bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of CD45RO in antithymocyte globulin's stimulation of primitive haemopoietic cells
Authors: Huang Andrew T. , Mold Nelda G.
Published: March 2008

Summary. We have found that antithymocyte globulin (ATG), an equine antibody with proven efficacy in aplastic anaemia (AA), has a direct stimulatory effect on primitive haemopoietic cells from normal donors. This growth stimulation may be mediated via anti‐CD45RO activity present in the ATG preparation. Addition of unabsorbed ATG enhanced colony growth at 21 d in the blast colony forming cell (Bl‐CFC) assay. Prior absorption of ATG by incubation with the CD45RO+ MOLT‐4 cell line resulted in the loss of enhancement. Absorption by MOLT‐4 cells preincubated with anti‐CD45RO mAb, UCHL‐1, restored ATG's stimulatory effect. The Bl‐CFC could also be stimulated to grow by the addition of UCHL‐1 directly. Incubation of the primitive haemopoietic cells for 4 h with ATG was associated with a decline in the antigenic density of CD45RO, a tyrosine phosphatase. This down‐regulation may upset the balance between growth factor‐induced tyrosine kinase activation and tyrosine phosphate dephosphorylation resulting in increased growth of primitive cells. a possible factor in the sustained recovery of haemopoiesis seen in AA patients after ATG treatment.
Haemophagocytic lymphohistiocytosis, interferon‐gamma‐naemia and Epstein‐Barr virus involvement
Authors: Imashuku Shinsaku , Hibi Shigeyoshi , Fujiwara Fumihiro , Ikushima Satoshi , Todo Shinjiro
Published: March 2008

Summary. To clarify the correlation between Epstein‐Barr virus (EBV) involvement and hypercytokinaemia in haemophagocytic lymphohistiocytosis (HLH), we analysed serum interferon‐gamma levels and EBV‐DNA in biological specimens obtained from 25 HLH cases (23 children and two adults). We found that HLH patients showed a wide range of serum IFN‐gamma levels from 0.2 to 1300 U/ml, with a median 126U/ml for EBV‐DNA‐positive (n = 9) and 4.5 U/ml for EBV‐DNA‐negative (n = 16) groups. The latter group could be classified further into a group with hyper‐IFN‐gamma‐naemia (> 4.5 U/ml) (n = 8) and a group without hyper‐IFN‐gamma‐naemia (n = 8). The survival of the hyper‐IFN‐gamma‐naemic cases was significantly poorer than non‐hyper‐IFN‐gamma‐naemic cases. We conclude that EBV is probably involved in one third of the HLH cases, all of whom show hyper‐IFN‐gamma‐naemia, and in the half of the HLH cases with hyper‐IFN‐gamma‐naemia who have a rapidly fatal outcome.
Dysplastic haemopoiesis following orthotopic liver transplantation: comparison with similar changes in HIV infection and primary myelodysplasia
Authors: Clatch Richard J. , Krigman Hannah R. , Peters Marion G. , Zutter Mary M.
Published: March 2008

Summary To validate scientifically our prior empiric observations that patients develop significant haemopoietic dysplasia following solid organ transplantation, we developed a quantitative lineage‐specific scoring system to evaluate dysplastic features of bone marrow aspirates and core biopsies. We used this scoring system to compare retrospectively randomly selected bone marrow aspirates and core biopsies from 19 patients undergoing orthotopic liver transplantation (OLT), 21 with a known history of human immunodeficiency virus (HIV) infection, and 18 with primary or chemotherapy‐related myelodysplastic syndromes (MDS). Our results show that the OLT patient group developed significant but milder haemopoietic dysplastic changes than the HIV or MDS groups, and that the MDS group developed more severe dysplasia of the myeloid lineage than the other groups. The possible roles for drugs and infectious agents in the pathophysiology of dysplastic changes are discussed.
Effects of rhG‐CSF, 5‐fluorouracil and extramedullary irradiation on murine megakaryocytopoiesis in vivo
Authors: Scheding Stefan , Media Joseph E. , Kraut Michael , Valdivieso Manuel , Nakeff Alexander
Published: March 2008

Summary The aim of this study was to systematically characterize possible rhG‐CSF effects on the murine megakaryocyte‐platelet system (untreated and recovering from chemotherapy or extramedullary irradiation). In untreated, splenectomized male B6D2F1 mice rhG‐CSF treatment (50μg/kg/d for up to 8d) markedly decreased femoral megakaryocytopoiesis. CFU‐Meg, small acetylcholinesterase‐positive (SAChE) cells, and megakaryocytes were significantly reduced to 35–70%: platelets, however, were not affected. Peripheral CFU‐Meg and CFU‐GM increased up to 200‐fold. Following a single injection of 5‐FU (150mg/kg) on day O, rhG‐CSF (50 μg/kg/d) on days 1–8 suppressed the megakaryocytopietic recovery as indicated by significantly lower platelet numbers on day 9. Granulopoietic recovery was accelerated by rhG‐CSF. When rhG‐CSF treatment was started on day 5, no beneficial effect on granulopoietic recovery was observed, but again platelet levels were significantly lower on day 9, indicating that within the first 4 d of rhG‐CSF application, recruitment or lineage competition was not a critical event. To test for the effects of extramedullary irradiation on circulating progenitors, mice pretreated with 50 μg/kg/d of rhG‐CSF for 8 d received irradiation to the chest with 500 cGy resulting in a substantial kill of circulating CFU‐Meg and CFU‐GM of up to 99%. However, this striking decrease of blood progenitors did not significantly affect their total body contents. This sutdy indicates that rhG‐CSF treatment can impair bone marrow megakaryocytopoiesis, which might be an important consideration for those clinical situations that carry a high potential for treatment‐induced thrombocytopenia.
Haemoglobin inhibits GTP‐hydrolysis and GDP/GTP‐exchange activities of a low Mr GTP‐binding protein, ras p21
Authors: Nagata Koh‐Ichi , Nozawa Yoshinori
Published: March 2008

