haemoglobinuria (PNH) has been inaccurately viewed as a late clonal complication of aplastic anaemia (AA). To clarify the relationship between PNH and marrow failure, we tested for the presence of glycosylphosphatidyl‐anchored protein‐deficient (GPI‐AP) granulocytes in large cohorts of patients with AA, myelodysplasia (MDS), and pure haemolytic PNH. A PNH clone was detected in 32% of new AA patients and 18% of MDS patients. In serial studies, this proportion did not change up to 15 years after diagnosis, suggesting that expansion of aberrant cells is an early event (i.e. prior to initial presentation). For all patients with a PNH clone, on average 14% of PNH granulocytes were found on presentation and 37% at 10 years. Patients with PNH but without cytopenia showed higher percentages of GPI‐AP‐deficient cells than did those with the AA/PNH syndrome. After immunosuppression, there was no change in the contribution of PNH clone to blood production, arguing against the ‘immune‐escape’ theory in PNH. Clinically, a high proportion of GPI‐AP‐deficient cells correlated with marrow hypercellularity. GPI‐AP‐deficient cells were similarly present in patients with and without karyotypic abnormalities. Our results indicate that the GPI‐AP‐deficient clones show quantitative and kinetic differences between classic haemolytic PNH and PNH with marrow failure, in which the evolution rate is low later in the course of the disease.
Primary effusion lymphomas exhibit complex and recurrent cytogenetic abnormalities
Authors: Wilson Kathleen S. , McKenna Robert W. , Kroft Steven H. , Dawson D. Brian , Ansari Qasim , Schneider Nancy R.
Published: March 2003

Summary. Cytogenetic findings in a few primary effusion lymphoma (PEL) cell lines have been reported, but only three complete karyotypes of primary specimens from patients with this neoplasm have been published. In this study, cytogenetic analysis was performed on 11 effusion specimens from 10 patients with PEL. We corroborate data obtained from the cell line studies that trisomy 7, trisomy 12 and aberrations in the proximal long arm of chromosome 1 (1q) are recurring cytogenetic aberrations in PEL and also identify breakpoints at 3q23, 7p22, 7q22, 10q24, 12q24, 13q22, 14q24, 14q32, 15p11.2 and Xq22 as well as +8, +15, +19, +X and –Y as recurring chromosome abnormalities. The identification of recurring cytogenetic aberrations may lead to delineation of the genetic events in PEL.
Mechanisms responsible for the failure of protamine  to inactivate low‐molecular‐weight heparin
Authors: Crowther Mark A. , Berry Leslie R. , Monagle Paul T. , Chan Anthony K. C.
Published: March 2003

Summary. Protamine is unable to completely reverse the anticoagulant effect of the low‐molecular‐weight heparins (LMWH), a fact of clinical importance given the rapid increase in use of LMWH in clinical practice. This investigation sought to determine the mechanism by which LMWH were able to resist protamine‐mediated inactivation. Affinity fractionation of LMWH by passage through a protamine column, with subsequent determination of molecular mass and sulphate charge density, demonstrated that the protamine‐resistant fraction in LMWH is an ultra‐low‐molecular‐weight fraction with low sulphate charge density. This group of molecules was not found in unfractionated heparin, even when species of similar molecular mass were compared. We then determined that different commercially available LMWH varied in their ability to be neutralized by protamine, and that this variability correlated with the total sulphate content of the LMWH. We conclude that reduced sulphate charge, not molecular mass, is the principle reason that protamine is unable to fully inactivate LMWH. Furthermore, different LMWH vary in their ability to be neutralized by protamine, suggesting that product‐specific recommendations for neutralization might be developed.
Factor VIII inhibitor in a patient with mild haemophilia A and an Asn618→Ser mutation responsive to immune tolerance induction and cyclophosphamide
Authors: Vlot André J. , Wittebol Shulamiet , Strengers Paul F. W. , Turenhout Ellen A. M. , Voorberg Jan , Van Den Berg H. Marijke , Mauser‐Bunschoten Eveline P.
Published: March 2003

