bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody engagement of intercellular adhesion molecule 3 triggers apoptosis of normal and leukaemic myeloid marrow cells
Authors: Stucki Anne , Hayflick Joel S. , Sandmaier Brenda M.
Published: March 2003

Intercellular adhesion molecule 3 (ICAM‐3, CD50) is an immunoglobulin (Ig) domain‐containing cell–cell adhesion receptor that binds to the lymphocyte function antigen 1 (LFA‐1; CD11a/CD18) integrin. It is constitutively expressed on haematopoietic precursors and differentiated leucocytes, as well as on most leukaemic cells. ICAM‐3/LFA‐1 binding during a lymphocyte‐mediated cellular immune response has been well established; however, its role in the marrow compartment is unclear. In this study, marrow cells from normal and acute leukaemic donors, as well as leukaemic cell lines, were cultured in the presence of various monoclonal antibodies (mAbs) to ICAM‐3, and apoptosis was subsequently measured by annexin V binding. Anti‐ICAM‐3 mAb ICR 1.1 engagement triggered increased percentages of apoptosis among normal and leukaemic marrow myeloid cells. Fab fragments of ICR 1.1 mimicked the intact mAb, suggesting that the apoptotic signal was independent of Fc receptor interactions and did not require bivalent epitope engagement. In addition, the apoptotic signal was found to be independent of ICAM‐1/LFA‐1 binding interactions, as well as Fas/FasL and tumour necrosis factor alpha (TNF‐α)/TNF receptor‐activated pathways, as neutralizing antibodies to CD11a/CD18, Fas and TNF‐α failed to abrogate the response.
High levels of antiphospholipid antibodies are associated with cytomegalovirus infection in unrelated bone marrow and cord blood allogeneic stem cell transplantation
Authors: Mengarelli Andrea , Minotti Clara , Palumbo Giovanna , Arcieri Paolo , Gentile Giuseppe , Iori Anna Paola , Arcese William , Mandelli Franco , Avvisati Giuseppe
Published: March 2003

Antiphospholipid antibodies (APA) are a family of autoimmune and alloimmune immunoglobulins recognizing protein–phospholipid complexes in in vitro laboratory test systems. These antibodies have been associated with several conditions (malignancies, autoimmune diseases, infections, use of drugs); moreover, a syndrome capable of inducing thromboembolic disease has recently been associated with the presence of these antibodies. The aim of this prospective study was to investigate the levels of APA in subjects affected by haematological malignancies undergoing allogeneic haematopoietic stem cell transplantation (ASCT). Between March 1996 and December 1997, 32 patients undergoing ASCT were studied prospectively until day +180 from transplant. The mean values of IgG and IgM anticardiolipin antibodies (ACA) increased in recipients of stem cells from anunrelated donor, and a statistically significant difference inACA IgG mean value between unrelated and related transplanted patients was demonstrated between days +95 and +180. All of the subjects who received stem cells from an unrelated donor had APA levels higher than the mean normal value +3 SD vs. 35% of those receiving stem cells from a related donor (P < 0·01). The reason for such a difference may be a result of the different incidence in documented cytomegalovirus (CMV) infection in the two groups (83% vs. 23%; P < 0·01), as indicated by the significant correlation between APA positivity and CMV infection (P < 0·05). No relationship was found between APA, conditioning regimen and acute or chronic graft vs. host disease (GVHD). Moreover, we did not observe any thromboembolic disorder or veno occlusive disease (VOD).
Features of essential thrombocythaemia in childhood: a study of five children
Authors: Randi  , Putti  , Fabris  , Sainati  , Zanesco  , Girolami
Published: March 2003

Essential thrombocythaemia (ET) is usually considered a disease of the middle‐aged but, with the advent of automated platelet counting, ET is diagnosed with increasing frequency in young adults and, even more rarely, in children. We report five paediatric patients (four girls and one boy, mean age 89 months) diagnosed with ET in agreement with Polycythaemia Vera Study Group criteria. The patients had a persistent thrombocytosis over 900 × 109/l and, at the time of diagnosis, their platelet count ranged between 1112 and 3178 × 109/l. A 9‐month‐old girl had thrombosis of the inferior cava vein, two children had headaches and two others remained asymptomatic throughout the follow‐up period. Megakaryocytes in the bone marrow were increased in number. The chromosomal analysis was normal, and bcr rearrangement was always negative. None of the patients had spontaneous BFU‐E or altered levels of serum erythropoietin and thrombopoietin. Two patients showed alteration of platelet aggregation, and all had decreased levels of intraplatelet serotonin. In spite of the diagnosis of ET in our patients, we are not sure that the cases reported here are really myeloproliferative disorders. The features could suggest that the cases observed may be affected by an ‘idiopathic thrombocytosis’ without myeloproliferation. Possible variants of ET are described in young adults, and the heterogeneous nature of ET is also suggested by our paediatric patients. Only careful long‐term follow‐up of patients such as these will clarify the natural history of these disorders and suggest therapeutic management.
Induction of remission after donor leucocyte infusion for the treatment of relapsed chronic idiopathic myelofibrosis following allogeneic transplantation: evidence for a ‘graft vs. myelofibrosis’ effect
Authors: Byrne  , Beshti  , Clark  , Ellis  , Haynes  , Das‐Gupta  , Russell
Published: March 2003