Summary Haemoglobin was observed to inhibit the GDP/GTP‐exchange activity of ras protein (ras p21) by measurement of [3H]GDP‐dissociation activity in time‐ and dosedependent manners. Haemoglobin also inhibited the [32P]GTP‐hydrolysis activity of ras p21 time‐ and dose‐dependently. These inhibitory effects of haemoglobin were lost after incubation of haemoglobin at 80°C for 3 min. Globin showed limited inhibition on the [32P]GTP‐hydrolysis activity of ras p21, and haemin had no effect, indicating that the ternary tetrameric structure of haemoglobin is essential for the inhibitory effects on ras p21 activities. Methaemoglobin also inhibited both [3H]GDP‐dissociation and [32P]GTP‐ hydrolysis activities of ras p21 in a very similar manner to that by haemoglobin. The obtained results strongly suggest that haemoglobin suppresses the physiological functions(s) of ras p21 in vivo inhibiting both [32P]GTP‐hydrolysis and GDP/GTP‐dissociation of ras p21 in erythrocytes.
Primary plasma cell leukaemia
Authors: Dimopoulos Meletios A. , Palumbo Antonio , Delasalle Kay B. , Alexanian Raymond
Published: March 2008

Summary. Among 750 previously untreated patients with multiple myeloma, 27 (4%) presented with plasma cell leukaemia. All but one patient had high tumour mass and, when compared with comparable patients without leukaemia, more frequent extraosseous involvement, thrombocytopenia, high serum lactate dehydrogenase and hypodiploid plasma cells. Most patients also had complex cytogenetic abnormalities. Treatment with standard melphalan‐prednisone was ineffective, with a median survival of 2 months, but more intensive chemotherapy induced responses in approximately one‐half of the patients, with a median survival of 20 months. Primary plasma cell leukaemia usually results from the proliferation and extramedullary expansion of immature plasma cells and requires prompt and intensive chemotherapy.
Cyclic thrombocytopenia associated with IgM anti‐GPIIb‐IIIa autoantibodies
Authors: Kosugi Satoru , Tomiyama Yoshiaki , Shiraga Masamichi , Kashiwagi Hirokazu , Nakao Hiroshisa , Kurata Kanayama Yoshiyuki , Matsuzawa Yujr
Published: March 2008

Summary. We studied a female patient with cyclic fluctuation in platelet count following splenectomy for autoimmune thrombocytopenia. The cyclical fluctuation appeared to be in phase with her menstrual cycle and her platelet count was low during menses. Bone marrow examinations performed at the peak as well as the bottom of the platelet count showed normal or increased numbers of megakaryocytes. The patient's platelet count increased rapidly after intravenous gamma‐globulin (IVIgG) therapy, suggesting that a failure of platelet production is unlikely to account for the cycle. Platelet‐associated IgM (PAIgM) was markedly elevated, whereas PAIgG was normal at any stage of the cycle. MACE assay demonstrated that PAIgM contained IgM anti‐glycoprotein (GP) IIb‐IIIa autoantibodies. Comparison between MACE assay using untreated and EDTA‐treated platelets at 3 7°C demonstrated that the platelet‐associated IgM autoantibodies mainly recognized divalent cation‐dependent conformation(s) of GPUb‐IIIa. No antibodies were, however, detected in her serum. The levels of IgM anti‐GPIIb‐IIIa showed an inverse relationship with the platelet count. In spite of the marked increase in platelet count after IVIgG, however, the levels of IgM anti‐GPIIb‐IIIa remained elevated. These findings suggest that plateletassociated IgM anti‐GPIIb‐IIIa autoantibodies are of pathogenic significance in this patient.
Role of ADP in platelet aggregation at high shear: studies in a patient with congenital defect of platelet responses to ADP
Authors: Cattaneo Marco , Zighetti Maddalena L. , Lombardi Rossana , Mannuccio Pier
Published: March 2008

Summary. The in vitro measurement of platelet aggregation (PA) at the high shear levels that can be found in the microcirculation may provide useful informations on primary haemostasis, which is usually explored in vivo with the skin bleeding time (BT). PA at high shear requires von Willebrand factor (vWf) and the platelet glycoprotein (GP) complexes Ib/IX/V and IIb/IIIa; controversial results have been reported on its requirement of released adenosine diphosphate (ADP). Due to its dependence on vWf, PA at high shear may be affected by the vasopressin analogue DDAVP, which increases the plasma vWf levels and shortens the prolonged BT of patients with congenital or acquired defects of platelet function. We studied PA at high shear, BT and plasma vWf levels in a patient with congenital impairment of platelet responses to ADP before and after the i.v. infusion of 0.3 μg/kg DDAVP. Two methods to study PA at high shear were used: shear‐induced PA (SIPA) and the filter aggregation test. With both methods, PA at high shear of the patient was impaired. The infusion of DDAVP increased plasma vWf levels, shortened the prolonged BT and potentiated PA at high shear of the patient. In conclusion, PA at high shear is impaired in a patient with congenital defect of platelet responses to ADP and prolonged BT and is potentiated by DDAVP. Our results suggest that released ADP plays an important role in PA at high shear and that potentiation of PA at high shear by DDAVP may be one mechanism by which the drug shortens the prolonged BT of patients with congenital or acquired defects of platelet function.
Platelet shape change and Ca2+ mobilization induced by collagen, but not thrombin or ADP, are inhibited by phenylarsine oxide
Authors: Greenwalt Dale E. , Tandon Narendra N.
Published: March 2008