Summary.  We describe a patient with mild haemophilia A (original value of factor VIII activity 0·30 U/ml) who developed an inhibitor (36·1 Bethesda U/ml) which cross‐reacted with his endogenous factor VIII. This caused a decline in basal factor VIII level (< 0·01 U/ml) and severe haemorrhagic events. Treatment to induce immune tolerance was started with factor VIII/von Willebrand factor (VWF) concentrates, but inhibitor levels increased progressively and the patient suffered serious bleeding. Cyclophosphamide was administered and, after 8 months treatment, factor VIII levels increased to 0·20 U/ml and the inhibitor could no longer be detected. Screening of his factor VIII gene revealed a missense mutation in exon 13 that predicts substitution of Asn618→Ser in the A2 domain of factor VIII. Immunoprecipitation analysis showed that the antibodies present in the patient's plasma reacted with metabolically labelled A2 domain and, to a lesser extent, with factor VIII light chain. Inhibitory antibodies were completely neutralized by recombinant A2 domain, whereas no neutralization was observed after the addition of factor VIII light chain (A3–C1–C2) and C2 domain. More detailed analysis showed that the majority of inhibitory antibodies were directed against residues Arg484–Ile508, a previously identified binding site for factor VIII inhibitors. Our findings suggest that immune tolerance therapy and cyclophosphamide were successful in eradicating inhibitory antibodies against a common epitope on factor VIII.
Characterization of two novel splice site mutations in human factor VII gene causing severe plasma factor VII deficiency and bleeding diathesis
Authors: Borensztajn Keren , Chafa Ouerdia , Alhenc‐Gelas Martine , Salha Siham , Reghis Abderrezak , Fischer Anne‐Marie , Tapon‐Bretaudière Jacqueline
Published: March 2003

Summary. The molecular basis of severe type I factor (F)VII deficiency was investigated in two Algerian patients. One patient, a 13‐year‐old‐girl who has suffered from severe bleeding since birth, was homozygous for a 7‐bp deletion (nt 7774–7780) and a 251‐bp insertion (nt 7773–7781) of mitochondrial origin, in IVS 4 acceptor splice site. The other patient, an infant who died from massive intracranial haemorrhage, was homozygous for a transversion in the IVS 7 donor splice site (T9726+2→G) and a missense mutation in exon 8 (G10588→A; Arg224→Gln). In both cases, the deleterious mutations are probably the splice site junction abnormalities impairing mRNA processing. These three lesions have not yet been reported.
Cultured autografting for juvenile myelomonocytic leukaemia
Authors: Grainger John D. , Will Andrew M. , Stevens Richard F.
Published: March 2003

Summary. A case of juvenile myelomonocytic leukaemia (JMML) associated with a chromosomal translocation (1;5) is described. Initial cytoreductive therapy failed to control the disease. In the absence of a matched family or unrelated donor, a Dexter‐type long‐term bone marrow culture (LTBMC) was established. The LTBMC showed preferential growth of normal stem cells over the abnormal clone, allowing a cultured autologous stem cell transplantation to be performed. Despite detection of the t(1;5) from 5 months to 7 years following cultured autograft, the patient remained in haematological remission. Currently the patient is alive and well at 10 years in full cytogenetic remission.
Congenital leukaemia: the Dutch experience and review of the literature
Authors: Bresters Dorine , Reus Angelino C. W. , Veerman Anjo J. P. , Van Wering Elizabeth R. , Van Der Does‐van den Berg Anna , Kaspers Gertjan J. L.
Published: March 2003

Summary. We reviewed Dutch patients and those described in the literature with congenital leukaemia in the past 25 years, with the intention to obtain an overview of the characteristics of this rare disease. Among the 117 patients reviewed, acute myeloid leukaemia (AML) was more frequent (64%) than acute lymphoblastic leukaemia (ALL, 21%). Most patients had a high leukaemic cell load with hepatosplenomegaly, leukaemia cutis and hyperleucocytosis. Cytogenetic abnormalities were found in the majority of the patients tested (72%); 11q23 abnormalities were found in less than half of them (42%). The probability of overall survival at 24 months was only 23%. When congenital AML and ALL were compared, clinical characteristics and overall survival were not significantly different. However, in patients at risk, the probability of event‐free survival (EFS) and disease‐free survival (DFS) were significantly higher in AML than in ALL, 43% versus 13% and 68% versus 0% respectively. Among the congenital AML cases, six spontaneous remissions have been described. In conclusion, the clinical characteristics of congenital leukaemia differ from those of leukaemia in older children and prognosis is generally poor. Once complete remission is achieved, patients with AML fare better than those with ALL. Chemotherapy for congenital leukaemia needs improvement to increase the sustained remission rate.
Gene mutations and three‐dimensional structural analysis in 13 families with severe factor X deficiency
Authors: Peyvandi Flora , Menegatti Marzia , Santagostino Elena , Akhavan Sepideh , Uprichard James , Perry David J. , Perkins Stephen J. , Mannucci Pier M.
Published: March 2003