A 54‐year‐old man showed evidence of disease progression and a reduction in donor chimaerism by molecular microsatellite analysis 6 months after an allogeneic peripheral blood stem cell transplant for chronic idiopathic myelofibrosis. He was treated with a single infusion of donor leucocyte infusions (DLI), which led to the development of mild acute graft versus host disease (GVHD) and the rapid restoration of full donor haemopoiesis. This subsequently led to a progressive reduction in marrow fibrosis from grade IV to grade I over the following 6 months. We believe that this is the first report to provide clear evidence for the efficacy of DLI in this setting, which also provides evidence for the existence of a T‐cell‐mediated ‘graft vs. myelofibrosis’ effect similar to that seen against other haematological malignancies.
Human myeloma cells promote the production of interleukin 6 by primary human osteoblasts
Authors: Karadag  , Oyajobi  , Apperley  , Russell  , Croucher
Published: March 2003

Interleukin‐6 (IL‐6) is an important growth and survival factor for myeloma cells. However, the identity of the cells producing IL‐6 in vivo remains unclear. Myeloma cells are found closely associated with sites of active bone turnover, and cells of the osteogenic lineage, including bone marrow osteoprogenitors, osteoblasts and bone lining cells, may therefore be ideally placed to synthesize IL‐6. We have examined the possibility that human osteogenic cells may produce IL‐6 in response to stimulation by myeloma cells. Primary human osteoblasts (hOBs) were isolated from normal donors, co‐cultured with the human myeloma cell lines, JJN‐3, RPMI‐8226 and NCI‐H929, and the amount of IL‐6 released was determined by enzyme‐linked immunosorbent assay (ELISA). All myeloma cells stimulated a significant increase in the production of IL‐6 when cultured with hOBs (P < 0.05). Prior fixation of hOBs completely abrogated release of IL‐6 in the co‐cultures. In contrast, fixed myeloma cells retained the ability to induce IL‐6 production, suggesting that hOBs were the principal source of IL‐6. Physical separation of myeloma cells from hOBs using transwell inserts caused a partial inhibition of IL‐6 release (P < 0.05), whereas the addition of media conditioned by myeloma cells to cultures of hOBs stimulated a significant increase in IL‐6 production (P < 0.05). hOBs secreted greater amounts of IL‐6 than human bone marrow stromal cells (hBMSCs) (2.2‐ to 3.5‐fold, P < 0.05), but incubating hBMSCs with dexamethasone to stimulate osteoblastic differentiation resulted in an increase in their ability to produce IL‐6 (1.7‐ to 4.8‐fold, P < 0.05) and to respond to myeloma cells (P < 0.05). These data clearly indicate that cells of the osteoblast lineage release significant amounts of IL‐6 in response to stimulation by myeloma cells and may contribute to the IL‐6 that promotes the proliferation and survival of myeloma cells in vivo.
Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity
Authors: Faioni  , Hermida  , Rovida  , Razzari  , Asti  , Zeinali  , Mannucci
Published: March 2003

Two mutations in exons 3 and 9 of the protein C gene were identified by amplification and sequencing from symptomatic probands referred for venous thromboembolism and thrombophilia screening. The phenotype associated with the mutations is a type II protein C deficiency with normal amidolytic activity. In one family, the mutation in exon 3 (G3545→A), which predicts an R9 to H substitution in the Gla domain, was identified. A mutation in exon 9 (G10899→A), which predicts an R352 to W substitution in the catalytic site, was identified in the second family and has been reported previously in association with type II deficiency with low amidolytic activity. Western blotting of the purified proteins from the probands' plasma did not show any abnormal migratory pattern. Molecular modelling suggested a possible impairment in the recently described Na+ binding pocket for the R352→W mutant. No conclusions could be drawn relative to the R9→H mutant.
The effect of granulocyte colony‐stimulating factor administration in healthy donors before bone marrow harvesting
Authors: Machida Utako , Tojo Arinobu , Takahashi Satoshi , Iseki Toru , Ooi Jun , Nagayama Hitomi , Shirafuji Naoki , Mori Shin‐Ichiro , Wada Yuka , Ogami Kazuo , Yamada Yoshitsugu , Sakamaki Hisashi , Maekawa Taira , Tani Kenzaburo , Asano Shigetaka
Published: March 2003

To investigate whether granulocyte colony‐stimulating factor (G‐CSF) administration to donors before harvest may lighten the burden imposed on them and accelerate the bone marrow (BM) recovery, we administered 2 μg/kg/d of G‐CSF for five consecutive days before the marrow harvest. All of the donors tolerated the G‐CSF administration well without severe adverse events. After 5 d of G‐CSF treatment, CD34+ cells and granulocyte–macrophage colony‐forming units (GM‐CFU) in the donors' BM exceeded baseline values by 4.2‐fold (range 0.71–316) and 1.6‐fold (0.28–118) respectively. The concentration of total nucleated cells (× 107/ml) in the graft increased from 1.61 (0.95–3.23) to 2.44 (1.27–4.01). Although we collected 1020 ml of BM and obtained 1.50 × 1010 nucleated cells from unprimed donors, 940 ml of BM were sufficient to obtain 2.14 × 1010 nucleated cells from primed donors. However, G‐CSF‐primed BM did not shorten the time to tri‐lineage engraftment and the duration of hospitalization compared with unprimed BM, although primed BM contained more CD34+ cells than baseline values. We consider that the advantages of BM priming are not the acceleration of BM recovery but rather the reduction of blood loss during BM harvesting.
Apoptosis in factor‐dependent haematopoietic cells is linked to calcium‐sensitive mitochondrial rearrangements and cytoskeletal modulation
Authors: Garland John , Brown Gillian , Beasley Julian , Brown Robin
Published: March 2003

