bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐term follow‐up of severe aplastic anaemia patients treated with antithymocyte globulin
Authors: de Planque Mariet M. , Bacigalupo A. , Würsch A. , Hows Jill M. , Devergie Agnes , Frickhofen N. , Brand Anneke , Nissen Catherine ,   for the Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation (EBMT)
Published: March 2008

Summary. 468 severe aplastic anaemia (SAA) patients registered in the EBMT‐SAA registry who did not undergo bone marrow transplantation and were treated with immunosuppressive therapy (IS; 96% of patients received ATG) were evaluated. Their median age was 23 years (range 1–73) at initial IS therapy, 59% were males; in 69% the aetiology of SAA was idiopathic. Of these 468 patients, 245 had a follow‐up of <2 years after IS 166/245 died, 71/245 are still alive, 8/245 are lost to follow‐up. Of 223 patients who survived ≥2 years (LTS long‐term survivors), 191 are alive, 21 died >2 years and 11 are lost. Median follow‐up of 223 LTS was 4.1 years (range 2.0–10.9). Comparison of 166 patients who died <2 years and 223 LTS revealed no difference at time of initial IS therapy as regards sex, duration of AA, or its aetiology, but the age distribution and, in particular, severity of SAA differed significantly: more LTS were between 21 and 40 years old (44%v. 32%, P<0.02), less LTS had reticulocytes <20 × 109/l (63%v. 80%, P<0.001), polymorphonuclear granulocytes (PMN) <0.2 × 109/l (30%v. 57%. P<0.001), haemorrhages (58%v. 79%, P<0.002) and infection (30%v. 49%, P<0.005) at time of IS. A gradual improvement of blood counts was seen in patients alive ≥ 2 years after IS. At 2 years after IS 80% had a normal haemoglobin and PMN >0.5 × 109/l, but only after 5 years 80% of cases had platelets > 50 × 109/l. Development of clonal disease was reported of 31 LTS: 19 developed paroxysmal nocturnal haemoglobinuria (PNH), one acute leukaemia, 11 myelodysplastic syndromes and of these 11 five subsequently acute leukaemia. The majority of these patients (23/31) are still alive. Actuarial mortality of LTS is 22% at 8 years, but so far no plateau was achieved. It is concluded that SAA patients who become LTS following IS, show an improvement in haematological status but are probably not cured and are prone to develop clonal (malignant) disease.
Plasma from patients with severe Lassa fever profoundly modulates f‐met‐leu‐phe induced superoxide generation in neutrophils
Authors: Roberts Pamela J. , Cummins David , Bainton Adrian L. , Walshe Karen J. , Fisher‐Hoch Susan P. , McCormick Joseph B. , Gribben John G. , Machin Samuel J. , Linch David C.
Published: March 2008

A recurrent theme in studies of the pathology of fatal Lassa fever in man is the lack of histological lesions to explain disordered cell function and death. Recently, we demonstrated the existence of a factor in the plasma of patients with Lassa fever which markedly inhibits the aggregation responses of normal platelets in vitro. To assess whether this factor could mediate more global cellular dysfunction, we studied the effects of Lassa plasma on the respiratory burst of neutrophils.

Thirteen of 15 samples from patients in the acute phase of Lassa fever profoundly inhibited the amount of superoxide generated by normal neutrophils in response to the chemotactic peptide, f‐met‐leu‐phe (FMLP) (mean superoxide generated = 54.7 ± 6.1% of control). In contrast, eight of nine samples from patients who had infections other than Lassa fever enhanced the neutrophil response to the peptide. All Lassa samples which inhibited the ADP‐induced aggregation responses of normal platelets inhibited the neutrophil response to FMLP. Unlike the effect on platelets, however, the inhibition of neutrophils was only apparent when the cells were stimulated within 5 min of exposure to the plasma. The inhibition of neutrophils is not due to either interference with FMLP‐neutrophil binding or an effect on the NADPH‐oxidase, suggesting a suppression of signal transduction. Our data suggest the inhibitory factor in Lassa plasma has global effects on cellular function, and may play a central role in the pathogenesis of this often fatal illness.
Immunochemical analysis of platelet autoantibodies in HIV‐related thrombocytopenic purpura: a study of 68 patients
Authors: Bettaieb Ali , Oksenhendler Eric , Fromont Patricia , Duedari Najib , Bierling Philippe
Published: March 2008

Serum antiplatelet IgG and platelet‐associated IgG (PAIgG) were studied in 68 AIDS‐free human immunodeficiency virus (HIV)‐infected patients with severe immunologic thrombocytopenic purpura (ITP), for the presence of platelet autoantibodies. Serum IgG with antiplatelet activity was found in 72% of the sera. However, the presence of autoantibodies against platelet surface glycoproteins was not found in these sera by means of Western blot and immunoprecipitation procedures. Nevertheless, an immunoblot immuno‐assay and an indirect immunofluorescence test against semi‐permeabilized platelets demonstrated the presence of antibodies in the patient sera, that reacted with intracyto‐plasmic platelet components, and which might participate in the elimination of platelet fragments. Direct immunofluorescence tests demonstrated an increased amount of PAIgG in 75% of the patients; the bound antibodies could be eluted with ether in 44% of the cases. These eluates were found to bind to normal platelets but not to Glanzmann type I platelets. Finally, immunoprecipitation procedures demonstrated the presence of platelet autoantibodies in six of the 35 eluates studied. These antibodies recognized GPIIb in two cases. GPIIIa in one case, and an unidentified platelet protein of 150 kDa in the three other cases. The discrepancy between sera and platelet eluates was interpreted as being due to the low titre of the antibodies and to their dilution in polyclonal hypergammaglobulinaemia.