Summary. In this report we have examined the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO) on receptor‐mediated platelet shape change, secretion and aggregation. PAO was found to inhibit platelet aggregation induced by collagen, thrombin, ADP and epinephrine at IC50 values of 0.35 μmol/l, 2.5 μmol/l, 0.2 μmol/l and 0.3 μmol/l, respectively. Agonist‐induced secretion of ATP was inhibited at similar or lower concentrations of PAO. The specificity of the interaction of PAO with platelet proteins was demonstrated by the ability of the disulfhydryl compound 2,3‐dimercaptopropanol, which abstracts PAO from proteins to form a stable cyclic adduct, to reverse PAO inhibition of both agonist‐induced platelet secretion and aggregation. Dimercaptopropanesulphonic acid, a membrane‐impermeable analogue of dimercaptopropanol, did not reverse inhibition of collagen‐induced shape change or aggregation by PAO, thereby demonstrating that PAO acted intracellularly. PAO inhibited collagen‐induced shape change and internal Ca2+ mobilization but had no effect on these two phenomena when induced by thrombin or ADP. PAO was also unable to prevent arachidonic acid‐induced shape change, indicating that PAO acts at a site prior to the phospholipase A2‐mediated release of arachidonic acid to inhibit collagen‐induced shape change. PAO induced the accumulation of a number of phosphotyrosine‐containing proteins and inhibited the collagen‐induced phosphorylation of a 40 kD protein. The potency and agonist‐specific effects of PAO on platelet activation suggest that this inhibitor will be of value in elucidation of signal transduction pathways involved in receptor‐mediated platelet function.
Biological effect of desmopressin in eight patients with type 2n (‘Normandy’) von Willebrand disease
Authors: Mazurier Claudine , Gaucher Christine , Jorieux Sylvie , Goudemand Maurice
Published: March 2008

Summary It is generally thought that the plasma increase in factor VIII (FVIII) after desmopressin (dDAVP) infusion is related to the plasma increase in von Willebrand factor (vWF), which is the plasma carrier for FVIII. The aim of this study was to evaluate the FVIII and vWF responses in patients with type 2N vWD, characterized by the mild FVIII deficiency related to markedly decreased affinity of vWF for FVIII. At different times after one intravenous dose of dDAVP (0.3 or 0.4 μg/kg) we measured the FVIII coagulant activity, FVIII antigen, vWF antigen and ristocetin cofactor activity, in eight patients with either Arg91Gln or Arg53Trp amino acid substitution in mature vWF. In all the patients, whatever their mutation, the dDAVP infusion resulted in a 2.3. ± 0.7 ‐fold increase of vWF and a variable rise (9.5 ± 7.7 times) of FVIII, whereas the vWF capacity to bind FVIII was not improved. The FVIII response was more transient than vWF response, and FVIII half disappearance time was evaluated to the approximately 3h. The data indicate that the stabilizing effect of vWF on FVIII is not responsible for the FVIII increase induced by dDAVP. The clinical implication of this study is that, in the 2N vWD patients, dDAVP may be a useful prophylactic or curative treatment when the test dose has been shown to be effective to reach a haemostatic FVIII level.
Isolation and culture of endothelial cells from human bone marrow
Authors: Masek Lisa C. , Sweetenham John W.
Published: March 2008

Summary : Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to re‐establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin‐1 (UEA‐1) to endothelial cells. Magnetic Dynabeads coupled with UEA‐1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum‐free medium together with 5% fetal calf serum for 24h. Cells were then cultured in endothelial serum‐free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplemented with 5% fetal calf serum, endothlial cell growth supplement and heparin. After 2–4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle‐shaped morphology with a rapid growth rat, the other a rounded morphology and a slow growth rate. Both population have a vasiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM‐1, VCAM‐1, E‐wlectin, CD31 and BMA120. Weibel‐Palade bodies were observed by electron microscopy.

Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.
The reliability of the mean normal prothrombin time of fresh plasmas and of the normal value from a lyophilized ‘normal’plasma in prothrombin ratio determination
Authors: Poller L. , Houghton D. , Carroll J.
Published: March 2008

Summary. The mean normal prothrombin time (MNPT) based on the fresh plasma of 20 healthy individuals is the recommended way to derive a normal value for the prothrombin ratio. This is generally believed to give a reasonable representation of the normal in the local population, but there may be difficulty in obtaining a good representative sample in some centres. The alternative of a uniform lyophilized ‘normal’plasma has been sugested. The comparative value of a widely used commercial ‘normal’plasma and the MNPT has been assessed in an international study at 37 centres. Two common thromboplastins were tested using the local coagulometer methods. The variability of the results and their relative performance in local thromboplastin calibration to derive the International Sensitivity Index (ISI) were assessed. The reliability of the two types of normal in local system ISI calibration with different lyophilized plasma calibrants has also been tested. When the lyophilized ‘normal’was substituted for the MNPT in ISI determination no appreciable difference in the degree of correction for caogulometers effects on International Normalized RAtios was found.
In vitro culture of colony forming unit‐megakaryocyte (CFU‐MK) in fetal alloimmune thrombocytopenia
Authors: Warwick R. M. , Vaughan J. , Murray N. , Lubenko A. , Roberts I.
Published: March 2008

