bluuebunny/crossref_metadata_2025 huggingface dataset:
BCR‐ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation
Authors: Ten Bosch George J. A. , Kessler Jan H. , Blom Johanna , Joosten Antonia M. , Gambacorti‐Passerini Carlo , Melief Cornelis J. M. , Leeksma Onno C.
Published: March 2003

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR‐ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR‐ABL mRNA and with maximal platelet counts ranging from 149 to 3069 × 109/l. P2l0BCR‐ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph‐positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR‐ABL‐negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p2l0BCR‐ABL‐positive platelets lysates with anti‐CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR‐ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR‐ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR‐ABL‐mediated CrkL tyrosine phosphorylation.
A domain mutations in 65 haemophilia A families and molecular modelling of dysfunctional factor VIII proteins
Authors: Liu MiaoLiang , Murphy Michael E. P. , Thompson Arthur R.
Published: March 2003

A variety of mutations are found in haemophilia A families. Those with circulating, dysfunctional protein can provide insights into structural determinants of factor VIII function. A molecular model based upon the crystal structure of the homologous A domains in caeruloplasmin enables predictions of molecular consequences of mutations. To identify haemophilic mutations in coding regions for three A domains of factor VIII and predict amino acid substitutions important for coagulant cofactor function, amplified DNA fragments from 188 unrelated haemophilia A families were screened for heteroduplex formation. Exons 1–19 were examined. 65 families were positive for 58 distinct mutations (39 novel) on DNA sequencing. 12 were non‐missense mutations. 38 missense mutations were found in patients that circulate or potentially circulate dysfunctional factor VIII protein and are in an A domain molecular model. Of these 38, 12 have identical residues among all known species of factors V, VIII and caeruloplasmin. These 38 mutations have been localized onto a factor VIII A domain molecular model. Of these, 19 are in coiled, 15 in β‐pleated sheet, and two each in turns and α‐helical structures. 15 substituted residues are on the surface, nine are partially on the surface and 14 are buried within the model structure. Mutant side‐chain substitutions were inserted to predict changes in surface groups or, for buried residues, potential surface areas whose structure is probably disrupted by the substitution.
Down‐regulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin‐stimulated human platelets
Authors: Kawano Hironori , Suzuki Hidenori , Tanoue Kenjiro , Kimura Akiro , Fujimura Kingo
Published: March 2003

We have studied down‐regulation and redistribution of glycoprotein V (GPV) and its fragment GPVf2, a subunit of a receptor for von Willebrand factor (VWF), on the surface membrane of thrombin and thrombin receptor activating peptide (TRAP) stimulated platelets by using a newly developed GPVf2‐specific monoclonal antibody (1D9). Immunoelectronmicroscopical studies revealed that about 50% each of total GPV and GPIX were expressed on the surface membrane of the resting human platelets, and about 83% of GPlbα was expressed on the surface. In thrombin‐stimulated platelets, the surface GPIbα, GPIX and GPV, after hydrolysis by thrombin, was converted to GPVf2, translocated from the surface to the intraplatelet pool and then again redistributed to the surface. In TRAP‐stimulated platelets, GPIbα, GPIX and GPV, without conversion to GPVf2, were translocated from the surface to the intraplatelet pool and then returned to the surface. Ristocetin‐induced agglutinations of both the thrombin‐ and TRAP‐stimulated platelets were lowered during the decreased surface expressions of GPIbα, GPIX and GPV/GPVf2 and then normalized when these GPs were again redistributed onto the surface, indicating that the redistributed GPIb/IX/Vf2 complex on the surface can act as a VWF receptor as efficiently as an intact GPIb/IX/V.
Examination of a rheological profile for blood using micropore filters
Authors: Jones J. , Adams Rachel , Cook Alison , Evans Shelley‐Ann
Published: March 2003

