bluuebunny/crossref_metadata_2025 huggingface dataset:
Radioimmunoassay for quantitative measurement of factor VIII‐heavy chain
Authors: Nordfang O. , Ezban M. , Nilsson P. , Knudsen J. B.
Published: March 2008

Summary An immunoassay has been developed for the measurement of factor VIII heavy chain (FVIII‐HC). IgG from a human inhibitor plasma with specificity for FVIII‐HC and FVIII‐light chain (FVIII‐LC) was coated to microplates with loose wells. After washing, the plates were incubated with sample and after another wash 125I‐FVIII‐HC was added so that the amount of bound tracer was dependent on the amount of FVIII‐HC in sample. When EDTA was included in the assay buffer the assay response was increased 3‐fold for normal human plasma. This indicated that the antibody was reactive with a determinant hidden in the FVIII‐HC/FVIII‐LC complex as EDTA splits the complex. The sensitivity of the assay was 0.004 units/ml (1 unit/ml in normal human plasma pool).

Together with a previously published assay for FVIII‐LC it has now become possible to measure the relative amounts of FVIII‐HC and FVIII‐LC in haemophilia A plasma and to study the separate expression of FVIII‐HC and FVIII‐LC by recombinant DNA technology. Patients with severe haemophilia A had FVIII‐HC levels below 0.01 units/ml. However, inhibitor patients in high dose FVIII‐treatment showed up to 50 times higher levels of FVIII‐HC than FVIII‐LC and FVIII:C, indicating the presence of FVIII/anti‐FVIII‐LC immune complexes. Thus, dependent on assay specificity plasma samples can show very variable content of FVIII:antigen.
Variation in clinical severity among patients with Hb Lepore‐Boston‐β‐thalassaemia is related to the type of β‐thalassaemia
Authors: Efremov D. G. , Efremov G. D. , Zisovski N. , Stojanovski N. , Kutlar F. , Diaz‐Chico J. C. , Kutlar A. , Yang K. G. , Stoming T. A. , Huisman T. H. J.
Published: March 2008

Summary Clinical and haematological observations, made for 10 Yugoslavian patients with the Hb Lepore‐β‐thalassaemia condition, suggested a considerable variation from severe disease and complete blood transfusion dependency to a moderate, compensated, anaemia without major complications and without a need for regular blood transfusions. As the type of Hb Lepore was the same in all patients (Lepore‐Boston‐Washington) and an a‐globin gene deficiency was absent, it was concluded that the type of β‐thalassaemia determined the severity of the disease. Six patients with severe disease had one of the following three β‐thalassaemia determinants: IVS‐1 position 110 G→A, exon 2 codon 39 C→T, and IVS‐1 position 1 G→A, while the three patients with mild disease had the Portuguese type of thalassaemia which is caused by the T→C substitution at position 6 of the IVS‐1. In one patient with severe disease the β‐thalassaemia determinant remained unknown. Our observations are consistent with those made for thalassaemia patients with a homozygosity for these determinants.
In vivo studies on the relationship between intestinal iron (Fe3+) absorption, hypoxia and erythropoiesis in the mouse
Authors: Raja K. B. , Simpson R. J. , Pippard M. J. , Peters T. J.
Published: March 2008

Summary The effect of hypoxia and changes in erythropoiesis on the absorption of 59Fe3+ from in situ tied‐off duodenal segments was studied in the mouse. Hypoxia led to an increase in mucosal uptake within 6 h, whilst mucosal transfer was unaffected for about 20 h, suggesting independent regulation of these two processes. Hypoxia (3 d) stimulated erythropoiesis and resulted in a 2–3‐fold increase in the total mucosal uptake of 59Fe. Conversely, hyperoxia (100% O2) caused a decrease in reticulocyte counts and the total mucosal uptake. The changes in the transfer of 59Fe from the mucosa to the body were more marked than changes in uptake in both hypoxia and hyperoxia.

Mice subjected to subtotal nephrectomy showed a normal increase in the total mucosal uptake of 59Fe3+ following hypoxic exposure, despite the absence of any changes in the reticulocyte count. Obliteration of the erythroid tissue of animals by splenectomy and 89Sr treatment was accompanied by a marked decrease in the transfer of 59Fe from mucosa to the carcass. However, exposure of splenectomized 89Srtreated mice to hypoxia resulted in an increase in the total mucosal uptake and carcass transfer of 59Fe, without any change in erythropoiesis. These results indicate that hypoxia enhances mucosal iron uptake by a mechanism which is independent of stimulated erythropoiesis, but that changes in the rate of erythropoiesis have an additional effect, particularly on the transfer phase of iron absorption.
Cytoskeleton organization of normal and neoplastic lymphocytes and lymphoid cell lines of T and B origin
Authors: Zauli Daniela , Gobbi M. , Crespi Cristina , Tazzari P. L. , Miserocchi F. , Tassinari Angela
Published: March 2008

Summary An anomalous organization of the cytoskeleton has been described in lymphocytes from chronic lymphatic leukaemia and in only few cell lines. We have now studied normal and neoplastic lymphocytes and lymphoid cell lines of both T and B lineage in order to detect morphological differences in the expression of microfilaments and intermediate filaments. Microfilaments appear to be well expressed by all the B cells, whereas a rich network of intermediate filaments is present in T cells and plasma cells. Most prominent changes occur in the latter system, which is almost lacking in cells of B chronic lymphatic and hairy cell leukaemia. Although the significance of the present findings is not yet clear, one might speculate that such alterations account for some of the aberrant functions and peculiar biologic properties of neoplastic lymphocytes.
Changes in the plasma levels of vitamin K‐dependent proteins C and S and of C4b‐binding protein during pregnancy and oral contraception
Authors: Malm Johan , Laurell Martin , Dahlbäck Björn
Published: March 2008