Summary. Factor X (FX) deficiency is a rare autosomal recessive disorder. The phenotype and genotype of 15 Iranian patients with FX deficiency from 13 unrelated families with a high frequency of consanguinity were analysed. Five different assays identified four patients from three families with a discrepancy between low‐FX coagulant activity (FX:C) and higher‐FX antigen (FX:Ag) (a type II deficiency). The remaining 11 patients had parallel reductions of FX:C and FX:Ag (a type I deficiency). Nine different homozygous candidate mutations were identified, of which eight were novel. The four type II cases were associated with an Arg(−1)Thr missense mutation in the prepropeptide: Arg(−1) is highly conserved in all vitamin K‐dependent proteins. Four type I mutations (Gly78Asp, Cys81Tyr, Gly94Arg and Asp95Glu) were localized to the EGF‐1 and EGF‐2 domains, for which molecular views showed that the protein folding would be disrupted. The type I mutation Gly222Asp was localized in the catalytic domain of FX, and is sufficiently close to the Asp‐His‐Ser catalytic triad to disrupt its correct protein folding. The two type I splice site mutations were IVS1+3, A→T and IVS2–3, T→G. These novel homozygous FX mutations were consistent with their phenotypes and agree with experimental data from knockout mice, indicating that FX is an essential protein for survival.
Autologous bone marrow transplantation with 4‐hydroperoxycyclophosphamide purging for acute myeloid leukaemia beyond first remission: a 10‐year experience
Authors: Smith B. Douglas , Jones Richard J. , Lee Shing M. , Piantadosi Steve , Vala Milada S. , Fuller Don , Gore Steven D. , Noga Steven J. , O'Donnell Paul V. , Braine Hayden , Vogelsang Georgia B. , Fuchs Ephraim J. , Flinn Ian W. , Brodsky Robert A. , Ambinder Richard F. , Miller Carole B.
Published: March 2003

Summary. Between January 1987 and January 1997, 69 eligible patients with acute myeloid leukaemia (AML) in either second (CR2) or third (CR3) complete remission (CR2 = 60, CR3 = 9) underwent 4‐hydroperoxycyclophosphamide‐purged autologous bone marrow transplantation (BMT) at the Johns Hopkins Oncology Center. The patients' median age was 27 years (range 1–62) and all received busulphan and cyclophosphamide as their preparative regimen. The probability of event‐free survival (EFS) at 5 years was 30%[95% Confidence Interval (CI): 19–42%] for CR2 patients and 22% (3–51%) for those in CR3, with a median follow up of 8 years in the surviving group. The median time to an absolute neutrophil count of 0·5 × 109/l was 45 d (range 20–185). Relapse was the major cause of failure with a relapse rate of 55% in CR2 and 44% in CR3, while the non‐relapse, transplant‐related mortality rate was 15% in CR2 and 33% in CR3. In univariate analysis, patient age, cytogenetics, white blood cell count at presentation, CR1 duration and the sensitivity of clonogeneic leukaemia (CFU‐L) in the graft to 4HC were all prognostic for EFS. Using each of these significant variables in multivariate modelling, patient age and sensitivity of CFU‐L to 4HC were determined to be predictors of EFS. 4HC‐purged autologous BMT produced results similar to allogeneic BMT for AML patients beyond first remission.
Transforming growth factor‐β1 causes transcriptional activation of CD34 and preserves haematopoietic stem/progenitor cell activity
Authors: Pierelli Luca , Marone Maria , Bonanno Giuseppina , Rutella Sergio , De Ritis Daniela , Mancuso Salvatore , Leone Giuseppe , Scambia Giovanni
Published: March 2003