Summary . Perinatal alloimmune thrombocytopenia (PAITP) causes intracranial haemorrhage in the fetus and neonate. However, the severity of the thromobocytopenia correlates poorly with maternal anti‐platelet antibody titres. To test the hypothesis that reduced platelet production contributes to fetal thrombocytopenia in PAITP, maternal sera from three HPA‐la‐negative mothers whose pregnancies were complicated by anti‐HPA‐la (two severe cases, one mild case) were added to colony forming unit‐megakaryocyte (CFU‐MK) cultures from HPA‐la positive and negative individuals. Sera from the two severely affected pregnancies containing anti‐HPA‐la caused 66–100% inhibition of HPA‐la‐positive fetal and neonatal CFU‐MK, whereas CFU‐MK from two HPA‐la‐negative mothers were not inhibited by the anti‐HPA‐la‐containing sera. Maternal serum from the case of mild PAITP caused only mild inhibition of HPA‐la‐positive cord and adult CFU‐MK and did not inhibit HPA‐la‐positive fetal CFU‐MK. Taken together, these findings suggest that reduced megakaryocyte production contributes to fetal thrombocytopenia due to maternal anti‐HPA‐la antibodies and also that the degree of CFU‐MK inhibition correlates with severity of fetal thrombocytopenia.
Phosphorylation of 2‐chlorodeoxyadenosine (CdA) in extracts of peripheral blood mononuclear cells of leukaemic patients
Authors: Arner Elias S. J. , Spasokoukotskaja Tatjana , Juliusson Gunnar , Liliemark Jan , Eriksson Staffan
Published: March 2008

2‐Chlorodeoxyadenosine (CdA) is an antileukaemic agent used in treatment of hairy cell leukaemia (HCL) and chronic lymphocytic leukaemia of B‐ and T‐cell type (B‐CLL and T‐CLL). The aim of this study was to elucidate the interpatient variability of CdA phosphorylation and its relation to response to CdA treatment. In extracts of peripheral blood mononuclear cells of patients with B‐CLL (n= 39), CdA phosphorylation was significantly higher than in HCL (n= 19) when calculated per protein (391 ± 155 pmol CdA phosphorylated/mg protein/min versus 288 ± 166 pmol/mg/min, P < 0.001), but was the same when calculated per cell (12 ± 5.9 pmol/106 cells/min versus 14 ± 5.9 pmol/106 cells/min) due to a larger cell volume in HCL. In T‐CLL (n= 6), CdA phosphorylation was significantly lower than in B‐CLL, both when calculated per protein (128 ± 68 pmol/mg/min, P < 0.001) or per cell (5.7 ± 2.7 pmol/106 cells/min, P < 0.05). This low CdA phosphorylation in T‐CLL was unexpected because normal B‐ and T‐lymphocytes contain equal amounts of CdA phosphorylation. With B‐CLL, 21 patients who responded (complete and partial response) to CdA treatment showed a significantly higher CdA phosphorylation than 13 patients not responding to CdA treatment (456 ± 170 pmol/mg/min versus 309 ± 97 pmol/mg/min, P < 0.01). We conclude that the level of CdA phosphorylation is correlated with the response of leukaemias to CdA treatment.
Adult T‐cell leukaemia with various abnormalities in endocrine and metabolic systems
Authors: Adachi Mitsunobu , Shiomura Tadahiko , Shimada Hideto , Kishimoto Norihumi , Iida Kei , Ikeno Humiaki , Nishimura Haruo , Inoue Tatsuhide , Sako Yoshiyasu
Published: March 2008

Adult T‐cell leukaemia (ATL) is a unique type of T‐cell malignancy closely associated with human T‐cell leukaemia virus‐1 (HTLV‐1). Despite frequent descriptions of hypercalcaemia, cases accompanied by diabetes insipidus or syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in ATL patients have rarely been reported. We present an unusual case of ATL with various abnormalities in his endocrine and metabolic systems involving anterior pituitary function, thyroid function, lipid metabolism and Ca metabolism. Some of these abnormalities were considered to arise from infiltration or leukaemic cells into systemic organs after elimination of the above symptoms. Clinical and haematological data showing improvement following chemotherapy are also presented.
Propagation of large numbers of T cells with natural killer cell markers
Authors: Schmidt‐Wolf I. G. H. , Lefterova P. , Johnston V. , Huhn D. , Blume K. G. , Negrin R. S.
Published: March 2008

Summary. Previously, a subset of T cells co‐expressing the NK cell antigen CD56 has been described. These CD3+CD56+ cells are rare in peripheral blood collections and have been poorly characterized. We have developed culture conditions which allow for the rapid expansion of CD3+CD56+ cells. The protocol for cellular expansion includes the addition of interferon‐gamma on day O, interleukin‐1, interleukin‐2 and a monoclonal antibody against CD3 on day 1 to peripheral blood lymphocytes. Cells of the CD3+CD56+ phenotype increased up to 6000‐fold using this protocol after 16 d in culture. These cells have been characterized by flow cytometry and have been found to express the alpha, beta T cell receptor, co‐express the CD5 and CD8 antigens and do not express the CD16 antigen. Morphologically, these cells cannot be distinguished from NK cells. CD3+CD56+ killer cells lyse a variety of tumour cells with intermediate activity between CD3−CD56+ NK cells and CD3+CD56− T cells.
Spontaneous megakaryocyte colony formation in myeloproliferative disorders is not neutralizable by antibodies against IL3, IL6 and GM‐CSF
Authors: Li Yan , Hetet Gilles , Maurer Anne‐Marie , Chait Yasmine , Dhermy Didier , Briere Jean
Published: March 2008