The present study provides direct evidence that isolated ITP in some HIV‐positive patients is due to the presence of platelet autoantibodies. These results, however, do not exclude either direct or indirect involvement of HIV in the platelet destruction.
Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics
Authors: McNeil H. Patrick , Chesterman Colin N. , Krilis Steven A.
Published: March 2008

Autoantibodies to phospholipid antigens can be characterized using solid phase immunoassays to detect anticardiolipin antibodies (ACA) or in phospholipid‐dependent clotting tests where lupus anticoagulant (LA) activity can be demonstrated. It has not been established whether each activity is due to the same or separate antibody subgroups. Plasma from two patients with high levels of both activities were used for purification of ACA and LA using sequential ion‐exchange, gel‐filtration, and anti‐Ig affinity chromatography. Plasma could be separated into fractions containing each activity in the absence of the other. In these fractions, antibodies responsible for LA activity do not bind to isolated phospholipids in solid phase immunoassays, and conversely antibodies binding in these assays (ACA) do not possess LA activity, suggesting LA are directed against a more complex lipid epitope. In addition, in one patient ACA was of IgG isotype only, whilst LA was due to IgG and IgM isotypes. In this patient, the IgG‐ACA was heterogenous with three peaks clearly separated on ion‐exchange chromatography. Affinity purified antiphospholipid antibodies have been previously prepared from a number of patients using a phospha‐tidyl‐serine column and antibodies purified in this manner possess ACA but not LA activity. Taken together, these data indicate that tests for ACA and LA define separable subgroups of phospholipid binding antibodies, thus explaining the discordance often seen between the two activities.
The rapid and decremental change in haem oxygenase mRNA during erythroid differentiation of murine erythroleukaemia cells
Authors: Fujita Hiroyoshi , Sassa Shigeru
Published: March 2008

Changes in mRNA for haem oxygenase (HO), the rate‐limiting enzyme for haem catabolism, were examined in murine Friend‐virus transformed erythroleukaemia (MEL) cells while they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulfoxide (DMSO). When MEL cells were treated with 1.5% (v/v) DMSO, a rapid decrease in HO mRNA content was observed (< 12 h) which reached the lowest value at 18 h (18% of the untreated control). HO mRNA levels remained at substantially lower levels (< 50%) than those in untreated controls thereafter. A rapid decline in HO mRNA may be involved in the cellular events that determine the onset of erythroid differentiation.
The beneficial effects of αIFN in CGL are probably not mediated by NK cells
Authors: Galvani D. W. , Owens W. , Nethersell A. B. W. , Cawley J. C.
Published: March 2008

Summary Natural killer (NK) cells in CGL were measured phenotypically (by Leu7 and CD16 Mab staining) and functionally (by a standard chromium‐release cytotoxicity assay) in eight patients before and during α‐IFN therapy.

Before αIFN therapy, phenotypic NK cells were normal in relative and absolute numbers but were consistently defective functionally; this defect was partially corrected by in vitro exposure to αIFN.

During αIFN therapy, there was no change in NK function in five patients and enhanced (two patients) or reduced (one patient) activity was observed in the other three. Cold‐target inhibition experiments showed no evidence of NK binding to normal or CGL myeloid progenitors.

It is concluded that αIFN‐enhanced NK function, a known anti‐tumour mechanism in animal models, is probably not the basis of the responsiveness of CGL to αIFN therapy.
Vitronectin (S protein) is associated with platelets
Authors: Parker C. J. , Stone O. L. , White V. F. , Bernshaw N. J.
Published: March 2008

Summary Vitronectin is a plasma glycoprotein that has regulatory activity in the complement and the coagulation systems, in cell‐cell and cell‐substrate interactions, and in monocyte/macrophage function. Because of its potential to participate in several of the processes of inflammation and repair, the association of vitronectin with platelets was investigated. Immunochemical studies demonstrated that the majority of the platelet associated vitronectin was intracellular, while a relatively modest amount was localized to the ectoplasmic portion of the plasma membrane. Analysis by Western blot showed that the electrophoretic mobility of platelet associated vitronectin was indistinguishable from that of vitronectin isolated from plasma. In response to thrombin, approximately 1 μg of vitronectin was released into the supernate of 109 platelets, while somewhat less than one‐half of the total platelet vitronectin remained cell associated. The binding of vitronectin to platelets was investigated by comparing the capacity of unlabelled vitronectin and fibronectin to inhibit binding of radiolabelled fibronectin to thrombin stimulated platelets. On a weight basis, inhibition by the two proteins was equivalent, suggesting that vitronectin competes with fibronectin for binding to platelet glycoprotein IIb/IIIa. These results demonstrate that vitronectin is a platelet specific protein which, because of its multifunctional properties, may participate in physiological and pathophysiological events associated with thrombosis and haemostasis.
Platelet aggregation induced by type IIb platelet von Willebrand factor
Authors: Gralnick Harvey R. , McKeown Laurie P. , Williams Sybil , Jenneau Christine , Sultan Yvette , Mourik Jan van
Published: March 2008

Summary Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet‐rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard‐Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate‐induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.
Immune thrombocytopenia more than a year after allogeneic marrow transplantation due to antibodies against donor platelets with anti‐PIA1 specificity: evidence for a host‐derived immune reaction
Authors: Panzer Simon , Kiefel Volker , Bartram Claus R. , Haas Oskar A. , Hinterberger Wolfgang , Mueller‐Eckhardt Christian , Lechner Klaus
Published: March 2008