Various techniques have been used to assess the flow properties of blood and blood cells in a range of clinical situations. Filtration through microfilters offers a single technique for measuring the flow properties of all cellular components of blood in one experiment but depends on an assumed ability to recognize cells that make up <10% of leucocytes. The remaining leucocytes, labelled fast leucocytes, were previously presumed to be lymphocytes and granulocytes. This study confirmed the identities of these fast leucocytes as those of lymphocytes and granulocytes in undiluted blood. The transit time for lymphocytes (1.2 s) and granulocytes (1.6 s) is close to that recorded for fast leucocytes (1.7 s). The resistance of each type of blood cell to flow through 5 μm filters was defined in this study as the product of the concentration of that cell in blood and its transit time through a pore in the filter. The total resistance to flow of healthy blood through the filter is 4.46 × 107 s/ml and is attributed to plasma (2.7%), red cells (25.9%), fast leucocytes (25.3%) and slow leucocytes (46.1%). In a cohort of 21 men with peripheral arterial disease the total resistance was increased to 7.82 × 107 s/ml and attributed to plasma (1.5%), red cells (14.5%), fast leucocytes (21.0%) and slow leucocytes (63.0%). This analysis therefore provides a single test for assessing the flow properties of all the cellular components of blood and plasma.
Mouse peritoneal cells as a reservoir of late dendritic cell progenitors
Authors: Rezzani Rita , Rodella Luigi , Zauli Giorgio , Caimi Luigi , Vitale Marco
Published: March 2003

Despite the key role played by dendritic cells (DCs) in the physiology of immunity and related diseases, their differentiation pathway has not yet been fully elucidated. In this study we demonstrated that cells obtained from mouse peritoneal cavity lavage can be induced to differentiate in vitro along the dendritic lineage by the addition of optimal concentrations of murine recombinant GM‐CSF (50 U/ml) for 6 d. At morphological analysis, GM‐CSF‐treated peritoneal cells appeared loosely adherent to plastic and showed cytoplasmic protrusions and veils typical of DCs. A de novo expression of the DC phenotypic markers MIDC8, DEC205, CD11c and relB with up‐regulation of surface MHC‐II and complete down‐regulation of non‐specific esterase (NSE) was also observed in peritoneal cells upon GM‐CSF treatment. Functionally, GM‐CSF‐treated peritoneal cells were highly stimulatory in a mixed lymphocyte reaction, showed a reduced phagocytosis of latex particles and enhanced pinocytic activity. Moreover, tumour necrosis factor (TNF)‐α (5–10 ng/ml) was able to synergize with GM‐CSF in the induction of DC differentiation. On the other hand, when peritoneal cells were induced to differentiate into macrophages by treating in vivo the animals with thioglycollate before peritoneal harvesting, they completely lost the ability to acquire in vitro the dendritic phenotype in response to GM‐CSF, either used alone or in combination with TNF‐α. These results were confirmed by limiting dilution experiments which demonstrated the differentiation of peritoneal cells into DCs at the single cell level. Taken together, these data suggest that resting peritoneal cells in the mouse represent an immature population, capable of further differentiation along either the dendritic or the macrophagic pathway, depending on the type of stimuli they receive.
Dermatan sulphate in patients with heparin‐induced thrombocytopenia
Authors: Taliani Maria Rita , Agnelli Giancarlo , Nenci Giuseppe , Gianese Francesco
Published: March 2003

Patients with thromboembolic diseases who develop heparin‐induced thrombocytopenia (HIT) type II require an alternative anticoagulation strategy. Dermatan sulphate (DS) was administered to five patients with thromboembolic diseases who developed HIT type II and showed an in vitro cross‐reactivity with low molecular weight heparins. The platelet count and the extension of thrombosis were monitored during DS administration. In four of the five patients the platelet count rapidly increased after heparin was discontinued and DS started. A low platelet count persisted in the single patient with cross‐reactivity to DS, up to 4 d after its discontinuation. None of the patients experienced thrombus extension, haemorrhagic side‐effects or other adverse events.
Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia
Authors: Pallis M. , Turzanski J. , Harrison G. , Wheatley K. , Langabeer S. , Burnett A. K. , Russell N. H.
Published: March 2003

We have used a combination of flow cytometric assays to define multidrug resistance (MDR) positive and negative blasts in cryopreserved samples from 47 MRC trial patients with acute myeloblastic leukaemia (AML). Our primary test is a standardized assay for daunorubicin accumulation. Confirmatory assays for MDR comprised the cyclosporin modulation assay for rhodamine‐123 uptake as a measure of functional P‐glycoprotein and the measurement of lung resistance protein and multidrug resistance associated protein (with LRP‐56 and MRPr1 respectively).