Summary The plasma concentrations of protein S, protein C and C4b‐binding protein (C4BP) were analysed during pregnancy, in the postpartum period and in women using oral contraceptives. Free protein S. measured after precipitation of the C4BP‐protein S complexes with 5% PEG 6000, was found to be 8.3 mg/l in the control group, which represents 36.3% of the total plasma protein S content (average 23.5 mg/l). The concentration of protein S was significantly decreased during pregnancy, the lowest levels occurring in the second trimester (14.8 mg/1). The values returned to normal within a few days after delivery. The concentration of free protein S was also decreased, down to an average of 3.7 mg/l at delivery, and did not return to normal within the first week postpartum. The mean concentration of protein S in women using oral contraceptives decreased to 17.7 mg/l and the free fraction went down to 6.6 mg/l. Unlike that of protein S, the plasma concentration of protein C increased during pregnancy, reaching a maximum of 135% in the second trimester. Also, it was significantly higher in the postpartum period and in women using oral contraceptives, than in controls. The level of C4BP was increased throughout pregnancy, with a maximum of 143.4% at delivery. These changes in the plasma levels of proteins C and S during pregnancy indicate that the two proteins differ in the regulation of their synthesis. The major decrease in the level of free protein S may predispose to thrombotic episodes during pregnancy, whereas the increased level of protein C may have the reverse effect. These results indicate the importance of taking into account the normal changes in the plasma levels of protein C and S during pregnancy and the use of oral contraceptives, when evaluating patients with increased risk of thromboembolic disease.
Production of a multipotential cell (CFU‐S) proliferation inhibitor by various populations of mouse and human macrophages
Authors: Pojda Z. , Dexter T. M. , Lord B. I.
Published: March 2008

Some bone marrow macrophages from a variety of animal species synthesize a proliferation inhibitor which specifically inhibits the flow of murine CFU‐S into DNA‐synthesis. Macrophages from other sources have now been assayed for their capacity to produce this inhibitor. Macrophages from mouse blood, marrow, spleen and peritoneum and human blood were cultured and their ability to produce inhibitor assayed against proliferating CFU‐S by the tritiated thymidine suicide assay. Inhibitor was observed in all mouse and most human preparations though with varying degrees of activity, showing functional heterogeneity in the development of macrophages in different tissues. Peripheral blood from both mouse and human showed activity approaching that of fresh marrow. This confirms that mouse assay systems can be used to study the human counterpart of the mouse CFU‐S proliferation inhibitor and that blood is a convenient source of material for purification and characterization studies.
Dendritic reticulum cell pattern as a microenvironmental indicator for a distinct origin of lymphoma of follicular mantle cells
Authors: Manconi Riccardo , Poletti Alessandro , Volpe Rachele , Sulfaro Sandro , Carbone Antonino
Published: March 2008

Follicular dendritic reticulum cells (DRCs) are known to be normally present in primary follicles and both follicular centres and mantle zones of secondary follicles of peripheral lymphoid tissue. Involved frozen biopsy tissue specimens from eight cases of intermediate lymphocytic lymphoma/mantle zone lymphoma (ILL/MZL); eight cases of follicular centre cell lymphomas (FCCL) of the centroblastic/ centrocytic type; and seven cases of well‐differentiated lymphocytic lymphoma (WDLL) consistent with chronic lymphocytic leukaemia (CLL) were analysed immunohistologically with R4/23 (DRC‐1) monoclonal antibody reactive with ‘bystander’ DRCs. As opposed to FCCL and most WDLL/CLL cases, the DRCs consistently formed a loose, ill‐defined meshwork with a radiating or blurred outline in all MZL cases and one ILL. On the basis of the observed findings as well as from those reported in literature, the hypothesis is proposed that ILL/MZL originates from the follicular mantle zone and represents a distinct lymphoma entity owing to its peculiar immunostaining pattern of DRCs that allows it to be separated from both FCCL and WDLL/CLL. Moreover, the absence of DRCs in the microenvironment of other B‐cell malignancies such as prolymphocytic leukaemia and hairy cell leukaemia, analogously with most CLL cases, would speak in favour of their different—possibly extrafollicular— compartment of origin within lymphoid tissue.
Acquired type II von Willebrand's disease: demonstration of a complexed inhibitor of the von Willebrand factor‐platelet interaction and response to treatment
Authors: Goudemand J. , Samor B. , Caron C. , Jude B. , Gosset D. , Mazurier C.
Published: March 2008

An acquired von Willebrand's disease developed in two patients in association with a monoclonal gammopathy plus a Sjögren's syndrome and a chronic lymphocytic leukaemia (CLL). In both cases a plasma inhibitor to von Willebrand factor (vWf) was suspected and characterized after plasma gel filtration. The inhibitor was shown to be entirely complexed with vWf and was only demonstrated after complex dissociation by heating. The inhibitor was able to inhibit the binding of125I‐vWf to platelets in the presence of ristocetin in both cases and to thrombin‐stimulated platelets in one case. In the two patients, the highest molecular weight multimers (HMWM) of vWf were absent when assessed by sodium dodecyl‐sulphate agarose plasma electrophoresis. Intravenous infusion of 1‐deamino‐(8‐D‐arginine) vasopressin (DDAVP) resulted in the appearance of the HMWM in both cases and of the satellite bands of each multimer subunit which were lacking prior to the infusion in one patient. After transfusion of a VIII/vWf concentrate containing a significant amount of HMWM, there was a rapid plasma clearance of the vWf‐related activities and of the HMWM when compared to that seen in a patient with type III constitutional vWD. We conclude that in the two patients studied the coagulation defect was related to the presence of a circulating inhibitor to vWf which could be responsible for the disappearance of the HMWM from plasma.
Improvement in rejection, engraftment rate and survival without increase in graft‐versus‐host disease by high marrow cell dose in patients transplanted for aplastic anaemia
Authors: Niederwieser Dietger , Pepe Margaret , Storb Rainer , Loughran Thomas P. , Longton Gary
Published: March 2008