Summary. Megakaryocyte progenitor growth in 42 patients with myeloproliferative disorders (MPD), including 23 essential thrombocythaemia (ET), eight polycythaemia vera (PV). six chronic myelogenous leukaemia (CML) and five primary myelofibrosis (PMF), was studied in vitro using plasma clot assay and serum‐free agar culture. Spontaneous megakaryocyte colonies (CFU‐MK) were found in 34/40 (80%) blood and 14/18 (77.8%) bone marrow plasma clot cultures, and also observed in 27/35 (77.1%) blood and 10/18 (55.6%) bone marrow serum‐free agar cultures. In the blood of 27 patients with MPD (15 ET, four PV, four CML and four PMF) and the bone marrow of 10 patients (five ET, four CML and one PV), spontaneous colony formation was observed in both plasma clot and serum‐free agar cultures. However, spontaneous CFU‐MK was only found in plasma clot culture, but not in agar culture in two blood (one ET and one CML) and four bone marrow cultures (one ET, two PV, one CML). The colony numbers were greatly increased in the presence of aplastic anaemia serum (AAS) under both conditions. In 17 patients (12 ET, two CML and three PV) with spontaneous megakaryocyte colonies, anti‐cytokine antibody neutralizing experiments were carried out in blood cultures. Anti‐IL3, anti‐IL6 and anti‐GM‐CSF antibody, alone or in combination, at different concentrations (1, 5 and 10 μg/ml), were added into plasma clot or agar cultures without exogenous stimulating growth factors. The results showed that the numbers of spontaneous megakaryocyte colonies were not significantly decreased in the presence of these monoclonal antibodies in the cultures. The data indicated that the megakaryocyte progenitor growth in MPD under in vitro conditions was heterogenous, and independent of exogenous stimulatory factors in most patients and that optimal megakaryocyte colony development in MPD still requires exogenous growth factors.
The proliferative activity of myelopoiesis in myelodysplasia evaluated by multiparameter flow cytometry
Authors: Jensen Inger Marie , Hokland Peter
Published: March 2008

Double staining of bone marrow cells for CD13 and CD33 leucocyte differentiatian antigens and for DNA content has allowed us to evaluate the proliferative capacity of myelopoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. By analysing 39 patients (15 RA/RAS, 14 RAEB and 10 RAEB‐t) and eight normal controls, we found significant differences in both the percentage of cells positive for these immature myeloid antigens between the FAB groups as well as in the fractions of CD13 and CD33 positive cells in S or S‐G2M phase of the cell cycle. Moreover, a clear decrease in the immature myeloid cell proliferative activity upon progression within the FAB groups was evident. Finally, we found a significant negative association between the percentage of myeloblasts in the bone marrow and the proliferative activity of the immature myeloid cells. indicating that the block in differentiation in MDS patients might be coupled to a simultaneous block in proliferation, especially in advanced stages. These data suggest that the use of double parameter assays in the longitudinal follow‐up of MDS patients might yield new information about the biology of MDS.
Elevated urinary levels of thromboxane and prostacyclin metabolites in sickle cell disease reflects activated platelets in the circulation
Authors: Kurantsin‐Mills Joseph , Ibe Basil O. , Natta Clayton L. , Raj J. Usha , Siegel Robert S. , Lessin Lawrence S.
Published: March 2008

There is evidence for increased factor VII turnover and the associated increased thrombin generation and fibrinolytic activities in sickle cell disease (SCD) that may affect in vivo platelet and endothelial cell reactivity. We studied the release of specific eicosanoids that are indicative of in vivo platelet activation and endothelial cell injury. The circulating and urinary levels of 2,3‐dinor thromboxane B2(2,3‐dinor‐TxB2), TxB2, PGI2 [as 6‐keto‐PGFlα], and PGE2 were measured in 15HbSS patients, eight HbAA nonhaemolytic anaemic individuals and 12 healthy HbAA controls using specific RIAs. The mean urinary 2,3‐dinor‐TxB2 in the HbSS patients was significantly higher than in both the healthy HbAA and the anaemic controls. 6‐keto‐PGFlα was undetected in the urines of the healthy HbAA controls, but was measured insignificant amounts in the HbSS and the HbAA anaemic patients. The urinary concentrations of PGE2 and TxB2 in HbSS patients' samples were also significantly higher than those of both control groups (P < 0.05). PGE2 and TxB2 levels were below the detection limit in the plasmas of the HbAA subjects, but were measurable in the HbSS and HbAA anaemic plasmas. The plasma level of 6‐keto‐PGFlα in the HbSS patients was also significantly higher than in the control groups. The data indicates a persistent inflammatory process in the HbSS patients, and is consistent with the hypothesis that there is platelet and endothelial cell activation in SCD.
Childhood onset cyclic neutropenia: G‐CSF therapy restores neutrophil count but does not influence superoxide anion and cytokine release by neutrophils
Authors: Marcolongo Renzo , Zambello Renato , Trentin Livio , Cassatella Marco , Gasparotto Giuseppe , Agostini Carlo
Published: March 2008

Summary. In this paper we describe the case of a 16‐year‐old boy with childhood onset cyclic neutropenia (CN) with a 21d cycle who was successfully treated with recombinant granulocyte‐colony stimulating factor (G‐CSF). Cyclic therapy with G‐CSF (5μg/kg/d s.c. for 1 week every 21d) maintained peripheral neutrophil count above the normal range, reduced the incidence of severe infections and significantly improved the patient's performance status throughout an 18‐month follow‐up. Phenotypic analysis of circulating lymphocytes demonstrated that G‐CSF treatment does not modify the phenotypic profile of circulating B. T and NK cell populations. Circulating neutrophils released normal amounts of cytokines (including IL‐1/3, IL‐8, TNFα) and superoxide anion during G‐CSF therapy.
Anti β2 glycoprotein I antibodies: detection and association with thrombosis
Authors: Martinuzzo M. E. , Forastiero R. R. , Carreras L. O.
Published: March 2008