Summary We report on a male patient transplanted from his HLA‐matched sister for Ph1‐chromosome positive chronic myelogenous leukaemia who developed immune thrombocytopenia more than 1 year after transplantation. The platelet antibody reacted with the platelet specific antigen PIA1 on donor platelets, and also on recipient platelets after engraftment. A presumed host‐versus‐donor induced thrombocytopenia was supported by Southern blot analysis using a Y‐chromosome specific probe demonstrating residual hostorigin cells in the patient's excised spleen.
The inhibitory effect of serum from hairy‐cell leukaemia patients on normal progenitor cells may disappear following prolonged treatment with α‐interferon
Authors: Lauria Francesco , Bagnara Gian Paolo , Catani Lucia , Gaggioli Licia , Guarini Anna , Raspadori Donatella , Foa Robert , Bellone Graziella , Buzzi Marina , Gugliotta Luigi , Zauli Giorgio , Giovannini Marco , Luigi zinzani Pier , Tura Sante
Published: March 2008

Summary The possibility that serum from hairy‐cell leukaemia (HCL) patients at diagnosis may show an inhibitory effect on the in vitro colony growth of normal haemopoietic progenitor cells has been suggested. Several studies have documented the efficacy of α‐Interferon (α‐IFN) in inducing a complete restoration of peripheral blood values and, in some cases, a complete clinical remission. In this study we have evaluated the regulatory effect of serum, collected before and after 3 and 12 months of a‐IFN tretment, from 10 patients with untreated HCL, on the in vitro growth of normal bone marrow CFU‐GM, BFU‐E and CFU‐MK. The effect of conditioned media, prepared from enriched hairy cells (HC) cultured in synthetic medium, on the growth of normal haemopoietic progenitors was also investigated. The results obtained confirm that sera from untreated HCL patients display a variable degree of inhibitory activity in the progenitor cell compartments analysed. Disappearance of the inhibitory activity, particularly evident for the erythroid compartment, was found only in patients who displayed a disappearance of circulating HC and a good haematological response after prolonged (12 months) treatment with a‐IFN. The possibility that the serum of patients with HCL may contain a haemopoietic inhibitory factor, released by the neoplastic HC population, is suggested.
The possible mode of escape of adult T‐cell leukaemia cells from antibody‐dependent cellular cytotoxicity
Authors: Kozuru Mitsuo , Uike Naokuni , Takeichi Naohisa , Nakagawa Setsuko , Takahira Hiroyuki , Shibata Keisuke , Sato Hiroyuki
Published: March 2008

Summary Antibody‐dependent cellular cytotoxicity (ADCC) was measured using 51Cr‐labelled ATL derived cell lines as the target, peripheral mononuclear cells (PMNCs) from disease‐free persons as the effector cells and heat‐inactivated serum from patients with ATL or the IgG purified from this. The release of 51Cr was not usually demonstrated in the HTLV‐1 non‐producing ATL derived cell line (MT‐1), but was evident in the HTLV‐1 producing ATL derived cell lines (KT 252 and IT 607). The 51Cr‐labelled MT‐1 cells after induction of HTLV‐1 retrovirus by 5‐iodo‐2′‐deoxyuridine (IdUr), showed a remarkable target sensitivity in the ADCC assay. On the other hand, the 51Cr‐labelled MT‐1 cells after culture with IdUr and ATL patient's serum, had no ADCC sensitivity, The fresh ATL cells immediately separated from ATL patient's blood did not express HTLV‐1 virus in the cell and had no ADCC sensitivity as the target. Based on these findings, an antigenic modulation on the ATL cell surface by ATL patient's serum is considered to be the possible mode of escape of ATL cells from ADCC.
Liquid culture and cryopreservation of marrow cells of leukaemic patients prior to autologous bone marrow transplantation
Authors: Stoppa A. M. , Maraninchi D. , Lafage M. , Mannoni P. , Novakovitch G. , Blaise D. , Gaspard M. H. , Michel G. , Carcassonne Y.
Published: March 2008

Summary The feasibility of marrow cryopreservation for autologous bone marrow transplantation after 7 d in liquid culture was assessed in 10 leukaemic patients. A median of 0·17 × 108 nucleated cells/kg and 0·4 × 104 CFU‐GM/kg could be collected after the complete procedure, with overall a consistent cell loss. Long‐term cultures could be established from these cultured and frozen marrows, showing the persistance of precursors of haematopoietic and stromal cells. In vitro a significant decrease in the proportion of leukaemic cells could be observed in only one out of nine evaluable patients. This patient, with refractory AML, received an autologous transplant and is alive in continuous complete remission after 600 d. One patient with chronic myeloid leukaemia in acute phase underwent an autologous BMT with a marrow collected and cultured while in chronic phase and failed to engraft. These results show the feasibility of cryopreservation of cultured marrow cells for autologous bone marrow transplantation. The procedure is associated with poor cell recovery and must be improved to have a more general clinical application. This technology may have a major application with the emergence of modulators of growth and differentiation of haematopoietic cell lines.
Erythropoiesis following bone marrow transplantation from donors heterozygous for β‐thalassaemia
Authors: Galanello Renzo , Barella Susanna , Maccioni Liliana , Paglietti Elisabetta , Melis Maria A. , Rosatelli Maria C. , Argiolu Francesca , Cao Antonio
Published: March 2008

Summary This study shows a marked and protracted activation of HbF synthesis in homozygous β°‐thalassaemia patients transplanted from HLA identical siblings heterozygous for β‐thalassaemia, as compared to patients transplanted from normal donors. HbF synthesis in recipients was much higher in relation to the corresponding bone marrow donor values either normal or heterozygous for β thalassaemia.