Summary. Two hundred and fifty‐two consecutive patients with severe aplastic anaemia were conditioned by cyclophosphamide and given marrow transplants from HLA‐identical siblings or parents. The results were analysed to examine the influence of marrow cell dose on graft rejection, speed of engraftment, survival and acute and chronic graft‐versushost disease (GVHD). Sixty patients received a corrected marrow cell dose below 2.2 × 108 nucleated cells per kg body weight, 73 patients between 2.2 and 3.2 × 108 cells/kg, 60 patients between 3.3 and 4.2 × 108 cells/kg, and 59 patients more than 4.2 × 108 cells/kg. The significant association between high marrow cell dose and low risk of graft rejection previously reported was confirmed. In addition, high marrow cell dose resulted in small but statistically significant reductions in time to granulocyte and platelet recoveries in patients with sustained engraftment. High marrow cell dose was also associated with a significant improvement in patient survival, a result solely due to the reduction in the incidence of graft rejection. Neither acute nor chronic GVHD were influenced by marrow cell dose. We conclude that the maximum marrow cell dose should be transplanted in patients with severe aplastic anaemia. Maximum marrow cell doses are likely to decrease the incidence of graft rejection, shorten the time to engraftment thereby decreasing the need for blood products and antibiotic support, and improve overall patient survival.
IIB von Willebrand's disease: pathogenetic and therapeutic studies
Authors: Kyrle P. A. , Niessner H. , Dent J. , Panzer S. , Brenner B. , Zimmerman T. S. , Lechner K.
Published: March 2008

Summary. Infusion of 1‐deamino‐(8‐D‐arginine)‐vasopressin (DDAVP) into patients with IIB von Willebrand's disease (vWD) has been reported to induce thrombocytopenia. In some families with this disorder thrombocytopenia is present even in the resting state. We have investigated the basis of this chronic thrombocytopenia in one such patient by performing platelet recovery and survival studies. Increased platelet consumption was suggested by a decrease in platelet recovery (40.5%, normal 45%) and mean platelet survival (112 h, normal range 144–224 h). In addition, we have administered test infusions of DDAVP and observed the effect on bleeding time and platelet count. DDAVP caused a decrease in the median platelet count from 86 × 109/1 (range 30–221) to 60 × 109/1 (range 5‐144), the individual decline in the nine subjects ranging from 12% to 84% compared to the pretreatment values. Formation of platelet aggregates was observed in all patients following DDAVP. The bleeding time was prolonged before DDAVP in all patients and lengthened further in two after the infusion. However, partial correction of the bleeding time was seen in three and normalization in one patient following DDAVP infusion.

Two IIB vWD patients were treated with virus‐inactivated cryoprecipitate (Ristofact®). Infusion of cryoprecipitate was followed by rapid appearance of all but the largest vWF multimers in plasma and did not affect the platelet count. The bleeding time was normalized in one patient but remained prolonged in the other.

In conclusion, in IIB vWD patients virus‐inactivated cryoprecipitate is the treatment of choice in the case of bleeding. Infusion of DDAVP might be effective in a subset of IIB vWD patients that has yet to be characterized.
The in vivo ageing of red cell enzymes: direct evidence of biphasic decay from polycythaemic rabbits with reticulocytosis
Authors: Zimran Ari , Torem Shlomo , Beutler Ernest
Published: March 2008

Summary. The rate at which the activities of red cell enzymes decay during maturation of the reticulocyte and ageing of the erythrocyte is poorly understood. It has recently been suggested that loss of enzyme activity may be rapid during reticulocyte maturation and occur slowly thereafter. We have now devised a rabbit model in which reticulocytosis is combined with transfusional polycythaemia. This makes it possible to follow the enzymatic activity of a cohort of reticulocytes without the interfering effect of newly formed cells.

In this model the reticulocyte count of the experimental animals falls from about 60% to subnormal levels within 4 d. Red cell hexokinase activity declined rapidly with more than half of the activity being lost within 7 d, little change occurring thereafter. Glucose‐6‐phosphate dehydrogenase activity declined more gradually, but also followed a biphasic course. The fall of pyrimidine 5’nucleotidase activity was the slowest of the three age‐dependent enzymes studied. This is consistent with our observations in children with transient erythroblastopenia of childhood, in which this enzyme, alone of the age‐dependent enzymes, was found to be decreased in a senescent red cell population. In contrast to the three age‐dependent enzymes studied, the activity of lactate dehydrogenase remained unchanged during the course of the experiment.