Apoptosis in murine haematopoietic interleukin (IL)3‐dependent cell lines is induced within 6–8 h by IL‐3 withdrawal. Direct introduction of cytochrome c by electroporation induces apoptosis within 2 h and was inhibited by caspase inhibitors, such as Z‐VADfmk and Z‐Dfmk. We report here that apoptosis induced by IL‐3 withdrawal was refractory to these inhibitors but was accompanied by striking redistribution of mitochondria, which aggregated into an area associated with centrioles without loss of Δψm. Both mitochondrial redistribution and apoptosis were inhibited by the calcium ionophore, ionomycin. Nocodozole, an inhibitor of microtubule assembly, also induced apoptosis, which was unaffected by caspase inhibitors. Although nocodozole did not alter mitochondrial distribution, it significantly reduced Δψm, and both reduction of Δψm and apoptosis were inhibited by ionomycin. Oligomycin, which inhibits the mitochondrial FoF1 ATPase, similarly induced apoptosis, which was unaffected by caspase inhibitors but was inhibited by ionomycin. Further, oligomycin stimulated the novel formation and release of surface membrane‐derived vesicles containing mitochondria with intact Δψm; ionomycin also inhibited their production. In all these conditions, Bcl‐2 protected cells from apoptosis. Our studies show that apoptosis induced by three very different agents shares insensitivity to caspase inhibitors, suppression by ionomycin and effects on mitochondria, which all appear to be linked to cytoskeletal/microtubule activity. They suggest that microtubules and the cytoskeleton play an important role in apoptosis through mechanisms affecting mitochondria but which are independent of cytochrome c release.
No evidence for a major role of heterozygous deletion 657del5 within the NBS1 gene in the pathogenesis of non‐Hodgkin's lymphoma of childhood and adolescence
Authors: Stanulla Martin , Stümm Markus , Dieckvoss Björn‐Ole , Seidemann Kathrin , Schemmel Verena , Müller Brechlin Annette , Schrappe Martin , Welte Karl , Reiter Alfred
Published: March 2003

Nijmegen breakage syndrome (NBS) is an autosomal recessive DNA repair disorder with a high predisposition for lymphoid malignancies. The majority of NBS patients carry a homozygous founder mutation (657del5) within the NBS1 gene. The observation of a high incidence of cancer in close relatives of NBS patients suggests a potential pathogenetic role of NBS1 mutations in heterozygotes as well. We assessed the frequency of the 657del5 mutation in 109 paediatric patients with non‐Hodgkin's lymphoma (NHL). None of the patients analysed carried a NBS1 allele with the 657del5 mutation, suggesting that this mutation does not play a major role in the pathogenesis of NHL of childhood and adolescence.
Treatment of anaemia in myelodysplastic syndromes with prolonged administration of recombinant human granulocyte colony‐stimulating factor and erythropoietin
Authors: Mantovani Luisa , Lentini Giuseppe , Hentschel Bettina , Wickramanayake Premaratne Dias , Loeffler Markus , Diehl Volker , Tesch Hans
Published: March 2003

Treatment with recombinant human erythropoietin (rhEPO) improves anaemia in ≈ 20% of the patients with myelodysplastic syndromes (MDS). Recent reports suggest that a combination treatment with rhEPO plus recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) given for up to 18 weeks may result in a higher erythroid response rate than with rhEPO alone. We investigated the potential advantage of an even more prolonged schedule of combined rhG‐CSF and rhEPO treatment to obtain and maintain stable responses. In a phase II study, 33 patients with MDS [17 with refractory anaemia (RA), eight with RA with ringed sideroblasts (RARS), eight with RA with excess blasts (RAEB) with bone marrow blast counts less than 20%] were scheduled to receive at least 36 weeks of combined therapy with rhG‐CSF and rhEPO. Seventeen of 28 evaluable patients demonstrated an erythroid response [61%; 95% confidence interval (CI) 41–78] after 12 weeks of treatment. The erythroid response rate was 80% (20 of 25 evaluable patients; 95% CI 59–93) after 36 weeks. Seven of these responses developed between week 12 and week 36, whereas two initially responding patients became refractory. The cytokine therapy was generally well tolerated. Nineteen of the 20 patients responding after 36 weeks continued to be treated with both cytokines. After 1 year and 2 years of continuous combined treatment, 50% of the initially included patients showed a continuing response. Our results suggest that a prolonged combination treatment with rhG‐CSF and rhEPO is highly effective in achieving a stable and long‐lasting erythroid response in many patients with MDS and low blast count.
Thrombopoietin levels in cord blood plasma and amniotic fluid in fetuses with alloimmune thrombocytopenia and healthy controls
Authors: Sainio Susanna , Javela Kaija , Kekomäki Riitta , Teramo Kari
Published: March 2003