Summary. It has been demonstrated that antiphospholipid antibodies (aPL) recognize epitopes formed by anionic phospholipids and protein cofactors. β2 glycoprotein I (β2GPI) is accepted as the cofactor of anticardiolipin antibodies (aCL). In the present study we explored the presence and clinical associations of anti β2GPI antibodies of IgG isotype (aβ2GPI‐IgG), measured by ELISA. We studied sera from 169 patients with aCL and/or lupus anticoagulant (LA), including 52 patients with systemic lupus erythematosus and 49 with primary antiphospholipid syndrome (PAPS). We found 31.9% positive sera for aβGPI‐IgG in the whole population and 48.6% in the aCL‐IgG(+) group. There was a good correlation between the titre of aCL‐IgG and the optical density for aβGPI‐IgG (r= 0.69, P <0.01). The presence of aβ2GPI‐IgG was associated with the presence of aCL‐IgG (P <0.0001) and LA (P <0.0005). However, none of 23 LA (+) patients without aCL had aβ2GPI‐IgG.

We found a statistically significant association between the presence of aβ2GPI‐IgG and a history of venous thromboembolism (VTE) in our patients (P <0.005). This association was observed in PAPS (P <0.05) but not in secondary antiphospholipid syndrome (SAPS).

Our study confirms that some aPL(+) sera react with β2GPI in special experimental conditions. In addition, the presence of these antibodies is associated with a history of VTE.
Over‐expression and amplification of the CDC2 gene in leukaemia cells
Authors: FURUKAWA YUSUKE , TERUI YASDHITO , SAKOE KUMI , OHTA MASATSUGU , KITAGAWA SEIICHI , MIURA YASUSADA , SAITO MASAKI
Published: March 2008

The expression and structure of the cdc2 gene, one of the master regulators of the eukaryotic cell cycle, were investigated in fresh leukaemic cells from 51 cases of various types of leukaemia. Cdc2 mRNA transcripts were detectable in approximately 40% (21/51) of cases by Northern blotting. Over‐expression of cdc2 mRNA as compared to normal bone marrow cells was noted in 10/21 cases with detectable cdc2 mRNA transcripts. Amplification of the cdc2 gene was found in three cases. Cdc2 mRNA was over‐expressed in these three cases, suggesting that gene amplification is a direct cause of mRNA over‐expression in a subset of cases. Cell proliferative capacity was well correlated with the amount of cdc2 mRNA transcripts, i.e. 3H‐thymidine incorporation was highest in cases with cdc2 mRNA over‐expression and was significantly higher in cdc2‐positive cases than in cdc2‐negative cases. These results suggest that over‐expression of CDC2, which is due to the gene amplification in some cases, might play a role in altered growth of leukaemic cells.
Evidence for a light chain restriction of glycoprotein Ib/IX and Ilb/IIIa reactive antibodies in chronic idiopathic thrombocytopenic purpura (ITP)
Authors: STOCKELBERG DICK , HOD MING , JACOBSSON STEFAN , KUTTI JACK , WADENVIK HANS
Published: March 2008

To address the assumption of clonally restricted antibodies in immune thrombocytopenias we studied sera from 19 patients with chronic ITP known to possess antibodies reactive with glycoprotein (GP) Ib/IX and/or GPIIb/IIIa. These sera were re‐analysed using the standard monoclonal antibody immobilization of platelet antigens (MAIPA) assay and 16 patients exhibited IgG antibodies reactive with GPIIb/IIIa; seven patients showed also a reactivity with GPIb/IX. Employing a light‐chain‐specific MAIPA assay, 75% (12/16) of these sera displayed GPHb/ Ilia‐specific antibodies that were light chain restricted; only 13% (2/16) of the GPIIb/IHa reactive sera showed a mixed kappa and lambda phenotype. A light‐chain‐restricted phenotype was also seen for the GPIb/IX reactive antibodies. To further substantiate these findings, the MAIPA assay was modified in order to avoid interference from human anti‐mouse antibodies. A high frequency of light‐chain restricted platelet antibodies was also found using the modified MAIPA technique. These results support the hypothesis of a clonal B‐cell expansion in immune thrombocytopenias, producing antibodies with a restricted idiotype repertoire and reacting with a limited number of epitopes.
Acquired hypochromic and microcytic sideroblastic anaemia responsive to pyridoxine with low value of free erythrocyte protoporphyrin: a possible subgroup of idiopathic acquired sideroblastic anaemia (IASA)
Authors: TAKEDA YASUSHI , SAWADA HIROYOSHI , SAWAI HIROFUMI , TOI‐MATSUDA TOMOKO , TASHIMA MASARO , OKUMA MINORU , WATANABE SHIGEYA , OHMORI SEIICHI , KONDO MASAO
Published: March 2008

Patients with idiopathic acquired sideroblastic anaemia (IASA) usually show macrocytic or normocytic anaemia and increased free erythrocyte protoporphyrin (FEP). The mean cell haemoglobin concentration is normal or slightly low. Here we report a pyridoxine‐responsive IASA patient with microcytic and hypochromic anaemia and low FEL level; these features are usually seen in cases of hereditary sideroblastic anaemia. Microcytosis increased during therapy.