γ‐chain synthesis and Gγ/Aγ ratio were also studied in peripheral blood BFU‐E from recipients and their donors. BFU‐E from donors heterozygous for β‐thalassaemia showed higher γ chain synthesis as compared to normal donors. Peripheral blood BFU‐E γ/β+γ ratios and Gγ percentage were higher in recipients than in their corresponding donors both normal or heterozygotes.

The marked and protracted reactivation of HbF synthesis in recipients of heterozygous β‐thalassaemia bone marrow most likely results from an increased erythropoietic stress on erythroid progenitors. In order to obtain adequate Hb levels heterozygous β‐thalassaemia bone marrow should produce more red blood cells to compensate for the low MCH. The magnitude of activation of HbF synthesis was very variable. This variability may result from inherited differences in the capacity of reactivation of HbF synthesis of red cell progenitors from heterozygous β‐thalassaemia under stressed erythropoiesis.
Circulating activated suppressor T lymphocytes in hepatitis‐associated aplastic anaemia
Authors: Kojima Seiji , Matsuyama Kohji , Kodera Yoshihisa , Okada Junichi
Published: March 2008

Lymphocyte subpopulations were measured in the blood of 21 children with severe aplastic anaemia (SAA). Five children with hepatitis‐associated AA appeared to constitute a unique group. Compared with children with idiopathic AA, four of five children had a striking increase in percentage of activated suppressor T lymphocytes, which were defined by binding of both anti‐Leu 2a and anti‐HLA DR monoclonal antibodies (children with hepatitis‐associated AA, 21·8±19·9% [mean ±SD], children with idiopathic AA, 2·9±1·2%). However, there was no difference in the mean absolute number of the activated suppressor T lymphocytes between the two groups, because the mean number of lymphocytes was markedly less in children with hepatitis‐associated AA (0·28±0·06 × 109/1) than in those with idiopathic AA (1·64±0·74 × 109/1). The mean helper: suppressor T lymphocyte ratio was significantly lower in children with hepatitis‐associated AA (0·58±0·74) than in those with idiopathic AA (1·22±0·44). On the other hand, we did not find such a remarkable alteration of lymphocyte subpopulations in children with non‐A non‐B hepatitis who did not develop AA. These findings suggested that an imbalance of lymphocyte subpopulations and T lymphocyte activation may have pathogenetic relevance in some of the children with hepatitis‐associated AA.
Use of the day 6 bone marrow to alter remission induction therapy in patients with acute myeloid leukaemia: a leukemia intergroup study
Authors: Browman G. , Preisler H. , Raza A. , Syracuse K. , Azarnia N. , Benger A. , Chervenick P. , D'Arrigo P. , Doeblin T. , Goldberg J. , Gottlieb A. , Grunwald H. , Kirshner J. , Larson R. , Meyer R. , Miller K. , Priore R. , Stein M. , Vogler W. R. , Walker I. , Wilson W. E. C. , Barcos M.
Published: March 2008

Summary. Patients with acute myeloid leukaemia who fail to show substantial bone marrow cytoreduction by day 6 of induction therapy enter complete remission (CR) less frequently than patients with good bone marrow leukaemic cytoreduction. The objective of the current study was to determine whether an increase in the intensity of therapy on days 8, 9 and 10 (‘augmentation’ of remission induction therapy) for patients with poor bone marrow cytoreduction detected in the day 6 bone marrow could improve the complete remission rate without increasing the number of toxic deaths. Patients from six centres were entered and treated with standard dose ara‐C for 7 or 10 d and an anthracycline for the first 3 d. Patients aged less than 60 years and with > 30% bone marrow biopsy cellularity or > 10% abnormal cells on the aspirate obtained 6 d after the start of therapy were augmented with cytosine arabinoside 3 g/m2 every 12 h on days 8, 9 and 10. Therapy was augmented in 116 of the 252 patients <60 years. There was a highly statistically significant difference between augmented and nonaugmented patients (P<0.001) for the per cent biopsy cellularity and per cent abnormal cells in the day 6 marrow. The CR rate for augmented patients was 69% and for nonaugmented patients 60% suggesting that augmentation therapy abrogated the prognostic significance of more extensive residual leukaemia in the day 6 bone marrow. The results suggest that augmentation of remission induction for patients with poor bone marrow cytoreduction detected 6 d after initiation of therapy, may salvage patients who are destined to fail remission induction because of resistant disease without producing excessive toxicity.
Myelofibrosis in primary myelodysplastic syndromes: a clinico‐morphological study of 10 cases
Authors: Pagliuca A. , Layton D. M. , Manoharan A. , Gordon S. , Green P. J. , Mufti G. J.
Published: March 2008

Summary. We describe 10 cases of primary myelodysplastic syndrome in which marrow fibrosis was striking at presentation. All the cases showed trilineage dysplasia with increased megakaryopoiesis and marked reticulin fibrosis. Significant organomegaly was notably absent. This association has hitherto not been highlighted and it is important to distinguish these cases from those of idiopathic myelofibrosis with which they may be confused. Furthermore, their comparatively long survival distinguishes these cases from those previously described as acute myelodysplasia with myelofibrosis and malignant myelosclerosis. The pathogenesis of fibrosis in these cases may be related to disordered megakaryopoiesis and the platelet‐derived cytokines that may be released. The treatment of these fibrotic cases remains problematical and further investigation is required.
Long‐term study of chimaerism in bone marrow transplantation recipients for severe aplastic anaemia
Authors: Keable Hélène , Bourhis Jean‐Henri , Brison Olivier , Lehn Pierre , Schenmetzler Claudine , Devergie Agnès , Gluckman Eliane
Published: March 2008