57% of samples had both low accumulation and at least one positive confirmatory test. 32% were MDR negative in all four assays. 15% of patients had primary chemo‐resistant disease. Resistant disease rates were 22% for confirmed MDR‐positive patients and 0% for confirmed MDR‐negative patients (P = 0.07). Complete remission was achieved in 74% of patients, with rates of 63% in confirmed MDR‐positive patients and 93% in confirmed MDR‐negative patients (P = 0.06). The use of a standardized method for daunorubicin uptake, combined with the use of confirmatory tests, should reduce the uncertainty that is currently characteristic of MDR evaluation in leukaemia. In comparison with daunorubicin uptake, p‐gp expression, measured using MRK‐16 antibody, was more closely associated with remission rates (P = 0.01). This suggests an additional role for p‐glycoprotein in mediating drug resistance beyond that of a drug efflux pump.
Platelet‐bound immunogloblins before and after platelet transfusion: measurement of in vivo binding
Authors: Levin M‐D. , De Vries W. , De Veld J. , Doekharan D. , Van Der Holt B. , Van 'T Veer M. B.
Published: March 2003

In routine tests to investigate immunological mechanisms as a cause for enhanced destruction of transfused platelets, serum from the patient is tested against a panel of donor lymphocytes and platelets to demonstrate the presence of antibodies against HLA or platelet specific antibodies. Here we describe a flow cytometric technique in which in vivo binding of immunoglobulins (Ig) is measured. By comparing the histograms of the platelet suspensions before and after transfusion, four different patterns were obtained: no Ig binding before and after transfusion (pattern 1), pre‐existent Ig binding (patterns 2 and 3) or preferential Ig binding to the transfused platelets (pattern 4). This technique was tested in 164 random platelet transfusions and 34 HLA‐matched platelet transfusions. A statistically significant association was found between pattern 1 and a negative result of the indirect platelet immunofluorescence test for the detection of antibodies in the serum taken before the transfusion studied or a good platelet recovery and between pattern 4 and a positive indirect platelet immunofluorescence test or a low platelet recovery.
Different expression of glutathione S‐transferase α, μ and π in childhood acute lymphoblastic and myeloid leukaemia
Authors: Den Boer M. L. , Pieters R. , Kazemier K. M. , Janka‐Schaub G. E. , Henze G. , Creutzig U. , Kaspers G. J. L. , Kearns P. R. , Hall A. G. , Pearson A. D. J. , Veerman A. J. P.
Published: March 2003

Expression of three major classes of glutathione S‐transferases (GSTs), i.e. α, μ and π class, P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry. In vitro cytotoxicity of 4‐hydroxy‐ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay. Expression of α, μ and π class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTαP=0.26; GSTμP = 0.09; GSTπP = 0.13). The expression of GSTα (1.4‐fold, P = 0.0004), GSTπ (1.3‐fold, P = 0.001) and to a lesser extent also GSTμ (1.1‐fold, P = 0.03) was higher in ALL compared with normal peripheral blood lymphocytes. Expression of GSTμ and GSTπ was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3‐fold, P = 0.0005 and 2‐fold, P < 0.0001). In contrast, GSTα was median 2‐fold lower expressed in the AML samples (P < 0.0001). Expression levels of α, μ and π class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL. One exception was a remarkably low expression of GSTα in IFOS‐sensitive samples compared with a heterogenous expression in IFOS‐resistant samples (P = 0.02). Expression of GSTπ, but not of GSTα or GSTμ, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P‐gp. However, a high expression of both GSTπ and MRP was not associated with in vitro resistance to IFOS, DNR or PRED. The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL. Whether the high expression of GSTμ and GSTπ in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P  0.0001) warrants further study.
The potential of human mast cell progenitors to differentiate into mature mast cells remains after prolonged culture with flt3 ligand, interleukin‐3 or granulocyte‐macrophage colony stimulating factor
Authors: HJERTSON MALIN , SUNDSTRÖM CHRISTER , NILSSON KENNETH , NILSSON GUNNAR
Published: March 2003

Stem cell factor (SCF) plays a key role in the development of mast cells from haemopoietic progenitor cells. In this study we have investigated the effect of the early acting haemopoietic cytokines flt3 ligand (FL), IL‐3 and GM‐CSF on the SCF‐dependent differentiation of mast cells from cord blood mononuclear cells. By using delayed addition of SCF, we examined the potential of mast cell progenitors to keep their capacity to differentiate into mast cells after exposure to factors signalling differentiation into other lineages. Culture with either cytokine for 3 weeks before transfer to SCF‐containing medium resulted in the development of mast cells in all cultures. The appearance of mast cells was attenuated when the cells had been in culture with IL‐3 or GM‐CSF prior to culture in SCF, compared to cultures exposed to SCF alone for 7 weeks. However, a proportion of the cells had not lost the capacity to develop into mast cells. In contrast, in cultures initiated with FL and transferred to medium containing SCF, the same amount of mast cells developed as in the SCF cultures. Thus, cells committed to the mast cell lineage appear to be resistant to the lineage directives of IL‐3 and GM‐CSF and keep their potential to differentiate into mature mast cells.
Treatment of advanced myelodysplastic syndrome with a regimen including recombinant human granulocyte colony‐stimulating factor preceding allogeneic bone marrow transplantation
Authors: OKAMOTO SHINICHIRO , TAKAHASHI SATOSHI , WAKUI MASATOSHI , ISHIDA AKARU , TANOSAKI RYUJI , IKEDA YASUO , ASANO SHIGETAKA
Published: March 2003