These studies provide direct verification of the suggestion that the decline of red cell enzyme activities may be biphasic. They show, moreover, that enzyme decay is not only rapid in reticulocytes, but also in young erythrocytes.
Sickle cell‐hereditary persistence of fetal haemoglobin and its differentiation from other sickle cell syndromes
Authors: Murray N. , Serjeant B. E. , Serjeant G. R.
Published: March 2008

Summary. Some haematological indices were compared in 13 subjects with sickle cell‐hereditary persistence of fetal haemoglobin (S‐HPFH) and in 10 patients with homozygous sickle cell (SS) disease and four patients with sickle cell‐betaγ thalassaemia, all with fetal haemoglobin (HbF) levels of 20% or above. Sickle cell‐betaγ thalassaemia could be differentiated by a high HbA2 level. S‐HPFH could be distinguished from SS disease with a high HbF level by red cell count, HbF level, reticulocyte count, total haemoglobin and total bilirubin level in decreasing power of discrimination.
Presence of an inhibitor of glycosyltransferase activity in a patient following an ABO incompatible liver transplant
Authors: Barbolla L. , Mojena M. , Cienfuegos J. A. , Escartín P.
Published: March 2008

Summary. The contribution of the liver to plasma ABO glycosyltransferase activity has been studied in a group O individual transplanted with a liver from a group B donor. The B transferase activity present in the post‐transplantation plasma was negligible. However, a potent B transferase inhibitor, absent from the pretransplantation plasma, was present after transplantation. The inhibitor was present in the excluded fraction following Sephadex G‐25 gel filtration, but was retained by a protein A‐Sepharose column, suggesting that it was an IgG antibody. This inhibitor was also effective in reducing A transferase activity.
Marrow fibroblasts from patients with myeloproliferative disorders show increased sensitivity to human serum mitogens
Authors: Kimura Akiro , Katoh Osamu , Kuramoto Atsushi
Published: March 2008

Summary. The growth of marrow fibroblasts from patients with myeloproliferative disorders (MPD) was investigated using platelet derived growth factor (PDGF) and human serum as mitogens in the presence of human plasma derived serum. The proliferation of fibroblasts in MPD patients was increased compared to normal individuals, especially in patients with chronic myelocytic leukaemia and essential thrombocythaemia. This increment of proliferation might be due to higher sensitivity of the fibroblasts to plasma derived serum than to PDGF, because the ratio of proliferation with PDGF to that without PDGF, when compared between patients and normals, remained unchanged. These results suggest that MPD fibroblasts are more sensitive to some factor(s) in plasma, and this fact could partially explain the pathogenesis of myelofibrosis in MPD patients.
Cytokine activity after human bone marrow transplantation.
Authors: Bentel Jacqueline , Atkinson Kerry
Published: March 2008

Summary. The production of procoagulant activity by circulating monocytes and its regulation by a cytokine secreted by mitogen‐stimulated peripheral blood mononuclear cells was investigated in recipients of HLA‐identical sibling bone marrow transplants. Blood monocyte numbers reached the normal range within 3 weeks of transplant. After stimulation with lipopolysaccharide, macrophage procoagulant activity was found to be within the normal range in all patients at all times post transplant. It did not appear to correlate with the presence or absence of graft‐versus‐host disease. Surprisingly, and in marked contrast to our previously documented severe depression of interleukin 2 production by transplant recipients' peripheral blood mononuclear cells, the mitogen‐induced production of the cytokine that induces procoagulant activity production (macrophage procoagulant inducing factor, MPIF) was also normal in the majority of patients when assayed using the responsive myelomonocytic cell line RC‐2A. These findings suggest firstly that monocyte differentiation and function normalize rapidly post transplant; and secondly, when taken together with previous studies, that the ability of peripheral blood mononuclear cells to synthesize cytokines post transplant varies greatly according to the specific cytokine involved.
A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro
Authors: Millar B. C. , Bell J. B. G. , Lakhani A. , Aylifee M. J. , Selby P. J. , McEtwain T. J.
Published: March 2008

Summary. A double layer agar technique has been developed to grow myeloma colonies (MY‐CFUc) from human bone marrow aspirates and peripheral blood. Heavily irradiated HL60 cells (5 × 105/plate) are added to an agar underlay in growth medium containing 0.5% agar. Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60‐conditioned medium (HL60‐CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37°C in an atmosphere of 5% CO2, 10% O2 and 85% N2. Colonies (> 50 cells) form between 2 and 3 weeks. Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay. The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid. The two cell types are usually, but not always, present in separate colonies. Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1). This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte‐macrophage colonies (GM‐CFUc). The drug sensitivity of myeloma cells (MY‐CFUc) compared with normal haemopoietic cells (GM‐CFUc) can be measured using dose‐response curves in individual patients. Furthermore, this method can detect resistant subpopulations within a given myeloma sample.
Terminal deoxynucleotidyl transferase positive acute myeloid leukaemia: an association with immature myeloblastic leukaemia
Authors: Parreira A. , de Oliveira M. S. Pombo , Matutes E. , Foroni L. , Morilla R. , Catovsky D.
Published: March 2008