To extend our knowledge of the kinetics of fetal thrombopoietin (TPO), we studied TPO levels in cord blood plasma and amniotic fluid collected from 15 fetuses considered to be at risk of fetomaternal alloimmune thrombocytopenia and also from 10 healthy controls at caesarean delivery. In the plasma of all 25 fetuses and newborn infants studied, TPO was detected above the lower limit of detection (7 pg/ml) and correlated inversely with platelet counts (r = −0·53, P = 0·006). At term, TPO detected in amniotic fluid was at significantly lower levels (7 pg/ml; range 0–22 pg/ml) than simultaneously obtained cord plasma TPO (114 pg/ml; range 43–201 pg/ml; P < 0·001). There was no correlation between levels of TPO in amniotic fluid and cord plasma or platelet counts. In the serial samples collected from the five fetuses with HPA‐1a alloimmunization before 37 weeks' gestation, the TPO levels in amniotic fluid were significantly higher than at term (P = 0·013): from 22 to 28 weeks' gestation, 42 pg/ml (30–78 pg/ml); from 32 weeks', 24 pg/ml (17–33 pg/ml); at term, 8 pg/ml (4–13 pg/ml), correlating inversely with gestational age (r = −0·81, P = 0·003). Thus, TPO is present in amniotic fluid at levels apparently inversely related to gestational age. Whether these high levels seen early in pregnancy are normal or are associated with the HPA‐1 alloimmunization remains to be shown.
Absence of herpesvirus DNA sequences in the 5T murine model of human multiple myeloma
Authors: Oyajobi B. O. , Deng J.‐H. , Dallas S. L. , Jenson H. B. , Mundy G. R. , Gao S. J.
Published: March 2003

Kaposi's sarcoma‐associated herpesvirus (KSHV, also known as HHV‐8) has been found in patients with multiple myeloma (MM) and postulated to be aetiologically associated with the development of this common plasma cell malignancy. A murine model of MM was previously established in which intravenous transfer of 5T myeloma cells into C57BL/KaLwRij mice resulted in characteristic features of human MM. In the present study, we sought to identify herpesvirus DNA sequences in this murine model of MM through polymerase chain reaction (PCR) analysis using primers specific for KSHV, murine herpesvirus 68 (MHV68) and murine cytomegalovirus (MCMV) as well as consensus primers designed from the highly conserved DNA polymerase genes of the Herpesviridae family. None of the DNA samples from whole bone marrow (n = 6) or dendritic cells enriched by long‐term culture (n = 8) of 5T myeloma‐bearing mice as well as the 5T myeloma cell lines (n = 3) maintained in long‐term culture yielded specific amplification products in any of the PCR assays. Two KSHV‐specific serological assays measuring antibodies to KSHV latent and lytic antigens also failed to detect the presence of anti‐KSHV antibodies in mice that developed MM. These results suggest that the development of 5T murine MM is unlikely to be involved with KSHV or a KSHV‐like murine herpesvirus.
Respiratory failure during induction chemotherapy for acute myelomonocytic leukaemia (FAB M4Eo) with Ara‐C and all‐trans retinoic acid
Authors: Lester W. A. , Hull D. R. , Fegan C. D. , Morris T. C. M.
Published: March 2003

We report two cases of acute myeloid leukaemia FAB classification M4Eo with high white cell counts at presentation, who developed acute respiratory failure with pulmonary infiltrates on chest radiograph soon after commencing conventional cytotoxic chemotherapy plus all‐trans retinoic acid (ATRA). We suggest that in patients with M4Eo ATRA should be used with caution, perhaps delaying its commencement until the white cell count is < 10 x 109/l.
Allogeneic peripheral stem cell transplantation in a case of hereditary sideroblastic anaemia
Authors: González M. I. , Caballero D. , Vázquez L. , Cañizo C. , Hernández R. , López C. , Izarra A. , Arroyo J. L. , González M. , García R. , San Miguel J. F.
Published: March 2003

We report on a case of pyridoxine refractory hereditary sideroblastic anaemia (HSA) in a 19‐year‐old man who underwent peripheral blood stem cell transplantation (PBSCT) from his HLA‐identical brother. By using short tandem repeat polymorphism, 100% donor cells were observed in peripheral blood on day +21; bone marrow showed mixed chimaerism from day +21 to day +221, when 100% cells of donor origin were observed. The patient developed extensive chronic graft‐versus‐host disease with favourable response to treatment. When the haemoglobin range was normal, a programme of phlebotomies reduced serum ferritin levels. Three years after transplantation, the patient has an ECOG rating of 0, with completely normal haemoglobin values (15 g/dl). To our knowledge, this is the first PBSCT reported in a case of hereditary sideroblastic anaemia.
TNP‐470 and recombinant human interferon‐α2a inhibit angiogenesis synergistically
Authors: Minischetti Monica , Vacca Angelo , Ribatti Domenico , Iurlaro Monica , Ria Roberto , Pellegrino Antonio , Gasparini Giampietro , Dammacco And Franco
Published: March 2003