Summary. We used minisatellite probes to analyse by DNA fingerprints the long‐term engraftment (median 4.3 years, range 1–2) of 21 bone marrow transplantation recipients for severe aplastic anaemia. Patients received their graft from histocompatible siblings. They were conditioned with cyclophosphamide (150 mg/kg) and a 6GY thoracoabdominal irradiation and did not have ex‐vivo T cell depletion of marrow donor. DNA was extracted from peripheral mononuclear cells and analysed by Southern blotting with 32Plabelled single‐stranded RNA probes. Seven out of 21 donor‐recipient pairs were sex‐mismatched and additionally studied with a probe detecting a male specific repeated sequence on the Y chromosome. Red cell surface phenotype was also used as marker of engraftment in most cases. Long‐term engraft‐ment appeared complete for all patients studied with respect to the three methods.
N‐RAS mutations in T‐cell acute lymphocytic leukaemia: analysis by direct sequencing detects a novel mutation
Authors: Bar‐eli M. , Ahuja H. , And A. Foti , Cline M. J.
Published: March 2008

Summary. A novel mutation of the N‐RAS gene of T‐ALL blast cells was detected by a direct sequencing of in vitro amplified exon‐1 of the N‐RAS gene. Threonine (ACA) was substituted for alanine (GCA) at codon 11. This mutation would have been overlooked by conventional probe hybridization techniques.

A search for other mutations in N‐RAS exon‐1 in T‐ALL revealed a codon 13 mutation substituting aspartic acid (GAT) for glycine (GGT) in one of 18 patients. No mutations at codon 12 were detected.
Fetal and neonatal von Willebrand factor (vWF) is unusually large and similar to the vWF in patients with thrombotic thrombocytopenic purpura
Authors: Weinstein Mark J. , Blanchard Rita , Moake Joel L. , And Evan Vosburgh , MOISE Kenneth
Published: March 2008

Summary. We have investigated the distribution of vWF multimers in blood from the umbilical cord of infants delivered vaginally and by caesarean section, from heel‐stick blood collected 1 d post‐partum, and from fetuses undergoing evaluation for Rh compatibility. To examine vWF multimers. plasma was separated by electrophoresis on SDS‐agarose gels, overlaid with 125I‐anti‐vWF, and analysed by densitometry of autoradiographs. Neonatal and fetal plasma contained unusually large von Willebrand factor multimers (ULvWFM), not present in normal adult plasma, in shed blood from adults, in maternal plasma at the time of birth, or in plasma from adults deficient in vitamin K‐dependent coagulation proteins. We conclude that ULvWFM, similar in size to vWF present in the Weibel Paladie bodies of endothelial cells, the alpha granules of platelets, and the plasma of patients with TTP, is present in the fetal circulation, at birth, and shortly after delivery.
The fate of intravenously injected tissue ferritin in pregnant guinea‐pigs
Authors: Lamparelli R. D. V. , Friedman B. M. , Macphail A. P. , Bothwell T. H. , Phillips J. I. , Baynes R. D.
Published: March 2008

Summary. The organ distribution of intravenously injected hepatic ferritin either labelled with 59Fe or with 19Fe and 125I, was studied in pregnant guinea‐pigs. At 5 h 71.2% of injected 59Fe was present in the placenta and fetus. Transfer of 59Fe to the fetus was slow, with 11.2% present at 5 h and 38.6% at 21 h. Analysis of a placental cellular lysate for 59Fe and 125I revealed that the injected iron was present as intact ferritin at 2 h but by 21 h the ferritin had been catabolized the 125I excreted and the 59Fe incorporated into endogenous ferritin. Most of the fetal 59Fe counts were detected in the liver, with 35.3% of the transferred 59Fe in ferritin, 30.4% in haemoglobin and 10.6% in a low molecular weight pool. The uptake of labelled ferritin by the placenta was inhibited by a 300‐fold molar excess of unlabelled ferritin but not by albumin, asialofetuin or by the injection of carbon particles. A nonsignificant reduction in uptake was noted after injection of mannosylated bovine serum albumin. The mannosidase inhibitor swainsonine had no effect. Iron transfer to the fetus was not affected by various microtubular inhibitors. Presaturation of endogenous transferrin with oral carbonyl iron prevented iron release from the feto‐placental unit back into the maternal circulation. In consequence, marrow 59Fe uptake by the maternal marrow was reduced. The ferrous chelator 2,2′‐bipyridine significantly reduced 59Fe transfer to the fetus and this occurred irrespective of whether the chelator was given prior to or after 59Fe ferritin administration. The ferric chelator desferrioxamine had no such effect. Electron microscopy of placental tissues revealed endocytosis of ferritin molecules. These results indicate that the guinea‐pig placenta takes up homologous tissue ferritin and transfers the iron slowly to the fetus after reductive mobilization. The process is compatible with a receptor‐mediated pathway.
Morphologic and morphometric light and electron microscopic studies of the spleen in patients with hereditary spherocytosis and autoimmune haemolytic anaemia
Authors: Ferreira J. A. , Feliu E. , Rozman C. , Berga Ll. , Bombi J. A. , Marti M. , Vives‐Corrons J. Ll. , Climent C. , Ferrán M. J.
Published: March 2008

Summary.With the aim of contributing to a better understanding of the haemolytic function of the spleen, a morphologic and morphometric study of this organ fixed by arterial perfusion was performed in nine patients with hereditary spherocytosis (HS), three with autoimmune haemolytic anaemia (AHA) and six with Hodgkin's disease without splenic involvement (controls).