Summary. The morphology, membrane markers and ultra‐structural cytochemistry of 39 cases of acute myeloid leukaemia (AML) with variable proportion (10–99%) of terminal deoxynucleotidyl transferase (TdT) positive blasts was compared with that of 134 cases of TdT negative AML. The incidence of TdT positive AML was 22.5% and this was significantly higher in poorly differentiated myeloblastic (M0 and M1) types (54%) than in all other FAB subtypes (10%; P<0.001). Our findings suggest heterogeneity among TdT positive cases. Whilst the majority correspond to genuine TdT positive AML in which evidence for exclusive myeloid nature was demonstrated by phenotypic, cytochemcal and ultrastructural markers, a distinct minority (22%) of cases had mixtures of lymphoid and myeloid blasts. A change in phenotype occurred in three out of six cases studied in relapse. There was no difference in the incidence of immuno‐globulin (Ig) gene rearrangement between TdT positive (two out of 12) and TdT negative (one out of 11) cases, although published data suggests that Ig gene rearrangement is significantly more common in TdT positive cases. The determination of TdT in AML allows the identification of cases of mixed acute leukaemia which probably represent proliferations of multipotent progenitor cells. The majority of TdT positive cases, nevertheless, correspond to immature types of myeloblastic leukaemia which may constitute a clinically distinct subgroup.
Magnetic resonance imaging of bone marrow in lymphoproliferative disorders: correlation with bone marrow biopsy
Authors: Döhner H. , Gückel F. , Knauf W. , Semmler W. , van Kaick G. , Ho A. D. , Hunstein W.
Published: March 2008

Summary. In a prospective clinical study 10 normal volunteers and 30 patients with lymphoma—11 patients with Hodgkin's lymphoma and 19 patients with non‐Hodgkin's lymphoma (11 low grade, 8 high grade)–were examined. Proximal femora, pelvis and lumbar spine were imaged with a 1.5 tesla superconducting MR imaging system (Siemens Magnetom). Areas of malignant infiltration in the bone marrow were clearly detected by visual and/or quantitative assessment. In most cases bone marrow involvement was demonstrated by both magnetic resonance imaging and bone marrow biopsies. However, in three of 30 patients magnetic resonance imaging showed evidence of lymphomatous involvement despite normal bone marrow histology. In one patient bone marrow cytology revealed malignant infiltration in the absence of MRI abnormalities. Thus, MRI is a sensitive method for detecting bone marrow infiltration by lymphoma.
Isolation of human megakaryocytes by immunomagnetic beads
Authors: Tanaka Hiroyuki , Ishida Yoji , Kaneko Toshio , Matsumoto Noboru
Published: March 2008

Summary. A simple method was developed to purify human megakaryocytes to homogeneity from normal bone marrow aspirates. An initial separation of marrow between 1.020 and 1.050 g/ml. Percoll® density cut was used to enrich megakaryocytes. After washing, the cells were suspended with immunomagnetic beads which were coated with sheep anti‐mouse IgG antibody and treated with anti‐human glycoprotein (GP) IIb/IIIa monoclonal antibody, or the cells were treated with human platelet GP IIb/IIIa monoclonal antibody and suspended with the immunomagnetic beads which were coated with sheep anti‐mouse IgG antibody. Megakaryocytes were selectively separated using a magnet. All of the isolated cells were morphologically recognizable megakaryocytes. 1.5–3.1 × 104 megakaryocytes were obtained from 1.7–4.5 × 108 bone marrow nuleated cells. These cells were all positive in immunoenzymatic staining for GP IIb/IIIa. Megakaryocytes obtained by this method responded to recombinant human GM‐CSF (rhGM‐CSF) showing an increased 3H‐thymidine (3H‐dT) incorporation. These data show that this method is useful for obtaining pure megakaryocyte populations which can be submitted to comprehensive biological studies.
Absence of rearrangement of proto‐oncogene MET in 88 cases of myelodysplastic syndromes (MDS)
Authors: Collyn‐d'Hooghe M. , Fenaux P. , Lai J. L. , Bauters F. , Loucheux‐Lefebvre M. H. , Kerckaert J. P.
Published: March 2008

Summary. Abnormalities of chromosome 7 are among the most frequent cytogenetic aberrations found in MDS, including de novo cases and cases secondary to chemo‐ and/or radiotherapy. Since MET is located on 7q and as Cooper et al (1984) showed that MET proto‐oncogene could be activated by a chemical carcinogen, we tried to evaluate whether it could be implicated in some cases of MDS. With specific probes for MET we analysed the DNA of 88 MDS patients (81 de novo and seven secondary cases). In 17 of them the RNA was also studied. We found no rearrangement or aberrant expression of MET in any samples studied. Our results, however, do not rule out point mutations or rearrangement of other regions of MET or adjacent DNA regions.
Clonal B lymphocytes lack bcr rearrangement in Ph‐positive chronic myelogenous leukaemia
Authors: Ferraris Anna Maria , Canepa Letizia , Melani Cecilia , Miglino Maurizio , Broccia Giorgio , Gaetani Gian Franco
Published: March 2008

Summary. Philadelphia (Ph) chromosome‐positive chronic myelogenous leukaemia (CML) was studied in a subject heterozygous for the X chromosome‐linked alloenzyme system of glucose‐6‐phosphate dehydrogenase (G6PD). Determination of G6PD mosaicism showed homogeneous expression in granulocytes, erythrocytes and platelets. Cytogenetic studies showed the typical Ph translocation in all metaphases from bone marrow and peripheral blood myeloid cells, bcr rearrangement was detected in bone marrow and in granulocytes. B cells were stimulated with Epstein‐Barr virus (EBV) in order to evaluate involvement of lymphocytes, EBV‐transformed lymphoblastoid cells expressed a single G6PD pheno‐type and therefore probably derived from the leukaemic stem cell. However they had a normal karyotpye and a constitutional bcr restriction pattern. Molecular analysis in this case of CML clarifies the differentiative potential of cells belonging to the leukaemic clone, by demonstrating that clonal Phnegative B cells maintain normal differentiative capacity and have a bcr gene sequence which is not rearranged.
Tyrosine protein kinase activity in normal and leukaemic human blood cells
Authors: Punt C. J. A. , Rijksen G. , Vlug A. M. C. , Dekker A. W. , Staal G. E. J.
Published: March 2008