We treated 13 patients with morphologically advanced myelodysplastic syndrome using cytosine arabinoside and total body irradiation, followed by allogeneic marrow transplantation from HLA‐identical sibling donors. Granulocyte colony‐stimulating factor (G‐CSF) was added to the preparative regimen to selectively increase chemosensitivity of leukaemic cells and to improve transplant outcome. No regimen‐related deaths occurred, and no side‐effects related to the addition of G‐CSF were observed except for transient mild bone pain. At a median follow‐up time of 39 months the projected 5‐year disease‐free survival and 5‐year overall survival were 67.7% and 75.5%, respectively, with only one case showing cytogenetic relapse. The preparative regimen including G‐CSF is feasible, and preliminary results seem to be encouraging. However, a larger trial is clearly warranted to evaluate its efficacy.
An international collaborative study on the INR calibration of freeze‐dried reference plasmas
Authors: HUBBARD ANTHONY R. , MARGETTS SARAH M. L. , WELLER LYNNE J. , MACNAB JO , BARROWCLIFFE TREVOR W.
Published: March 2003

A study was carried out to calibrate potential European Reference Plasmas for prothrombin time (PT) standardization. The International Normalized Ratio (INR) values of three freeze‐dried candidate plasmas (one pooled normal and two pools from anticoagulated patients) were determined in 20 laboratories using six thromboplastin reagents comprising three International Reference Thromboplastins (human, rabbit and bovine), two recombinant human reagents and one placental human reagent. Inter‐laboratory variability of INR estimation was low with geometric coefficients of variation (gcv) <10% except in one case. Significant differences in mean INR were found between the different thromboplastins with lowest INR values found with the bovine reagent. INR values from the International rabbit and human reagents differed by <6% and were combined to give proposed assigned INR values. Significant differences in INR estimates from four thromboplastins of human origin may indicate that single assigned INR values are not applicable for use with all thromboplastin reagents. Field trials to assess the validity of single assigned INR values in clinical practice are required.
Haematological parameters of parvovirus B19 infection in 13 fetuses with hydrops foetalis
Authors: Forestier François , Tissot Jean‐Daniel , Vial Yvan , Daffos Fernand , Hohlfeld Patrick
Published: March 2003

Thirteen cases of fetal parvovirus B19 infection with hydrops foetalis are reported. Viral DNA was identified by polymerase chain reaction (PCR) of amniotic fluid sampled between the 19th and the 29th week of gestation. Haematological examination revealed severe anaemia in all cases and thrombocytopenia in 11/13 cases, which was severe in two cases. Six fetuses died in utero; two after intrauterine transfusion. Complete recovery was observed in seven fetuses; five cases were treated by intrauterine transfusions, and in two cases spontaneous recovery occurred. Upon follow‐up, no case of congenital anaemia was observed.
Functional lymphocyte immunophenotypes observed in thalassaemia and haemophilia patients receiving current blood product preparations
Authors: Hodge G. , Lloyd J. V. , Hodge S. , Story C. , Han P.
Published: March 2003

Immune abnormalities have been reported in recipients of cellular and plasma blood products. To document the effect of current transfusion practices, we performed ex vivo lymphocyte immunophenotypic studies on patients with thalassaemia major who had received multiple (leucocyte‐depleted) transfusions and patients with haemophilia A and B who had received heat viral‐inactivated factor concentrates. Patients with thalassaemia major showed a significant lymphocytosis, with mainly B‐cell changes consistent with ongoing B‐cell stimulation associated with chronic exposure to red cell antigens. Reduced T‐cell IL‐2Rα expression would be consistent with inhibition by desferrioxamine chelation therapy. In contrast, patients with haemophilia showed predominantly T‐cell changes. Patients with haemophilia A showed significantly elevated activated CD8+ cytotoxic T lymphocytes whereas those with haemophilia B showed an increase in CD8+CD11adim and CD4+CD45RA+ suppressor T cells. Several of the immune abnormalities found may be due to the presence of cytokines not removed by leucocyte filtration or destroyed by factor concentrate production (e.g. TGF‐β) causing a T‐helper‐2‐like response. The extensive lymphocyte characterization in this study has not previously been performed and has enabled a closer examination of the functional lymphocyte immunophenotypes seen in patients treated according to current transfusion practices.
Chromosome aberrations are restricted to the CD56+, CD3− tumour cell population in natural killer cell lymphomas: a combined immunophenotyping and FISH study
Authors: Zhang Yanming , Wong Kit Fai , Siebert Reiner , Matthiesen Peter , Harder Svetlana , Eimermacher Hartmut , Feller Alfred C. , Schlegelberger Brigitte
Published: March 2003