The spleen weight in HS and AHA (621 ± 429 g. mean ± SD) was significantly increased with respect to controls (168 ± 3 6 g) (P= 0·003). In HS the red cell retention in the cords of Billroth was significantly increased (203 ± 68 per 104μm2) with respect to the cases with AHA(93 ± 35 per 104μm2) and to the controls (57 ± 28 per 104μm2) (P= 0·004). In HS and AHA the number of macrophages per 104μm2of red pulp was significantly increased (5·41 ± 1·10 and 7·52 ± 2·91, respectively) with respect to the controls (3·25 ± 0·58) (P> 0·003). There was no statistically significant difference between the number of macrophages in HS and AHA.

The transmission (TEM) and scanning electron microscopy (SEM) studies demonstrated predominantly red cell retention in the cords of HS spleens, red cell phagocytosis by cordal macrophages in AHA spleens and in a lesser intensity in HS spleens, and phagocytosis of haematic corpuscles by sinus endothelial cells (SEC) in the cases of HS. These quantitative studies allow a better understanding of splenic red cell destruction in haemolytic syndromes.
Erythrocyte heterogeneity in sickle cell disease: effect of deoxygenation on intracellular polymer formation and rheology of sub‐populations
Authors: Keidan A. J. , Noguchi C. T. , Player M. , Chalder S. M. , Stuart J.
Published: March 2008

Summary. Erythrocytes from 12 patients with homozygous sickle cell disease in the steady state were fractionated on a Percoll‐Stractan density gradient. Erythrocyte deformability was measured by initial‐flow‐rate filtration through pores of 5 μm diameter and erythrocyte polymer content was calculated as a function of oxygen saturation. Density fractionated sub‐populations of sickle cells showed distinct rheological characteristics, the filterability of dense cells being impaired by minimal oxygen desaturation with the apparent formation of little or no intracellular polymer. Lighter cell fractions required a greater degree of deoxygenation and polymer formation to impair deformability, although this occurred prior to morphological sickling. Dense cells therefore exert a disproportionate effect on blood rheology in sickle cell disease and are likely to have an adverse rheological effect in vivo at arterial oxygen tension.
Relationship between the nicotinamide adenine dinucleotide redox potential and the 2,3‐diphosphoglycerate content in the erythrocyte in sickle cell disease
Authors: Lachant Neil A. , Zerez Charles R. , Tanaka Kouichi R.
Published: March 2008

Summary. The percentage of nicotinamide adenine dinucleotide (NAD) in the oxidized form [NAD+/(NAD+ and NADH); i.e. the NAD+/NADT ratio] is increased in the red cell (RBC) in sickle cell disease. We tested the hypothesis that the increased NAD+/NADT ratio was a determinant of the increased 2,3‐diphosphoglycerate (DPG) content of the SCD RBC. Using normal subjects and individuals with sickle cell disease or autoimmune haemolytic anaemia (AIHA), we observed an inverse relationship between the packed cell volume (PCV) and the RBC DPG concentration (r=−0·69) and a direct relationship between the RBC NAD+/NADT ratio and the DPG concentration (r= 0·74). When the effect of the PCV on DPG was removed using analysis of covariance [DPGady(PCV)], the NAD+/NADT ratio had a significant relationship with the DPGadj(PCV) (r= 0·50, P < 0·001). In in vitro incubation studies, increasing the NAD+/NADT ratio significantly increased the DPG content of both normal and AIHA RBC. Conversely, decreasing the NAD+/NADT ratio decreased the DPG content of normal, AIHA and SCD RBC. Thus, the increased DPG content in the SCD RBC appears to be due, in part, to the increased NAD+/NADT ratio and is not purely a physiologic response to decreased oxygen carrying capacity.
Cyclosporine therapy of aplastic anaemia, congenital and acquired red cell aplasia
Authors: Leonard Eileen M. , Raefsky Eric , Griffith Patricia , Kimball Janice , Nienhuis Arthur W. , Young Neal S.
Published: March 2008

Summary. We treated 22 patients with severe aplastic anaemia refractory to antithymocyte globulin (ATG) with cyclosporine, alone or in combination with prednisone. Eight patients showed significant clinical improvement, all but one to transfusion‐independence. Although cyclosporine alone was effective, the addition of prednisone resulted in prompter and fuller haematologic improvement. No patient with an absolute granulocyte count <0·2 × 109/l responded to treatment. Haematologic remissions were sustained beyond the treatment period. Of nine patients with Diamond‐Blackfan syndrome, one showed a complete response to two separate courses of cyclosporine and relapse with withdrawal of therapy, and a second achieved significant reduction in corticosteroid dose without relapse; however, seven cases failed to respond. Two of three adults with acquired pure red cell aplasia recovered. A combination of cyclosporine and corticosteroids may be effective therapy in patients with aplastic anaemia who have failed ATG treatment. Occasional cases of congenital and acquired pure red cell aplasia may also respond to cyclosporine.
Differential activity of recombinant colony‐stimulating factors in supporting proliferation of human peripheral blood and bone marrow myeloid progenitors in culture
Authors: Caracciolo Daniele , Clark Steven , Rovera Giovanni
Published: March 2008