Summary. Tyrosine protein kinase (TPK) activity was measured in subcellular fractions of normal granulocytes, lymphocytes and monocytes, and acute and chronic myeloid and lymphoid leukaemic cells. Of several tested tyrosine‐containing substrates, poly (glutamic acid: tyrosine = 4:1) (S1) proved to be the best synthetic substrate. High cytosolic TPK activity was found in every cell type. Different TPKs may exist in various cell fractions, as was indicated by the difference in Km values for S1 in cell fractions of normal granulocytes and lymphocytes. No significant difference was found in total TPK activity between normal and leukaemic cells, indicating that total TPK activity is not related to the leukaemic process itself. A highly significant difference was found in membrane fractions in normal granulocytes and M1–M2 AML cells versus normal monocytes and M1–M5 AML cells, suggesting an association between TPK activity and monocytic differentiation in these cell fractions.
Autografting for patients with chronic myeloid leukaemia in chronic phase: peripheral blood stem cells may have a finite capacity for maintaining haemopoiesis
Authors: Brito‐Babapulle Finella , Bowcock Stella J. , Marcus Robert E. , Apperley Jane , Th'ng K. H. , Dowding Charles , Rassool Fey , Guo Ai‐Pu , Catovsky Daniel , Galton David A. G. , McCarthy Donald , Goldman John M.
Published: March 2008

Summary. We treated 14 patients with Ph‐chromosome‐positive chronic myeloid leukaemia still in chronic phase by autografting with blood‐derived haemopoietic stem cells. Eleven patients were autografted electively after cytoreductive treatment with busulphan (16 mg/kg) and melphalan (60 mg/m2) and three were autografted after marrow cells from HLA‐identical sibling donors had failed to engraft. In 13 patients haemopoiesis recovered; one failed to engraft and died 114 d after autografting. Two other patients became pancytopenic and received further stem cell transfusions at 3 and 40 months respectively after first autografting. One patient entered lymphoid transformation and died 14 months after autografting. Twelve patients survive at a median of 41 months (range 24–53) after autografting. Nine of the survivors have required further chemotherapy after autografting and four of the nine were electively autografted on a second occasion. Three patients surviving after autografting for 28, 43 and 53 months respectively have not required further chemotherapy. In two of these patients haemopoiesis is now predominantly Ph‐negative. We conclude that autografting in chronic phase might prolong survival in some cases by reducing the size of the leukaemic stem cell population. The fact that initially successful grafts failed in two patients suggests that blood‐derived stem cells may have a finite potential for self‐replication.
Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size
Authors: Federici A. B. , Bader R. , Pagani S. , Colibretti M. L. , De Marco L. , Mannucci P. M.
Published: March 2008

Summary. We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS‐agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/I) of the individual fractions for ristocetin‐dependent binding to GP Ib and thrombin‐induced binding to GP IIb/IIIa. Direct binding studies of the 125I‐vWF pool gave mean Kd values of 2.02 ±0.05 × 10−8 for GP Ib and 1.15±0.02 × 10−8 for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3–10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin‐ and thrombin‐induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.
Human liquid bone marrow culture in serum‐free medium
Authors: Drouet Xavier , Douay Luc , Giarratana Marie‐Catherine , Baillou Claude , Gorin Norbert‐Claude , Salmon Charles , Najman Albert
Published: March 2008

Prolonged in vitro maintenance of human bone marrow progenitor cells was achieved using a serum‐free (SF) liquid culture system. Culture medium was based on Iscove's medium supplemented with bovine serum albumin, human transferrin, bovine insulin, soybean lecithin, cholesterol, hydrocortisone and alpha‐thioglycerol. Under these standardized culture conditions, CFU‐GM were maintained for up to 4 weeks, as is the case when using conventional serum‐dependent medium. Erythropoiesis exhibited a slower decline than that found using serum containing medium. Development of normal haematopoiesis was effective in spite of poor stromal cell development—a confluent adherent layer as classically described in serum conditions was never achieved. Our newly defined system provides a reliable technique for studying human haematopoietic stem cell proliferation and differentiation in vitro; it allows for rational utilization of currently available purified recombinant growth factors. It may be a promising tool in the clinical use of cultured haematopoietic stem cells.
Regulatory abnormalities in the marrow of patients with myelodysplastic syndromes
Authors: Merchav Shoshana , Nagler Arnon , Fleischer‐Kurtz Gila , Tatarsky Ilana
Published: March 2008

Regulation of haemopoiesis in the marrow of patients with myelodysplastic syndromes (MDS) was evaluated by assaying (1) the production of haemopoietic regulators acting upon multipotent and committed progenitors by MDS marrow cells, and (2) the responsiveness of MDS marrow progenitors to stimulation with granulocyte colony‐stimulating factor (G‐CSF). The levels of multipotent progenitor cell colony‐stimulating activity (CFU‐GEMMCSA) in 7 d bone marrow‐conditioned medium (BMCM) from MDS patients were markedly reduced as compared to controls. MDS BMCM also exhibited reduced levels of burst‐promoting activity (BPA) for primitive erythroid (BFU‐E) progenitors. Both CFU‐GEMMCSA and BPA detected in BMCM were completely neutralized by antibodies directed against interleukin‐3. MDS BMCM exhibited markedly reduced levels of murine‐active CSA. This activity was partially neutralized by anti‐CSF‐1 antibodies. Levels of regulators in BMCM of refractory anaemia (RA), sideroblastic anaemia, RA with excess blasts, and chronic myelomonocytic leukaemia were virtually the same. CFU‐GEMM and BFU‐E growth in MDS marrow (n= 9) was markedly reduced. A 5‐fold saturating dose of G‐CSF induced an approximately 2‐fold increase in CFU‐GEMM in four of eight MDS and a 1.5‐fold increase of BFU‐E in five of nine MDS, but not in control (n= 5) marrow cell cultures. Impaired haemopoiesis in MDS marrow may be related to abnormalities both in regulator production by marrow accessory cells and in regulator responsiveness of multipotent and committed progenitors.
Co‐incident N and K ras gene mutations in a case of AML, restricted to differing cell lineages
Authors: Hiorns Lynne R. , Cotter Finbarr E. , Young Bryan D.
Published: March 2008