Natural killer (NK) cell lymphomas are a newly recognized entity of non‐Hodgkin's lymphoma with a highly aggressive clinical course and strong association with Epstein‐Barr virus (EBV) infection. Although no recurrent chromosome aberrations have been identified in NK‐cell lymphoma, deletions of 6q and trisomy 7 have been described repeatedly in this type of lymphoma. In this study we attempted to determine the immunophenotypes of tumour cells with certain chromosome aberrations, i.e. deletions of 6q and trisomy 7, in three cases of NK cell lymphomas by means of combined immunophenotyping and fluorescence in situ hybridization (FISH). In all three cases clonal chromosome aberrations were detected only in CD56+ cells but not in CD3+ or CD5+ cells. However, not all CD56+ cells were shown to contain these chromosome aberrations. Double immunophenotyping combined with FISH confirmed that the chromosome aberrations occurred only in CD56+CD3− cells. This study indicates that chromosome aberrations in NK‐cell lymphomas are restricted to the CD56+, CD3− and CD5− cell population and that NK‐cell lymphomas are indeed derived from mature true NK cells and not from T lymphocytes.
Transport of 14C‐deferiprone in normal, thalassaemic and sickle red blood cells
Authors: Shalev Oded , Hileti Dona , Nortey Philip , Hebbel Robert P. , Hoffbrand Victor A.
Published: March 2003

The transport of deferiprone (L1) in normal (N), sickle (S) and thalassaemic (T) red blood cells (RBC) was determined by incubation with 14C‐L1 at 37°C. Following incubation with 0.5 mm14C‐L1 for 4 h, the intracellular concentration of L1 in T RBC was 3 times higher than was found extracellularly. In contrast, no concentration gradient across N and S RBC membranes was detected. Efflux studies showed that T RBC released only 17 ± 2% of 14C‐L1 into the extracellular space. We hypothesize that L1 accumulation in T RBC results from their high content of chelatable iron and formation of large, hydrophilic L1–Fe(III) complexes trapped within the cytosol.
c‐kit proto‐oncogene exon 8 in‐frame deletion plus insertion mutations in acute myeloid leukaemia
Authors: Gari Mamdooh , Goodeve Anne , Wilson Gill , Winship Peter , Langabeer Stephen , Linch David , Vandenberghe Elisabeth , Peake Ian , Reilly John
Published: March 2003

Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c‐kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in‐frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA → ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in‐frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in‐frame deletion plus insertion mutations (n = 7) involved the loss or repacement of the codon for Asp419 which is highly conserved cross species and is located in the receptor's extracellular domain. The high frequency of the c‐kit proto‐oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptor's extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.
Infection with TT virus, a novel transfusion‐transmissible DNA virus, in haemophiliacs and in blood products
Authors: Yokozaki Shoichi , Toyoda Hidenori , Nakano Isao , Katano Yoshiaki , Ebata Mieko , Fukuda Yoshihide , Takamatsu Junki , Saito Hidehiko , Hayakawa Tetsuo
Published: March 2003

We evaluated the characteristics and rate of infection with TT virus (TTV), a novel DNA virus, in Japanese haemophiliacs. TTV DNA was measured in 60 haemophiliacs by semi‐nested polymerase chain reaction. Co‐infection with hepatitis C virus (HCV), hepatitis G virus (HGV) and human immunodeficiency virus (HIV) was also evaluated. In addition, the rate of detection of TTV DNA in blood products was evaluated. TTV DNA was detected in 35/60 haemophiliacs (58.3%). There were no differences in the backgrounds or characteristics between haemophiliacs with and without TTV infection, except for higher levels of IgG and IgM in patients with TTV infection. In patients infected with TTV of types other than type 1, which are rarely detected in Japan, the rate of co‐infection with HCV of imported types was high; TTV of types other than type 1 in Japanese haemophiliacs were probably transmitted by imported blood products. TTV DNA was detected in over half of the blood products tested, but TTV DNA concentrations in these products were lower than in the serum of haemophiliacs.
Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1‐loop fragments
Authors: Miura Shuji , Sakurai Yoshihiko , Takatsuka Hideo , Yoshioka Akira , Matsumoto Masanori , Yagi Hideo , Kokubo Tetsuro , Ikeda Yasuo , Matsui Taei , Titani Koiti , Fujimura Yoshihiro
Published: March 2003