There may be a subgroup of IASA with microcytic and hypochromic anaemia, low normal FEP and some response to pyridoxine like hereditary sideroblastic anaemia.
Detection of the BCR‐ABL gene by reverse transcription/polymerase chain reaction and fluorescence in situ hybridization in a patient with Philadelphia chromosome negative acute lymphoblastic leukaemia
Authors: RHEE F. VAN , KASPRZYK A. , JAMIL A. , DICKINSON H. , LIN F. , CROSS N. C. P. , GALVIN M. C. , GOLDMAN I J. M. , SECKER‐WALKER L. M.
Published: March 2008

The Philadelphia (Ph) chromosome is detected in leukaemia cells in approximately 20% of adults with acute lymphoblastic leukaemia (ALL). When treated with chemotherapy alone, Ph‐positive ALL has a poor prognosis, and patients may benefit from bone marrow transplantation in first remission. Here we report a patient with chromosomally normal bone marrow, in all 60 cells analysed, who was found to have the p210‐type BCR‐ABL chimaeric transcript by RT/PCR. Fluorescence in situ hybridization was labelled cosmid probes for BCR and ABL showed the presence of BCR‐ABL juxtaposition on a normal chromosome 22 in leukaemia cell metaphases. We conclude that molecular and cytogenetic methods should be used in conjunction to detect the BCR‐ABL gene rearrangement in ALL.
Transcriptional and post‐transcriptional regulation of granulocyte‐macrophage colony‐stimulating factor production in human growth factor dependent M‐07e cells
Authors: BRIZZI MARIA FELICE , ROSSI PIER RICCARDO , ROSSO ARTURO , AVANZI GIAN CARLO , PEGORARO LOIGI
Published: March 2008

Summary. To elucidate the regulatory mechanisms of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) production in human myeloid leukaemic cells we studied GM‐CSF gene transcription, mRNA expression and GM‐CSF secretion in human growth factor dependent M‐07e cells. GM‐CSF transcript was detected in cells cultured in the presence of interleukin‐3 (IL‐3), GM‐CSF or mast cell growth factor (MGF), whereas it was undetectable in growth factor deprived cells. Growth factor re‐addition induced, within 2 h, the appearance of GM‐CSF mRNA. Nuclear run‐on experiments demonstrated that the increase of GM‐CSF mRNA levels depends on GM‐CSF gene transcription. The simultaneous addition, to deprived cells, of the growth factor, and of cycloheximide (CHX) for 2h inhibited GM‐CSF mRNA expression, suggesting the requirement for newly made proteins for GM‐CSF gene transcription. By means of the M‐07e bioassay, which allows the detection of GM‐CSF, IL‐3 and MGF activities, and neutralizing antibodies to each of these factors, GM‐CSF activity was detected in the cell‐free extract of both IL‐3‐ and MGF‐sustained cells and of cells deprived for 24 h. This finding demonstrates that M‐07e cells produce and store biologically active GM‐CSF in response to both IL‐3 and MGF. In contrast, analysis of the growth stimulatory activity present in the culture supernatants revealed that MGF, unlike IL‐3, is able to induce the secretion of consistent amounts of GM‐CSF. Taken together, our results suggest that, in M‐07e cells, GM‐CSF gene transcription and GM‐CSF production are mediated, unlike its secretion, by mechanisms shared by IL‐3 and MGF.
Polyclonal haemopoieses associated with long‐term persistence of the AML1‐ETO transcript in patients with FAB M2 acute myeloid leukaemia in continous clinical remission
Authors: GUERRASIO ANGELO , ROSSO CLAUDIA , MARTINELU GIOVANNI , COCO FRANCESCO LO , PAMPINELLA MARIA , SANTORO ALESSANDRA , LANZA CARLO , ALLIONE BERNARDINO , RESEGOTTI LUIGI , SAGLIO GIUSEPPE
Published: March 2008

Summary. The t(8;21) (q22;q22) translocation is a recurring chromosomal abnormality observed in about 20‐40% of AML patients with subtype FAB M2 (AML‐M2). The molecular facet of this translocation is represented by the formation of a new hybrid gene, the AML1‐ETO, which is regularly transcribed in a chimaeric mRNA and translated into a new fusion protein believed to have a key role in the pathogenesis of this type of leukaemia. We looked for the presence of AML1‐ETO transcripts, by RT‐PCR, in 49 unselected patients affected by AML‐M2 diagnosed at various Italian Institutions. A hybrid transcript was detected in 11 cases (23%). Minimal residual disease status was investigated in three patients in continuous complete remission (CCR) after a median follow‐up of 44 months; at least one sample from each subject was found positive for the AML1‐ETO transcript suggesting a long‐term persistence of t(8;21) leukaemic cells.

In two female patients in CCR a‘clonality’analysis was performed on peripheral blood DNA by exploiting the × chromosome inactivation pattern of the human androgen‐receptor gene (HUMARA); in both cases the results were consistent with the presence of a polyclonal haemopoiesis.

Our data confirm that the persistence of residual cells expressing the AML1‐ETO transcripts is a frequent occurrence even in patients with long‐term remission; on the other hand, clonality assays indicate that in t(8;21) leukaemias long‐term remission haemopoiesis is sustained by a polyclonal bone marrow reconstitution.
Comparative study of peripheral blood progenitor cell collection in patients with multiple myeloma after single‐dose cyclophosphamide combined with rhGM‐CSF or rhG‐CSF
Authors: DEMUYNCK HILDE , DELFORGE MICHEL , VERHOEF GREGOR , ZACHEE PIERRE , VANDENBERGHE PETER , BOOGAERTS MARC
Published: March 2008

Summary. Patients suffering from high‐risk multiple myeloma (MM) were randomized to receive single high‐dose cyclophosphamide followed by either rhGM‐CSF or rhG‐CSF in order to harvest circulating peripheral blood progenitor cells. The safety of the procedure, the mobilization kinetics, the relative efficacy of rhGM‐CSF and rhG‐CSF to mobilize progenitor cells and their relative toxicity were studied. Special attention was paid to the antigenic profile of CD34+ progenitor cells.