The hypothesis that the combination of two known antiangiogenic agents TNP‐470 and interferon (IFN)‐α exerts synergistic effects has been investigated in vitro and in vivo. In vitro, TNP‐470 and recombinant human IFN‐α2a (rhIFN‐α2a) resulted in a dose‐dependent inhibition of proliferation of human umbilical vein endothelial cells (HUVECs) and EA.hy926 endothelial cells. Compared with the two agents used singly at their lowest or ineffective doses, combined treatment with the same doses inhibited more intensely in the absence of cytotoxicity and displayed similar behaviour on cell chemotaxis and capillary morphogenesis on Matrigel. However, the secretion of matrix metalloproteinase 2 (MMP‐2) and MMP‐9 was not influenced by the two agents, either alone or in combination, even when they were applied at their lowest efficacious doses or at higher cytotoxic doses. Experiments in vivo with the chick embryo chorioallantoic membrane (CAM)–sponge assay revealed the same dose‐dependent inhibition and synergy. As the basic fibroblast growth factor (bFGF)‐induced angiogenesis in the CAM–sponge model was strongly inhibited by the combined treatment, TNP‐470 and rhIFN‐α2a would appear to exert antiangiogenesis synergistically, perhaps by interfering with the bFGF‐mediated pathway.
Screening for mutations of Bcl10 in leukaemia
Authors: Grimwade David , Du Ming‐Qing , Langabeer Stephen , Rogers Joanne , Solomon Ellen
Published: March 2003

The Bcl10 gene was identified through characterization of the t(1;14)(p22;q32) associated with mucosa‐associated lymphoid tissue (MALT) lymphoma. Bcl10 is implicated in the regulation of apoptosis and has been reported to be mutated in other subtypes of non‐Hodgkin's B‐cell lymphoma (B‐NHL) and leukaemic cell lines, raising the possibility that its deregulation could be implicated in other forms of haematological malignancy. We screened 226 cases, including 123 acute myeloid leukaemia (AML), 50 acute lymphoblastic leukaemia (ALL), 20 chronic myeloid leukaemia (CML), 10 chronic lymphocytic leukaemia–prolymphocytic leukaemia (CLL/PLL) and 23 cases with 1p abnormalities, for Bcl10 mutations by reverse transcription polymerase chain reaction–single‐stranded conformation polymorphism (RT‐PCR/SSCP). Three known polymorphisms and two common splice variants were identified; however, no mutations were detected. One splice variant led to a 33‐bp in frame deletion, whereas the other caused a 16‐bp deletion predicting C‐terminal truncation of Bcl10. However, both splice variants were also detected in normal bone marrow, suggesting that they are unlikely to be of pathogenetic significance. Furthermore, Southern blot analysis revealed no rearrangements of Bcl10 among 16 ALL and 11 cases of haematological malignancy with 1p abnormalities. Our results suggest that mutation of the Bcl10 gene as a mechanism of tumorigenesis is not associated with leukaemia.
Thrombopoietin in Plasmodium falciparum malaria
Authors: Kreil Anna , Wenisch Christoph , Brittenham Gary , Looareesuwan Sornchai , Peck‐Radosavljevic Markus
Published: March 2003

Thrombopoietin (TPO) is the key growth factor for platelet production and is elevated in states of platelet depletion. As thrombocytopenia is a common finding in malaria, we analysed TPO regulation before, during and after antimalarial treatment. Before treatment, TPO serum levels were significantly higher in patients with severe malaria (n = 35) than in patients with uncomplicated malaria (n = 44; P = 0·024), normalizing within 14–21 d of therapy. The rapid normalization of TPO levels and increase in low peripheral platelet counts after treatment indicate that the biosynthesis of TPO and its regulation in malaria patients are normal.
Inhibition of complement‐mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low‐molecular weight heparin
Authors: Ninomiya Haruhiko , Kawashima Yasuko , Nagasawa Toshiro
Published: March 2003

Complement (C′)‐mediated haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is mainly due to the deficiency of glycosyl phosphatidylinositol‐anchored membrane proteins with C′‐regulatory activities CD55 and CD59 in PNH‐affected red blood cells (RBCs). Hydrophobic insertion of C5b‐7 to RBC membranes, initiating the formation of a membrane attack complex, readily results in lysis of PNH RBCs due to the deficiency of CD59. We studied the significance of the electrostatic interactions between C5b‐6 and RBC membranes preceding the insertion of C5b‐7. In vitro, C′‐mediated lysis of PNH RBCs (assessed by sucrose haemolytic assay) was inhibited by heparin, low‐molecular weight heparin (LMWH) or protamine, indicating the significance of the electrostatic interactions between C′ components and RBC membranes in the process of C′‐mediated haemolysis. Neuraminidase‐treated PNH RBCs became resistant to C′ activation, suggesting that the sialic acid moieties on RBC membranes are involved in the interactions of RBC with C′ components. By using biotin‐labelled C7, we demonstrated that LMWH as well as heparin inhibited the insertion of C5b‐7 to RBCs, although they did not inhibit the incorporation of C7 into membrane‐associated C5b‐6. Neither heparin nor LMWH could inhibit the procoagulant alteration of PNH RBC membranes induced by C′ activation even at concentrations which inhibited the haemolysis completely. Because LMWH inhibited the C′‐mediated lysis of PNH RBCs in vitro at the range which induced a limited prolongation of activated partial thromboplastin time of normal plasma, we consider that LMWH may be useful for both the inhibition of haemolysis and the prevention of thrombosis, which often follow a haemolytic attack in PNH.
The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization
Authors: Westwood N. B. , Gruszka‐Westwood A. M. , Pearson C. E. , DeLord C. F. M. , Green A. R. , Huntly B. J. P. , Lakhani A. , McMullin M. F. , Pearson T. C.
Published: March 2003