Summary. Unlike bone marrow progenitor cells, human myeloid progenitors isolated from peripheral blood do not form colonies in semi‐solid medium in the presence of rhG‐CSF, rhM‐CSF or rhIL‐6, but do form colonies containing neutrophils, macrophages, eosinophils, basophils or mixed neutrophilic‐macrophages colonies in the presence of rhIL‐3 or rhGM‐CSF. Priming of blood progenitors by culturing them for several days in the presence of rhGM‐CSF resulted in a dramatic increase in the frequency of cells that proliferate in response to G‐CSF and IL‐6 and form neutrophilic granulocytic colonies. Suspension cultures maintained in the presence of IL‐3 yielded increased numbers of clonogenic cells responsive to GM‐CSF and G‐CSF, but not to M‐CSF or IL‐6. rhIL‐6 did not directly stimulate colony formation of peripheral blood progenitors but did prime them to respond to G‐CSF. These results are consistent with a hierarchical model of granulocytic differentiation in which circulating progenitors proceed sequentially through a programme of changing growth factor sensitivity with the following sequence: IL‐3, GM‐CSF, IL‐6 and/or G‐CSF.
Marked erythropoietin increase before fall in Hb after treatment with cytostatic drugs suggests mechanism other than anaemia for stimulation
Authors: Birgegård Gunnar , Wide Leif , Simonsson Bengt
Published: March 2008

Summary. Serum erythropoietin (Epo) was measured in 23 patients before, during and after intensive cytostatic treatment courses for acute leukaemia or before bone marrow transplantation. A marked increase was seen in all patients, starting 1 or 2 d after initiation of treatment. A peak was reached after about 7 d, at levels as high as 1450 IU/l, after which Epo fell rapidly, even in patients who were anaemic at that time. In 13 of the patients there was no fall in Hb level that could explain the increase in Epo. The increase was too large to be explained by an altered Epo metabolism or marrow utilization. Cytostatic drugs cause an increase in Epo production not mediated through anaemia, possibly initiated by a cytostatic effect on the kidneys or by an unknown stimulatory factor, responsive to bone marrow inhibition.
In situ hybridization detection of light chain mRNA in routine bone marrow trephines from patients with suspected myeloma
Authors: Akhtar N. , Rupral A. , Pringle J. H. , Lauder I. , Durrant S. T. S.
Published: March 2008

A method for the detection of immunoglobulin light chain mRNA by in situ hybridization has been applied to routine bone marrow trephines, from patients suspected of having multiple myeloma. The principle aim was to define monoclonal populations even in cases lacking a paraprotein or an obvious atypical plasma cell proliferation.

The expression of light chain mRNA in plasma cells in routine bone marrow trephines from 14 patients with suspected myeloma was studied. Plasma cells in the marrow ranged from 4% to 99%. Clonal populations based on light chain restriction were delineated in 12 patients, using biotinylated probes to kappa and lambda mRNA and visualized using an alkaline phosphatase/fast red naphthol capture method. Normal tonsil and bone marrow trephines were used as controls. In situ hybridization was found to be a specific, safe and rapid technique. It could be applied to both fixed and fresh tissue. It was found to lack the background staining of interstitial immunoglobulin, particularly in sclerotic cases, which is so often encountered with immunocytochemistry.

Additionally, this technique will type plasma cell neoplasms (undetected by routine immunocytochemistry) in post‐transcriptional block or non‐secretory myeloma.
Ultrastructural and ultracytochemical differences between transient myeloproliferative disorder and megakaryoblastic leukaemia in Down's syndrome
Authors: Eguchi Mitsuoki , Sakakibara Hitoshi , Suda Junko , Ozawa Takebumi , Hayashi Yasuhide , Sato Takeyuki , Kojima Seiji , Furukawa Toshiharu
Published: March 2008

Ultrastructural and ultracytochemical studies were performed on blast cells from 12 Down's syndrome neonates with transient myeloproliferative disorder (TMD) and 13 Down's syndrome patients with megakaryoblastic leukaemia (MKL), in order to clarify the cytological characteristics of these cells. Average platelet peroxidase‐positivity in blast cells of TMD patients was similar to that found in cases of MKL. Blast cells from subjects with TMD contained a number of different granules, namely, alpha granules, those that were myeloperoxidase (MPO)‐positive, electron‐lucent or basophil‐like, and those containing membrane components or ferritin particles. On the other hand, granules found in the blast cells of MKL patients with Down's syndrome included the electron‐lucent variety, those with membrane components and a few that were basophil‐like, but not alpha and MPO‐positive granules nor those containing ferritin particles. A demarcation membrane system was observed in blasts from the TMD group, but not in the MKL group.

These findings suggest that blast cells in TMD patients differentiate to megakaryocytes, neutrophils, basophils and erythroblasts, while those in cases of MKL show limited differentiation to immature megakaryocytes, erythroblasts and, sometime, basophils. Such results correspond well with those of culture studies, in which TMD blasts were found to be precursors of various types of blood cell.
Desferrioxamine ototoxicity: evaluation of risk factors in thalassaemic patients and guidelines for safe dosage
Authors: Porter John B. , Jaswon Mervyn S. , Huehns Ernst R. , East Charles A. , Hazell Jonathan W. P.
Published: March 2008

Forty‐seven patients with thalassaemia have been studied to define risk factors for development of sensorineural hearing loss, and to establish guidelines for safe chelation. Sensorineural hearing loss was only present in patients who had previously received desferrioxamine (DFO). The two most significant risk factors were the maximum dose of DFO previously received (P < 0.01), and a serum ferritin of < 2000 μg/1 at that time (P < 0.001). A therapeutic index obtained from the ratio of the mean daily dose of DFO mg/kg divided by the serum ferritin identifies patients with a ratio of > 0.025 as at risk of sensorineural hearing loss (P < 0.001) and can be used as a guideline for safe DFO dosage. Follow‐up audiometry of the affected patients over a 2‐year period indicated that adjustment of the dose to a therapeutic index of < 0.025 resulted in the stabilization of hearing loss in seven patients and improvement in two.
The position of the M‐BCR breakpoint does not predict the duration of chronic phase or survival in chronic myeloid leukaemia
Authors: Jaubert Jerome , Martiat Philippe , Dowding Charles , Ifrah Norbert , Goldman John M.
Published: March 2008