Group I patients (n=ll) were treated with cyclophosphamide 4 g/m2 i.v. followed by rhGM‐CSF at 10 /ig/kg/d by subcutaneous administration. Group II (n=ll) patients received rhG‐CSF s.c. at 10/zg/kg/d after the same dose cyclophosphamide. Both mobilization regimens appeared to be equally effective. No significant differences in absolute numbers of circulating progenitors, determined by CD34 expression or in yields of MNC, CFU‐GM, BFU‐E and CD34 subsets were observed. rhGM‐CSF administration resulted however in delayed haemopoietic recovery and an increased complication rate.

We conclude that rhG‐CSF may be preferred because of its markedly lower toxicity and lower in‐hospital costs.
Dapsone for refractory chronic idiopathic thrombocytopenic purpura
Authors: HERNANDEZ FERNANDO , LINARES MARIANO , COLOMINA PEDRO , PASTOR EMIUO , CERVERO ANTONIO , PEREZ AMALIA , PERELLA MATILDE
Published: March 2008

Summary. Fifteen patients with refractory chronic idiopathic thrombocytopenic purpura (ITP) were treated with dapsone (lOOmg/d) for 1‐31 months. The overall response rate to dapsone was 40%. Five patients responded in 1 month and one patient in 2 months. No pretreatment characteristics ‐sex, age, platelet count or duration of ITP ‐ were correlated with response to dapsone. Treatment was well tolerated. The most frequent adverse effect was dose‐related haemolytic anaemia. In our experience, dapsone provides an inexpensive and well‐tolerated alternative for patients with ITP who had inadequate reponses to conventional therapy.
A promoter mutation, C → T at position ‐92, leading to silent /3‐thalassaemia
Authors: ROSATELLI MARIA CRISTINA , FAA VALERIA , MELONI ALESSANDRA , FIORENZA FLAVIA , GALANELLO RENZO , GASPERINI DANIELA , AMENDOLA GIOVANNI , CAO ANTONIO
Published: March 2008

Summary. This study describes the clinical phenotype of the C?→? T mutation at position – 92 of the β‐globin gene. Excluding two cases with HbA2 levels within the range of the /3‐thalassaemia carrier state, heterozygotes for this mutation showed normal or borderline red blood cells count, Hb levels, MCV, MCH and HbA2 values, and unbalanced globin chain synthesis. Compound heterozygotes for the ‐ 92 C → T mutation and a β° thalassaemia mutation (β°39) (two cases) or severe β‐thalassaemia (p+ IVSII nt 745) (two cases) developed thalassaemia intermedia. According to these characteristics, the –92 promoter mutation should be added to the list of silent β‐thalassaemias.
The effect of haemopoietic growth factors on the cell cycle of AML progenitors and their sensitivity to cytosine arabinoside in vitro
Authors: SMITH M. A. , SINGER C. R. J. , PALLISTER C. J. , SMITH J. G.
Published: March 2008

Summary. This study examines the effect of pretreatment in liquid culture of acute myeloid leukaemic (AML) progenitors with recombinant human IL‐3 or G and GM‐CSF. Prior to and following cytokine priming, the sensitivity of cells to cytosine arabinoside (Ara‐C) at concentrations ranging from 10−12 to 1(T−4 M was assessed in clonogenic culture. In addition, the initial percentage of AML cells in S phase was assessed and their subsequent kinetic response to cytokine treatment evaluated by FACScan analysis. Light‐density marrow cells (LDMCs) from 19 AML patients were initially T‐cell and monocyte depleted in order to remove potential sources of endogenous cytokine production prior to in vitro investigation. LDMCs were incubated in liquid phase for 7 d in a chemically defined complete medium with or without cytokines.

Clonogenic data from fresh AML LDMCs not pretreated with growth factors demonstrated a heterogenous response to Ara‐C. In only 4/15 marrows tested clonogenically was there any improvement in sensitivity to Ara‐C following cytokine priming. S‐phase data on all 19 marrows were similarly variable either before or after cytokine preincubation. There was no discernible correlation between clonogenic and kinetic data, nor could any relationship be established between in vitro findings and the FAB subtypes of patients or clinical outcomes.

In summary, it would appear that the cell‐cycle status of AML cells is likely to be only one of many contributory factors governing the sensitivity of AML progenitors to Ara‐C. The clinical response of AML patients to cytokine therapy in association with cell‐cycle‐specific cytotoxic agents may therefore be variable and unpredictable.
Minimally differentiated erythroleukaemia (AML M6 Variant'): a rare subset of AML distinct from AML M6
Authors: GARAND RICHARD , DUCHAYNE ELIANE , BLANCHARD DOMINIQUE , ROBILLARD NELLY , KUHIEIN EMILIENNE , FENNETEAU ODILE , SALOMON‐NGUYEN FLORENCE , GRANGE MARIE JOSE , ROUSSELOT PHILIPPE , DEMUR CECILS , TALMANT PASCALINE , RADFORD ISABELLE , FLANDRIN GEORGES ,   the Groupe Francais d'Hematologie Cellulaire
Published: March 2008

Summary.We describe eight cases of erythroleukaemia distinct from FAB‐AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblasts component. Three infants (including a Down's syndrome) and two adults presented with ade novoleukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast‐like morphology. The four others were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 din vitroculture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast‐like morphology or an undifferentiated aspect and a HLA DR−, CD36++, B−, T−, My−(CD33±) phenotype. We propose the term AML M6 ‘variant’ for this rare type of AML.
Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glul02Lys) in the second EGF‐like domain
Authors: MARCHETTI G. , CASTAMAN G. , PINOTTI M. , LUNGHI B. , DI IASIO M. G. , RUGGIERI M. , RODEGHIERO F. , BERNARDI F.
Published: March 2008

Summary. The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glul02Lys mutation affects a residue of the second EGF‐like module also conserved in protein C. Both mutated residues are surface‐exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.