Peripheral blood from a patient with acute myeloid leukaemia (AML) of M5 FAB classification, was shown to have mutations to both the N and K ras genes. Leucophoresed blood was separated on a discontinuous Percoll density gradient to provide fractions enriched for different cell lineages. DNA extracted from these fractions was amplified using the polymerase chain reaction (PCR) technique, and hybridized with oligonucleotide probes specific for the single base mutations previously demonstrated. The N‐ras mutation was shown to be restricted to the blast and monocytic cell fractions, concordant with the FAB subtype of M5. The K‐ras mutation, however, was present in all fractions, suggesting it had occurred in a multi‐potential stem cell representing an earlier stage in the generation of the leukaemia, or possibly an incidental background phenomenon.
IL‐2 and myelopoiesis: IL‐2 induces blast cell proliferation in some cases of acute myeloid leukaemia
Authors: Carron J. A. , Cawley J. C.
Published: March 2008

Interleukin‐2 (IL‐2) stimulated H‐thymidine incorporation in the blasts of six of 21 cases of acute myeloid leukaemia (AML). An IL‐2 induced increase in cell numbers was directly demonstrated in the two patients studied in this way, and T‐cell contamination was rigorously excluded. The IL‐2‐induced proliferation was usually less marked than that caused by granulocyte‐macrophage colony stimulating factor (GM‐CSF), and IL‐2 moderately enhanced GM‐CSF‐induced stimulation in five of the six patients; in the sixth, IL‐2 and GM‐CSF were strongly synergistic. IL‐2‐induced proliferation was observed only in AML with a monocytic component (M4/M5), but not all M4/M5 leukaemias responded to IL‐2. There was no correlation between expression of the light‐chain of the IL‐2 receptor and IL‐2‐induced stimulation.

It is suggested that IL‐2 is involved at a restricted stage of early myelopoiesis, perhaps when cells are becoming committed to the monocytic lineage: and that IL‐2 is a growth factor for early myeloid cells in a proportion of cases of AML.
True histiocytic lymphoma: histopathologic, immunophenotypic and genotypic analysis
Authors: Hanson Curtis A. , Jaszcz Waclaw , Kersey John H. , Astorga Magdalena G. , Peterson Bruce A. , Gajl‐Peczalska Kazimiera J. , Frizzera Glauco
Published: March 2008

Five cases of true histiocytic lymphoma (THL) were analysed by immunophenotyping and immunoglobulin/T‐cell receptor genotyping. These cases showed striking morphologic diversity but a strong degree of immunophenotypic homogeneity. The malignant cells reacted with multiple histiocytic markers including CD11c (Ki‐M1, LeuM5), CD14, CD68 (Ki‐M6) and Ki‐M8; anti‐HLA‐DR and non‐specific esterase staining was also found in all cases. The malignant cells did not express monoclonal immunoglobulin and did not react with the B‐ or T‐cell monoclonal antibodies used except for those known to be cytoplasmically expressed in monocytes/histiocytes, such as CD4 and CD19; B‐ and T‐cell staining was otherwise limited to background small lymphocytes. By genotypic analysis, three cases showed rearrangements: one with Tβ, one with Tβ and immunoglobulin heavy chain (JH) and one with both JH and light chain: the remaining two cases retained their immunoglobulin and T‐cell receptor genes in germline configuration. The results not only suggest that certain subsets of the histiocyte/reticulum cell system may be capable of rearranging immunoglobulin or Tβ genes while simultaneously expressing multiple histiocytic surface antigens but also demonstrate the necessity of using multiple histiocytic‐specific monoclonal antibodies and cytochemical staining in diagnosing THL. Gene rearrangement studies must be interpreted in conjunction with immunophenotyping and morphology in the determination of cell lineage.
Effect of exchange transfusion of reticulocytes on in vitro and in vivo intestinal iron (Fe3+) absorption in mice
Authors: Raja K. B. , Simpson R. J. , Peters T. J.
Published: March 2008

The effect of acute changes in erythron demands and in plasma‐iron turnover (PIT) on the in vitro and in vivo absorption of 59Fe3+ was studied in the mouse. Hypoxic exposure for 20 h (a time at which intestinal iron absorption is markedly stimulated) induced reticulocytosis with a marked elevation in PIT. More prolonged exposure (3 d) further enhanced the plasma‐iron clearance, even though the absorption of 59Fe was not further increased.

Recipient mice, exchange transfused with whole blood from phenylhydrazine‐treated animals, had a marked reticulocytosis and elevated PIT. However, in vivo studies exhibited only a small enhancement in intestinal 59Fe absorption. In vitro studies, in contrast, showed no changes in the kinetic parameters for duodenal Fe3+ uptake in similarly‐transfused mice. Blood cells, rather than plasma, were responsible for the enhanced in vivo absorption in the transfused animals.