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.
Multiple myeloma with deletion of chromosome 13q is characterized by increased bone marrow neovascularization
Authors: Schreiber Susanne , Ackermann Jutta , Obermair Andreas , Kaufmann Hannes , Urbauer Eleonora , Aletaha Katharina , Gisslinger Heinz , Chott Andreas , Huber Heinz , Drach Johannes
Published: March 2003

Anti‐angiogenesis therapy with thalidomide has been reported to have marked activity in multiple myeloma (MM). As cytogenetics is an independent prognostic factor in MM, we analysed bone marrow (BM) angiogenesis and cytogenetic abnormalities in 34 patients with active MM. BM microvessel density (MVD), as determined by staining with anti‐CD34, was significantly higher in MM (MVD: 221 ± 94 per mm2) than in controls (80 ± 36; P < 0·0001). In patients with the presence of at least one unfavourable cytogenetic abnormality (deletion of 13q14, deletion of 17p13, aberrations of 11q), a significantly increased BM MVD was observed (254 ± 93 vs. 160 ± 60 in patients with absence of these abnormalities; P = 0·0035). Further analyses indicated that increased BM MVD was significantly correlated with deletion of 13q14 (259 ± 96 vs. 188 ± 80; P = 0·026), but not with other cytogenetic, clinical and laboratory MM parameters. We conclude that BM neovascularization is particularly high in MM with deletion of 13q14, which provides a rationale for use of anti‐angiogenic strategies in the treatment of MM with high‐risk cytogenetics.
A new PKLR gene mutation in the R‐type promoter region affects the gene transcription causing pyruvate kinase deficiency
Authors: Manco Licínio , Ribeiro M. Letícia , Máximo Valdemar , Almeida Helena , Costa Alice , Freitas Orquídea , Barbot José , Abade Augusto , Tamagnini Gabriel
Published: March 2003

Mutations in the PKLR gene responsible for pyruvate kinase (PK)‐deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A→G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK‐deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA‐A element) in the R‐type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R‐PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the −72A→G point mutation disables the binding of the erythroid transcription factor GATA‐1 to the GATA‐A element. Supporting these data, the two patients homozygous for the −72A→G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C→T had a mild condition.
Erythrocyte membranes from a patient with congenital dyserythropoietic anaemia type I (CDA‐I) show identical, although less pronounced, glycoconjugate abnormalities to those from patients with CDA‐II (HEMPAS)
Authors: Zdebska Ewa , Woźniewicz Bohdan , Adamowicz‐Salach Anna , Kościelak Jerzy
Published: March 2003

Congenital dyserythropoietic anaemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and multinuclearity of erythroblasts. Three main types of the disease have been described. Glycoconjugate abnormalities in erythrocyte membrane glycoconjugates, consisting of hypoglycosylation of band 3 and accumulation of certain glycosphingolipids including lactotriaosylceramide, neolactotriaosylceramide and polyglycosylceramides, have been described only in patients with CDA type II (CDA‐II). We report on identical, although less pronounced, abnormalities in erythrocyte glycoconjugates from a patient with CDA‐I. A low degree of hypoglycosylation of band 3 in our patient with CDA‐I suggests that hypoglycosylation is not a cause, but, most probably, a consequence of dyserythropoiesis.
Th1 and Th2 cytokines in a patient with Evans' syndrome and profound lymphopenia
Authors: Karakantza Marina , Mouzaki Athanasia , Theodoropoulou Maria , Bussel James B. , Maniatis Alice
Published: March 2003

A case of Evans' syndrome with IgM deficiency and lymphopenia was studied before and after splenectomy. The lymphopenia was as a result of profound reduction of CD4 and CD8 cells. Study of cytokine secretion before splenectomy revealed a spontaneous Th1‐ and Th2‐type cytokine production, and complete suppression of transforming growth factor (TGF)‐β. After splenectomy, the patient achieved clinical remission, the natural killer (NK) cell number increased and the pattern of cytokine production showed normalization of interleukin (IL)‐2, IL‐4, IL‐10, TGF‐β and abolition of interferon (IFN)‐γ production. We conclude that splenectomy had a beneficial effect owing to an increase in NK cells and an associated increase in TGF‐β production.
Diagnostic utility of fluorescence in situ hybridization in mantle‐cell lymphoma
Authors: Remstein E. D. , Kurtin P. J. , Buño I. , Bailey R. J. , Proffitt J. , Wyatt W. A. , Hanson C. A. , Dewald G. W.
Published: March 2003