Under high shear stress, the binding of von Willebrand factor (VWF) A1‐loop domain to platelet glycoprotein (GP) Ibα occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1‐loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1‐loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1–1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1–708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449–728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481–718 of the subunit] and His‐rVWF465–728 (a monomer of aa residues 465–728 of the subunit)], and assessed their potency as inhibitors of botrocetin‐induced VWF binding to GPIbα and high shear stress induced platelet aggregation mediated by intact VWF. All these fragments completely inhibited botrocetin‐induced VWF binding to GPIbα at a final concentration of 40–200 μm. The homodimeric A1‐loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45–2.0 μm in the following order: 315 kD Fr  220 kD Fr ≫ 116 kD Fr. In contrast, the monomeric A1‐loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 μm, and their IC50s were 13–39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1‐loop fragment is important for optimal molecular interaction with GPIbα under high shear stress.
Dynamic cell cycle kinetics of normal CD34+ cells and CD38+/− subsets of haemopoietic progenitor cells in G‐CSF‐mobilized peripheral blood
Authors: Holm Mette , Thomsen Mette , HØyer Morten , Hokland Peter
Published: March 2003

Using a recently developed flow cytometric assay for the simultaneous measurement of cell surface antigens, DNA content and bromodeoxyuridine incorporation, we have for the first time determined the labelling index (LI), the duration of the S‐phase (Ts) and the potential doubling time (Tpot) of purified CD34+ cells mobilized by G‐CSF from 10 normal donors. Although CD34+ cells were not actively cycling immediately following purification, up to 5% could nevertheless traverse cell cycle without exogenous stimulation during the first 24 h of culture. In addition, it was possible to induce CD34+ cells to enter cycling by stimulation with haemopoietic growth factors (IL‐3, IL‐6 and SCF), resulting in median Tpot values of 18.2 d at 21 h, 7.7 d at 29 h, and 4.5 d at 37 h. Importantly, stimulation of CD34+ cells was seen almost exclusively within the CD38+ subset (mean Tpot value 2.8 d), whereas CD38− cells were not recruited into cycle (mean Tpot value 35.9 d). In conclusion, although cell cycle entry and progression can easily be induced in differentiated CD34+/CD38+ cells, immature CD34+/CD38− cells will remain dormant in most of the clinical and laboratory stimulation protocols hitherto employed. This assay can be used to obtain detailed cell cycle kinetics in leucocyte subsets in health and disease.
Chronic lymphocytic leukaemia and neuroendocrine cancer
Authors: Russo F. , Guadagni S. , Corazzelli G. , Frigeri F. , Monda V. M. , Abate G.
Published: March 2003

We report a unique association between neuroendocrine cancer and chronic lymphocytic leukaemia (CLL) in a 63‐year‐old man. Neuroendocrine cancer was resistant to various conventional treatments and following loco‐regional progression we treated the patient with hypoxic pelvic perfusion of cisplatin 100 mg/m2 plus mitomycin 40 mg/m2, using the stopflow method, for three cycles: a dramatic and surprising reduction of > 75% in the evaluable lesions was observed. The cumulative effect of treatment produced a complete response from CLL.

At cytogenetic examination the neuroendocrine cells were diploid, whereas CLL cells showed trisomy 12. Moreover, deletion of the short arm of chromosome 3 was found in both neoplastic populations. Whether the abnormality seen on chromosome 3 in the two diseases represents a critical event is not known.
Dexamethasone does not counteract the response of acute promyelocytic leukaemia cells to all‐trans retinoic acid
Authors: De Ridder Marleen C. , Van Der Plas Arnout J. , Erpelinck‐Verschueren Claudia A. J. , Löwenberg Bob , Jansen Joop H.
Published: March 2003