These data indicate that acute changes in body iron demands and in PIT have only a small effect on iron absorption via a process independent of the adaptive increase in carrier‐mediated uptake following chronic (3 d) hypoxia. This regulatory process, however, is inadequate to explain the adaptive changes seen during acute (20 h) hypoxic exposure.
The development of spatial distributions of CFU‐S and in‐vitro CFC in femora of mice of different ages
Authors: Mason T. M. , Lord B. I. , Hendry J. H.
Published: March 2008

The radial distributions of spleen colony forming units (CFU‐S) and in‐vitro colony forming cells (in‐vitro‐CFC) were measured in the diaphyseal marrow cavity of femora removed from 3‐, 5‐ and 11‐week‐old mice. The distributions observed in 11‐week‐old mice confirm earlier findings that the highest concentrations of CFU‐S exist near bone surfaces whereas the concentration of in‐vitro‐CFC increases to a peak value approximately 300 μm from the femoral axis with a low value at the bone surface. The gradients of the distributions in all three age groups are very similar suggesting that spatial organization in marrow is established by 3 weeks at the latest and, as the marrow cavities grow, so the distributions extend into the new space following their respective gradients. The peak of CFU‐S concentration at the bone surfaces in all age groups coincides with increased rates of DNA synthesis and a low self‐renewal capacity. Conversely. CFU‐S nearer the centre of the cavity maintain a low turnover but have a high self‐renewal capacity. Measurements made on 1‐week‐old mice show that the marrow contains a lower average concentration of CFU‐S in the femoral cavity compared to older mice. However, these CFU‐S have both a high rate of turnover and a high self‐renewal capacity. It appears that these better quality CFU‐S remain in a central location while the rest of the population ages and expands in association with growing bone regions.
Slow evolution of chronic myeloid leukaemia relapsing after BMT with T‐cell depleted donor marrow
Authors: Hughes T. P. , Economou K. , Mackinnon S. , Vlitos M. , Arthur C. K. , Guo A. P. , Rassool F. , Apperley J. F. , Hows J. , Goldman J. M.
Published: March 2008

Thirty‐three patients with Philadelphia positive (Ph +) chronic myeloid leukaemia (CML) treated in chronic phase by bone marrow transplantation (BMT) with T‐cell depleted HLA‐identical sibling marrow were evaluable for relapse at a median follow up of 41 months (range 16‐59 months). Twenty‐six (78%) had Ph+ marrow metaphases demonstrated at some time post BMT. The subsequent pattern of disease was variable. In 15 of these cases haematological relapse occurred within 24 months of BMT. Four patients proceeded to haematological relapse more slowly. Seven patients had only cytogenetic evidence of relapse. Of the 19 patients with haematological relapse, five received second transplants and two survive; 13 of the other 14 survive in chronic phase at median times from allografting and from recognition of haematological relapse of 41 months (range 25‐59 months) and 18 months (range 5‐36 months) respectively. For these 13 patients disease progression after relapse seems to be relatively indolent. In the four patients we could study, blood lymphocytes were almost all of donor origin. We suggest that even in patients with cytogenetic or haematological evidence of relapse after T‐cell depleted BMT, leukaemic cell proliferation may still be restrained to some extent by a graft‐versus‐leukaemia effect mediated by donor‐derived lymphoid cells.
Cytotoxic potential of anti‐CD7 immunotoxin (WT1‐ricin A) to purge ex vivo malignant T cells in bone marrow
Authors: Preijers F. W. M. B. , Witte T. De , Wessels J. M. C. , Meyerink J. P. P. , Haanen C. , Capel P. J. A.
Published: March 2008

Summary With the perspective of bone marrow purging in autologous transplantation, we investigated the cytotoxicity of the anti‐T cell immunotoxin (IT) WT1‐ricin A (anti‐CD7) to malignant T cells obtained from patients with T cell acute lymphoblastic leukaemia or lymphoma. The cytotoxic efficacy of IT was based on the extent of protein synthesis inhibition. Cytotoxicity of IT to malignant T cells showed a dependency on antigen density comparable to the T cell lines GH1, CEM, Jurkat, HSB‐2 and HPB‐ALL and was enhanced considerably in the presence of 6 mm ammonium chloride. The ultimate proof of cell kill can only be obtained from clonogenic assays: however, culturing of malignant T cells was not feasible. Therefore these assays were performed with the cell line CEM that expresses comparable amounts of CD7 antigen as malignant T cells of most patients. More than 6‐logs of CEM appeared to be eliminated after incubation with 10‐8m WT1‐ricin A.

Immunotoxins are only effective after entering the target cell. The pattern of internalization of the IT was determined by means of 125I‐WT1. After internalization the CD7 antigen was re‐expressed on the cell membrane. This enables a long incubation period resulting in an increased elimination of malignant T cells. Even after 16 h the IT was still accumulated intracellularly. This pattern of continuous uptake of IT was reflected in a gradually increasing cytotoxicity with incubation time. Effective bone marrow purging can be carried out without adverse effects on progenitor cells with 10‐8m WT1‐ricin A. At that concentration the antibody binding capacity was saturated.

We showed that the protein synthesis inhibition in malignant T cells by WT1‐ricin A is comparable to the inhibition in T cell lines and that high amounts of CEM cells can be killed. These data suggest that cell lines can be used to test the efficacy of IT to malignant T cells. WT1‐ricin A appears to be very potent for the purging of autologous bone marrow from patients with T cell malignancies.