Mantle‐cell lymphoma (MCL) has a poorer prognosis than other small B‐cell lymphomas, thus a definitive diagnosis is essential. The t(11;14)(q13;q32) associated with MCL juxtaposes portions of CCND1 (11q13) and IGH (14q32), resulting in over‐expression of cyclin D1. In this study, a highly sensitive two‐colour fluorescence in situ hybridization (FISH) method was developed to detect t(11;14)(q13;q32) in nuclei isolated from paraffin‐embedded tissue. Twenty‐three MCLs, 13 normal controls and nine small B‐cell lymphomas other than MCL were studied by FISH. Each MCL had been previously investigated to detect genomic IGH–CCND1 fusion by polymerase chain reaction (PCR) using DNA extracted from frozen tissue. The IGH–CCND1 fusion detection rate in the MCLs was 96% by FISH compared with 35% by PCR. By FISH, one MCL and three small B‐cell lymphomas other than MCL harboured abnormalities involving only IGH. Less than 1% of cells showed false‐positive IGH–CCND1 fusion in normal specimens by FISH. Thus, this highly sensitive FISH assay is very useful in confirming the diagnosis of MCL, has wide applicability as it may be performed on both paraffin‐embedded and fresh tissue, and may also facilitate detection of translocations involving these loci in tumours other than MCL.
MDM2 gene amplification and lack of p53 point mutations in Hodgkin and Reed–Sternberg cells: results from single‐cell polymerase chain reaction and molecular cytogenetic studies
Authors: Küpper Manfred , Joos Stefan , Von Bonin Frederike , Daus Heiner , Pfreundschuh Michael , Lichter Peter , Trümper Lorenz
Published: March 2003

Hodgkin's disease (HD) is the most common haematological malignancy after chronic lymphocytic leukaemia, but very little is known about its pathogenesis or the genetic events that contribute to the malignant phenotype of the tumour cells. p53 is assumed to play an important role in the pathogenesis of HD, based on the observation that p53 protein is frequently accumulated in Hodgkin and Reed–Sternberg (H & RS) cells. We investigated single H & RS cells from five different HD patients for point mutations at the genomic level using multiplex polymerase chain reaction amplification and subsequent sequencing. No point mutations were detected in 50 single H & RS cells analysed. Hence, accumulation of p53 protein cannot be explained by mutations within the gene. A genome‐wide screening for genomic imbalances using comparative genomic hybridization revealed gain on chromosome 12q14, i.e. the mapping position of the MDM2 gene in several HD cases. Therefore, we assessed the copy number of the MDM2 gene using fluorescence in situ hybridization. In four out of six HD cases analysed, the copy number of the MDM2 gene was found to be increased. As gene amplification is frequently associated with protein overexpression, the observed accumulation of p53 in the nuclei of H & RS cells could be as a result of elevated MDM2 protein levels resulting in stabilization of p53 protein.
Apoptosis in refractory anaemia with ringed sideroblasts is initiated at the stem cell level and associated with increased activation of caspases
Authors: Hellström Lindberg Eva , Schmidt‐Mende Jan , Forsblom Ann Mari , Christensson Birger , Fadeel Bengt , Zhivotovsky Boris
Published: March 2003

Treatment with granulocyte colony‐stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas‐agonist, Fas‐blocking antibody 2 and caspase‐3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0·05) and caspase activity (P < 0·05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony‐forming units (CFU‐E) (< 10% of control) and granulocyte–macrophage CFU (CFU‐GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0·01). Fas‐blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase‐mediated dUTP‐biotin nick‐end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU‐E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.
The apoptogenic response of human myeloid leukaemia cell lines and of normal and malignant haematopoietic progenitor cells to the proteasome inhibitor PSI
Authors: Soligo Davide , Servida Federica , Delia Domenico , Fontanella Enrico , Lamorte Giuseppe , Caneva Lorenza , Fumiatti Rossella , Lambertenghi Deliliers Giorgio
Published: March 2003

Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin‐dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z‐Ile‐Glu(OtBu)‐Ala‐Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre‐treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34+ bone marrow progenitors because the 50% growth inhibition of colony‐forming unit granulocyte macrophage (CFU‐GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP‐ribose) polymerase (PARP) and of β‐catenin, and was antagonized by ectopic expression of Bcl‐2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl‐2, a decrease of Bax but no changes in Bcl‐XL protein expression at any time point. In Ph+ cell lines BCR‐ABL protein was only down‐regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti‐tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.
Combination therapy with tacrolimus and anti‐thymocyte globulin for the treatment of steroid‐resistant acute graft‐versus‐host disease developing during cyclosporine prophylaxis
Authors: Durrant S. , Mollee P. , Morton A. J. , Irving I.
Published: March 2003

We report our experience with the combination of anti‐thymocyte globulin (ATGAM) and tacrolimus in the treatment of 20 patients with steroid refractory and dependent acute graft‐versus‐host disease (GVHD) transplanted between August 1996 and February 2000. All patients received cyclosporine‐based GVHD prophylaxis. Thirteen patients developed a maximum of grade IV, five grade III and two grade II acute GVHD, with 15 patients being refractory to steroids and five dependent on steroids. Patients were treated with ATGAM (15 mg/kg for 5 d) and tacrolimus (0·025–0·1 mg/kg/d) in addition to continuation of their high‐dose steroids and cessation of their cyclosporine. Within 28 d of treatment, we observed eight complete responses (CR), six partial responses (PR) and six with no response. Overall response (CR + PR) was predicted by GVHD severity. Infectious complications occurred in 80% of patients. The median survival was 86·5 d (range, 21–1081 d) with 35% of patients remaining alive. Survival following combination therapy was significantly more likely in men (P < 0·001), skin‐only GVHD (P = 0·027), less severe GVHD (P = 0·048), and in responders to tacrolimus and ATGAM (P < 0·001). In conclusion, concurrent introduction of ATGAM and tacrolimus is a promising therapeutic combination for GVHD refractory to steroids and cyclosporine.
Complexes of anti‐prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces
Authors: Simmelink Marleen J. A. , Horbach Daniëlle A. , Derksen Ronald H. W. M. , Meijers Joost C. M. , Bevers Edouard M. , Willems George M. , De Groot Philip G.
Published: March 2003