Retinoic acid syndrome is a serious condition that may complicate the treatment of acute promyelocytic leukaemia patients. This syndrome may be treated effectively with high‐dose corticosteroid therapy and, as a result, many patients with acute promyelocytic leukaemia receive dexamethasone at some point during treatment. We investigated whether dexamethasone would also antagonize the beneficial effects of retinoic acid. In t(15;17)‐positive NB4 cells, dexamethasone did not affect the retinoic acid induced differentiation, normalization of PML‐nuclear bodies or the induction of thrombomodulin mRNA. Finally, dexamethasone did not inhibit the anti‐proliferative effect of retinoic acid but rather showed anti‐proliferative activity itself.
Neurolymphomatosis in Waldenström's macroglobulinaemia
Authors: Abad Sébastien , Zagdanski Anne‐Marie , Brechignac Sabine , Thioliere Brigitte , Brouet Jean‐Claude , Mariette Xavier
Published: March 2003

We report four patients with Waldenström's macroglobulinaemia with an unusual neurologic complication, neurolymphomatosis, characterized by meningeal and root nerve infiltration by lymphoplasmacytic cells. Patients presented with rapidly progressive leg proximal weakness. Examination of cerebrospinal fluid disclosed lymphoplasmacytic cells. Magnetic resonance imaging of the lumbar spine was suggestive of a tissular infiltration of leptomeninges and nerve roots. Chemotherapy and irradiation of involved tissues led to a remarkable and sustained neurological improvement.
Pseudohomozygosity for activated protein C resistance is a risk factor for venous thrombosis
Authors: Castaman Giancarlo , Tosetto Alberto , Ruggeri Marco , Rodeghiero Francesco
Published: March 2003

Pseudohomozygosity for activated protein C resistance (APC‐r) is a rare condition due to the association of heterozygous FV Leiden mutation and partial type I FV deficiency. To assess the risk of venous thromboembolism in these subjects, seven families including 11 pseudohomozygotes and 45 relatives were examined. Among the relatives, 16 were heterozygous FV Leiden carriers, nine showed partial FV deficiency and 20 no abnormalities. Deep vein thrombosis occurred in 4/11 (36.3%) pseudohomozygous patients versus 6/16 (37.4%) FV Leiden carriers and 1/20 (5%) normal relatives. Pseudohomozygotes and FV Leiden carriers had a significantly increased risk of venous thrombosis in comparison to normal relatives (RR 8.8 and 5.7, respectively). There was no difference between the thrombotic risk of pseudohomozygous subjects and of FV Leiden carriers (RR 1.6, 95% CI 0.43–5.7). Furthermore, there was no difference in thrombosis‐free survival between pseudohomozygotes and 45 consecutive FV Leiden heterozygous outpatients, suggesting that a referral bias may explain the apparent younger age of thrombosis in the pseudohomozygotes in comparison to relatives with FV Leiden heterozygosity (27 years v 54 years; P = 0.01). Pseudohomozygosity for APC resistance carries a significantly higher risk for venous thromboembolism in comparison to normal subjects, but probably not in comparison to heterozygous FV Leiden carriers.
Prognosis for fludarabine therapy of chronic lymphocytic leukaemia based on ex vivo drug response by DiSC assay
Authors: Bosanquet Andrew G. , Johnson Stephen A. , Richards Sue M.
Published: March 2003

The cytotoxic antimetabolite fludarabine is a widely used active agent in chronic lymphocytic leukaemia (CLL). However, cost and occasional adverse side‐effects necessitate careful use. Identifying before treatment patients not likely to benefit from fludarabine could advance disease management both clinically and financially.

We used the DiSC (differential staining cytotoxicity) assay, an ex vivo apoptotic drug response test, to identify the sensitivity or resistance to fludarabine of lymphocytes from B‐cell CLL patients and compared the results with subsequent patient treatment, response and survival. Patients were grouped thus: those receiving fludarabine within 1 year of assay (± other cytotoxic drugs), and those receiving other chemotherapy (excluding fludarabine) within 1 year of assay.

Fludarabine‐test‐resistance was found in 12/100 (12%) of untreated patients and 45/143 (31%) of previously treated patients (17/32 (53%) of patients previously treated with fludarabine). Treating fludarabine‐test‐resistant patients with fludarabine resulted in poor response compared with fludarabine‐test‐sensitive patients (7% v 69%) and short survival (median 7.9 v 41.7 months; relative risk (RR) = 14.8; P < 0.0001). 81% of fludarabine‐test‐resistant patients were test sensitive to other regimens. If treated with chemotherapy other than fludarabine, test‐resistant patients responded better and survived substantially longer than those treated with fludarabine (RR = 2.9; P = 0.001).