Summary. Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surface protein CD34. Transforming growth factor β1 (TGF‐β1) is one of the soluble molecules that regulate cell cycle and differentiation of haematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has previously been shown that TGF‐β1 maintains human CD34+ haematopoietic progenitors in an undifferentiated state, independently of any cell cycle effect. Here, we have shown that TGF‐β1 upregulates the human CD34, an effect that was evident in primary stem/progenitor cells (CD34+lin–) both at the transcriptional and protein levels, and was not associated with any relevant effect on cell growth. The presence of TGF‐β1 influenced differentiation, maintaining primary CD34+/Lin– in an undifferentiated state. This effect was associated with Smad activation and with a dramatic decrease in p38 phosphorylation. Moreover, blocking p38 phosphorylation by the SB202190 inhibitor increased CD34 RNA levels but did not enhance CD34 protein expression in CD34+/Lin– cells, suggesting that modulation of multiple signalling pathways is necessary to reproduce TGF‐β1 effects. These data establish the role that TGF‐β1 has in the modulation of the CD34 stem/progenitor protein and stem/progenitor functions, providing important clues for understanding haematopoietic development and a potential tool for the modulation of human haematopoiesis.
Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura
Authors: Kohda Kyuhei , Kuga Takashi , Kogawa Katsuhisa , Kanisawa Yuuji , Koike Kazuhiko , Kuroiwa Ganji , Hirayama Yasuo , Sato Yasushi , Niitsu Yoshiro
Published: March 2003

Summary. The prevalence of Helicobacter pylori infection and the effect of its eradication on platelet count in 48 Japanese patients with autoimmune thrombocytopenic purpura (AITP), including 40 chronic idiopathic thrombocytopenic purpura (ITP) and eight secondary AITP, were investigated. H. pylori infection was found in 25 ITP patients (62·5%) and in two secondary AITP (25%). H.pylori eradication was obtained in 19 of 19 infected ITP patients (100%), who were not in remission (platelets < 100 × 109/l) at the time of infection assessment. During follow‐up (median 14·8 months), 12 of 19 H. pylori‐eradicated patients (63·2%) showed a significant increase in platelet count accompanied by a significant decrease of platelet‐associated immunoglobulin G (IgG). This response was maintained in all responding patients throughout the follow‐up period. However, two infected patients with secondary AITP did not show platelet increase after eradication. The assessment of H. pylori infection and its eradication should be attempted in ITP as this approach could be an effective strategy, at least for some of these patients.
The anti‐apoptotic genes Bcl‐XL and Bcl‐2 are over‐expressed and contribute to chemoresistance of non‐proliferating leukaemic CD34+ cells
Authors: Konopleva Marina , Zhao Shourong , Hu Wei , Jiang Shuwei , Snell Virginia , Weidner Douglas , Jackson C. Ellen , Zhang Xin , Champlin Richard , Estey Elihu , Reed John C. , Andreeff Michael
Published: March 2003

Summary. In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti‐apoptotic genes Bcl‐2 and Bcl‐XL have been implicated in cell cycle regulation, we investigated the expression of these genes in non‐proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl‐XL and Bcl‐2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl‐XS and Bax expression was found. Furthermore, the Bcl‐XL/XS but not the Bcl‐2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0·001). An inverse correlation between Bcl‐2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all‐trans retinoic acid (ATRA) reduced Bcl‐2 and Bcl‐XL expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara‐C). These findings demonstrate overexpression of the anti‐apoptotic proteins Bcl‐XL and Bcl‐2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl‐2 and proliferation suggests a role for Bcl‐2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.
Isolation of differentially expressed genes in NPM‐ALK‐positive anaplastic large cell lymphoma
Authors: Villalva Claire , Trempat Pascal , Greenland Catherine , Thomas Charles , Girard Jean Philippe , Moebius Fabian , Delsol Georges , Brousset Pierre
Published: March 2003