We investigated the mechanism by which anti‐prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity‐purified anti‐prothrombin antibodies from LAC‐positive plasma samples (α‐FII‐LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified α‐FII‐LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when α‐FII‐LAC+ were added to prothrombin‐deficient plasma or when purified anti‐prothrombin antibodies from LAC‐negative plasma samples (α‐FII‐LAC−) were added. When a combination of prothrombin and α‐FII‐LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The α‐FII‐LAC+ alone or a combination of prothrombin and α‐FII‐LAC− did not show inhibition. Ellipsometry studies showed that, in the presence of α‐FII‐LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with α‐FII‐LAC−. Our results show that complexes of prothrombin and anti‐prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti‐β2‐glycoprotein I antibodies.
Red cell antibodies in patients with homozygous sickle cell disease: a comparison of patients in Jamaica and the United Kingdom
Authors: Olujohungbe Ade , Hambleton Ian , Stephens Lorna , Serjeant Beryl , Serjeant Graham
Published: March 2003

The transfusion history and frequency of red cell antibodies in patients with homozygous sickle cell (SS) disease have been compared in 190 subjects from the Jamaican cohort study and 37 patients attending a sickle cell clinic in Manchester, England. The proportion of patients transfused did not differ between the groups although the number of units transfused and the frequency of red cell antibodies were significantly greater in the Manchester group. Immune antibodies occurred in three Jamaicans (2·6% of those transfused) and 16 UK subjects (76% of those transfused). Multiple antibodies occurred in 10 (63%) UK subjects but in no Jamaicans. Indications for transfusion also differed between the groups, Jamaican patients typically receiving 1–2 units for acute anaemia or acute chest syndrome, whereas UK patients frequently had multiple transfusions in preoperative exchange or prophylaxis programmes. The greater red cell alloimmunization among UK patients probably reflects both the greater use of transfusion and the disparity between donor and recipient populations in the UK.
Estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic stem cell transplantation by the amount of BCR‐ABL fusion transcripts detected using a new real‐time polymerase chain reaction method
Authors: Elmaagacli Ahmet H. , Freist Annette , Hahn Meinhard , Opalka Bertram , Seeber Siegfried , Schaefer Ulrich W. , Beelen D. W.
Published: March 2003

We have used a new single‐step real‐time reverse transcription polymerase chain reaction (RT‐PCR) method to quantify BCR‐ABL transcripts, thereby estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic transplants. In 402 samples from 172 patients, BCR‐ABL expression was determined and normalized, using the GAPDH housekeeping gene product as an endogenous reference. In our real‐time RT‐PCR assay, serial dilutions of RNA of the K562 cell line remained positive down to 7·5 pg. The median normalized BCR‐ABL amount differed significantly (P < 0·001) between the various disease stages and was 0·06% (range 0·001–1·55%), 3·2% (range 1·4–5·6%) and 21·5% (range 6·8 −827%) in 17 patients with a molecular relapse, in eight patients with a cytogenetic relapse and in 10 patients with a haematological relapse respectively.
Anti‐tissue factor pathway inhibitor activity in patients with primary antiphospholipid syndrome
Authors: Adams Murray J. , Donohoe Siobhán , Mackie Ian J. , Machin Samuel J.
Published: March 2003

The association between antiphospholipid antibodies and an increased risk of thrombosis in antiphospholipid syndrome (aPS) patients is probably caused by numerous mechanisms, including the effects of antibodies to phospholipid‐binding proteins such as β2‐glycoprotein I and prothrombin. In this study, we investigated the inhibition of tissue factor pathway inhibitor (TFPI) in 33 patients with primary antiphospholipid syndrome (PAPS). TFPI was measured in PAPS patients using an amidolytic assay, dependent on the generation of activated factor X (Fxa), and this was compared with 55 healthy subjects. Functional levels of TFPI (mean ± SD) were significantly lower in PAPS patients (0·89 ± 0·37 U/ml) than the control group (1·05 ± 0·15 U/ml) (P = 0·02). The difference was caused by a subset of five patients who had TFPI levels below the lower 99% confidence interval of the normal reference range, representing increased FXa generation in the assay system. IgG fractions were isolated from these five patients and five control subjects, then incorporated into normal plasma to measure FXa generation in the TFPI assay system. FXa generation was increased when polyclonal rabbit anti‐human TFPI IgG (P < 0·0001) or PAPS IgG (P = 0·0001) were added to normal plasma, demonstrating inhibition of TFPI. The apparent anti‐TFPI activity demonstrated in the five subjects with PAPS in this study may represent a significant new mechanism for thrombosis in patients with aPS, as it implies that increased tissue factor FVIIa‐mediated thrombin generation might occur.