It has been reported that patients with chronic myeloid leukaemia (CML) with 5’breakpoints within the major breakpoint cluster region (M‐BCR) of the BCR gene have somewhat better prognoses than those with 3’breakpoints. We studied the position of the breakpoint in 67 patients with CML in chronic phase using conventional Southern blotting. Using restriction enzymes BglII, BamHI and HindIII and two genomic probes, a 0.6 kb (3’M‐BCR) probe hybridizing to a part of the intron between exons b3 and b4 and a 2.0 kb (5’M‐BCR) probe hybridizing to sequences including exon b1, we localized the breakpoint in M‐BCR as occurring 5’(n= 38) or 3’(n= 28) of the HindIII restriction site located just downstream of exon b3. We failed to localize the breakpoint in one patient. The median durations of chronic phase (37 versus 44 months respectively) and of survival (50 versus 51 months respectively) for patients with 5’and 3’breakpoints were not significantly different. When we analysed only patients whose DNA was collected within 4 weeks of diagnosis (5’breakpoints, n= 30; 3’breakpoints, n= 19), there was again no significant difference in duration of chronic phase or survival. The median survivals of patients divided into good, intermediate and poor prognosis categories in accordance with the prognostic index developed by Sokal and colleagues were 54, 50 and 26 months respectively. This study confirms the value of the Sokal prognostic index but provides no support for the notion that the precise genomic position of the breakpoint in M‐BCR correlates with prognosis.
Relevance of class, molecular weight and isoelectric point in predicting human light chain amyloidogenicity
Authors: Bellotti Vittorio , Merlini Giampaolo , Bucciarelli Eleonora , Perfetti Vittorio , Quaglini Silvana , Ascari Edoardo
Published: March 2008

The ability to predict the amyloidogenicity of certain light chains may facilitate an earlier diagnosis of AL amyloidosis and, possibly, lead to more effective treatment. Using current methods, available in clinical chemistry laboratories, we assessed the class, the relative molecular mass (Mr) and the isoelectric point of urinary monoclonal light chains from 35 patients with AL amyloidosis (A +) and 51 without amyloidosis (A –). The light chain class (LCC) was λ in 77% and 45% of A + and A – patients, respectively. Light chain fragments (LCF) with low Mr (12–18 x 103 were detected in the urine of 30/35 A + patients and in 15/51 A – ones. The mean (SD) isoelectric point (pI) of A + light chains was 4.8 (1.1) while in A – patients it was 6.2 (1.6). Univariate analysis showed significant differences between the two groups for the three parameters. Discriminant analysis gave a function which allowed a correct allocation of 81% of the cases between the two groups.
Antithrombin III and procoagulant activity: sex differences and effects of the menopause
Authors: Meade T. W. , Dyer Sandra , Howarth D. J. , Imeson J. D. , Stirling Yvonne
Published: March 2008

Among participants in the Northwick Park Heart Study, antithrombin III activity was lower in pre‐menopausal women than in men of the same age. In the women, however, the menopause was associated with a significant increase in antithrombin III, mean levels in these older women then exceeding levels in men of the same age. The occurrence of the menopause was also accompanied by large increases in factor VII coagulant activity, VIIc, and in plasma fibrinogen, these increases being greater in those experiencing a natural menopause than in those whose menopause was artificial. Sex differences in antithrombin III may form part of the explanation for the observed differences between men and women in their experience of ischaemic heart disease (IHD) and also for the contrasting effects of oral contraceptives and of hormone replacement therapy on the risk of thromboembolic disease.
Receptor–ligand complexes are cleared to the open canalicular system of surface‐activated platelets
Authors: Leistikow Elizabeth A. , Barnhart Marion I. , Escolar Gines , White James G.
Published: March 2008

Human platelets were incubated with gold particles coupled to fibrinogen to label the glycoprotein IIb–IIIa (GPIIb–IIIa) receptor after initial activation of the cells by contact with formvar‐coated grid and glass surfaces. Fibrinogen–gold (Fgn–Au) markers were absent on discoid platelets, but diffusely spread over the surface and extended pseudopods of early dendritic cells. Conversion to spread platelets resulted in movement of ligand‐receptor complexes away from the cell margin toward cell centres. However, Fgn–Au gold did not concentrate in the central region. Rather, the Fgn–Au, GPIIb–IIIa complexes in the middle of spread platelets appeared to move toward a belt‐like, intermediate zone, as did the ligand receptor complexes from the cell margin and pseudopods. The ultimate destination of the mobile receptor‐ligand complexes, however, appeared to be channels of the surface‐connected open canalicular system (OCS). Fgn–Au was concentrated in OCS channels of most dendritic and a small proportion of spread platelets. The decreased frequency of Fgn–Au filled channels in more transformed platelets may have been due to collapse or evagination of the OCS. Examination of platelets exposed to Fgn–Au after spreading on glass and then prepared for thin sections confirmed that the OCS was the final destination for mobile ligand receptor complexes on surface‐activated platelets. Findings of this study are consistent with previous work showing clearance of mobile receptor–ligand complexes to the OCS of platelets activated in suspension.