Summary. In this study, we used subtractive suppression hybridization to compare gene expression between an ALK‐positive anaplastic large cell lymphoma (ALCL)‐derived cell line and a clinical case of ALK‐negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1‐receptor, thymosin β4, thymosin β10, moesin and cytohesin‐1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis‐1 (HIAP‐1), Bax inhibitor‐1 and MCL‐1, or DNA repair, such as poly (ADP‐ribose) polymerase 1 (PARP‐1), X‐associated protein‐1 (XAP‐1), SUMO‐1 (sentrin‐1) and RanGTPase‐activating protein 1 (RanGAP‐1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM‐ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM‐ALK‐driven transformation process, and that moesin and cytohesin‐1 may be more specifically implicated in a signalling pathway involving PLCγ and PI3K.
The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial
Authors: Burnett Alan K. , Wheatley Keith , Goldstone Anthony H. , Stevens Richard F. , Hann Ian M. , Rees John H. K. , Harrison Georgina ,   the Medical Research Council Adult and Paediatric Working Parties
Published: March 2003

Summary. Patients under 55 years in the United Kingdom Medical Research Council Acute Myeloid Leukaemia 10 trial who entered complete remission were tissue typed (n = 1063). Four hundred and nineteen had a matched sibling donor and 644 had no match. When compared on a donor versus no donor basis the relapse risk was reduced in the donor arm (36%vs 52%; P = 0·001) and the disease‐free survival (DFS) improved (50%vs 42%; P = 0·01), but overall survival (OS) was not different (55%vs 50%; P = 0·1). Sixty‐one per cent of patients with a donor underwent transplantation. When patients were subdivided into risk groups based on cytogenetics alone or with the addition of blast response to course 1, a reduction in relapse risk was seen in all risk groups and in three age cohorts (0–14, 15–34 and 35+ years). Significant benefit in DFS was only seen in the intermediate‐risk cytogenetic group (50%vs 39%; P = 0·004). The OS benefit was only seen in intermediate‐risk patients (55%vs 44%; P = 0·02). The reduction in relapse risk in good‐risk patients was attributable to patients with t(15;17) and not to patients with t(8;21) or inv(16). Allogeneic transplantation given after intensive chemotherapy was able to reduce relapse in all risk and age groups. However, due to the competing effects of procedural mortality and an inferior response to chemotherapy if relapse does occur, there was a survival advantage only in patients of intermediate risk. This trial found no survival advantage in children, patients over 35 years or good‐risk disease.
Plasminogen activator inhibitor‐1 is an independent diagnostic marker as well as severity predictor of hepatic veno‐occlusive disease after allogeneic bone marrow transplantation in adults conditioned with busulphan and cyclophosphamide
Authors: Lee Je‐Hwan , Lee Kyoo‐Hyung , Lee Jung‐Hee , Kim Shin , Seol Miee , Park Chan‐Jeoung , Chi Hyun‐Sook , Kang Weechang , Kim Seung Taik , Kim Woo‐Kun , Lee Jung‐Shin
Published: March 2003

Summary. We attempted to identify the diagnostic markers and severity predictors of hepatic veno‐occlusive disease (VOD) in 115 adult patients who were uniformly conditioned with busulphan and cyclophosphamide and who underwent allogeneic bone marrow transplantation (BMT). A diagnosis of VOD was made according to clinical criteria. Severity of VOD was classified as mild, moderate or severe. Various haemostatic parameters were determined at five time points (d −7, 0, 7, 14 and 21). Using clinical and haemostatic parameters, we developed multivariate models to identify diagnostic markers as well as severity predictors of VOD. Of the 115 patients included in the study, 50 (43·5%) developed VOD. Twenty‐nine had mild VOD, 13 moderate and 8 severe. Multiple logistic regression models showed that plasminogen activator inhibitor‐1 (PAI‐1) antigen (P = 0·029), percentage change of body weight (P = 0·001) and bilirubin (P < 0·001) were independent marker variables for the occurrence of VOD, and PAI‐1 antigen (P = 0·030) and bilirubin (P = 0·049) were independent marker variables for the occurrence of severe VOD. Our study suggests that PAI‐1 antigen can be used as a diagnostic marker as well as a severity predictor of VOD after allogeneic BMT.