Not all CLL patients should receive fludarabine. Fludarabine‐test‐resistance by DiSC assay is a powerful independent prognostic factor. Pretreatment DiSC assay results could enable the toxic, clinical and financial costs of fludarabine treatment to be avoided in fludarabine‐test‐resistant patients. Disease management, response, survival and use of financial resources might be significantly improved if therapy choice in CLL patients was guided by DiSC assay.
Flt3L induces the ex‐vivo amplification of umbilical cord blood committed progenitors and early stem cells in short‐term cultures
Authors: De Felice Lidia , Di Pucchio Tiziana , Grazia Mascolo Maria , Agostini Francesca , Breccia Massimo , Guglielmi Cesare , Rosaria Ricciardi Maria , Tafuri Agostino , Screnci Maria , Mandelli Franco , Arcese William
Published: March 2003

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use.

Eight‐day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10‐fold amplification of CD34+ cells and the expansion of multipotent (CFU‐GEMM) and committed (CFU‐GM, BFU‐E) progenitors respectively of 5‐, 7‐ and 9‐fold over input cells. As to the early stem cell pool, the primitive CD34+Thy‐1+ cell fraction increased 6‐fold and the LTC‐IC were amplified 17‐fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long‐term reconstitution after transplant.
A monoclonal antibody, 3G12, reacts with a novel surface molecule, Hal‐1, with high expression in CD30‐positive anaplastic large cell lymphomas
Authors: Asanuma Hiroko , Takahashi Shuji , Ishikawa Masako , Kamiguchi Kenjiro , Sato Noriyuki , Poppema Sibrand , Fujimoto Jun‐ichiro , Kikuchi Kokichi
Published: March 2003

We established a monoclonal antibody, 3G12 (IgG1), with antiproliferative effects on a human T‐cell leukaemia cell line, SUP‐T13. Among haematolymphoid cell lines, 3G12 reacted with most T‐cell lines, Epstein‐Barr transformed B‐cell lines, some myelomonocytic cell lines and, most strongly with an anaplastic large cell lymphoma (ALCL) cell line, Karpas 299. The cell panel reactive with 3G12 was similar, but not identical, to that of the anti‐CD30 antibody Ber‐H2. 3G12 induced Fas‐independent apoptosis in SUP‐T13 and it also induced growth‐inhibition in a limited number of other cell lines, but not Karpas 299. Immunohistochemical studies on paraffin‐embedded tissue specimens demonstrated that 3G12 reacted with most CD30‐positive ALCL cases and some T‐cell lymphomas and some Hodgkin's lymphomas, but not with B‐cell lymphomas or non‐haematogeneic tumours. The immunoprecipitation study with 3G12 demonstrated a major band of 200 kD and a minor band of 100 kD, which were different from CD30. Thus 3G12 defines a novel antigen that shares a similarity to CD30 in terms of distribution among haemopoietic cells. The data suggest that the 3G12‐defined antigen, designated Hal‐1, is important as a marker for ALCL and may play a role in its pathogenesis.
Two‐colour FISH detection of the inv(16) in interphase nuclei of patients with acute myeloid leukaemia
Authors: Dauwerse Hans G. , Smit Elizabeth M. E. , Giles Rachel H. , Slater Rosalyn , Breuning Martijn H. , Hagemeijer Anne , Van Der Reijden Bert A.
Published: March 2003

The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two‐colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p‐arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p‐arm breakpoint which is present in 20% of all cases.
Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high‐dose therapy and peripheral blood stem cell transplantation
Authors: Rottenburger Christof , Kiel Katja , Bösing Tobias , Cremer Friedrich W. , Moldenhauer Gerhard , Ho Anthony D. , Goldschmidt Hartmut , Moos Marion
Published: March 2003

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high‐dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse.

The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele‐specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0–7.2 tumour cells/ml PB) higher than in the CD20− fractions (median 0; range 0–29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8–585; median 32 tumour cells/ml PB; CD20−: range 25–25527; median 334 tumour cells/ml PB). Enrichment with the anti‐CD19 antibody as a second pan B‐cell marker revealed comparable tumour cell numbers.

In conclusion, an anti‐CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B‐cell compartment and an almost complete absence of tumour cells in the CD20− fractions.
Haemochromatosis mutations in North‐East Scotland
Authors: Miedzybrodzka Z. , Loughlin S. , Baty D. , Terron A. , Kelly K. , Dean J. , Greaves M. , Pippard M. , Haites N.
Published: March 2003

The HFE gene and its mutations C282Y and H63D cause hereditary haemochromatosis (HH). Among 54 affected individuals from North‐East Scotland, 91% were homozygous for C282Y and 5.5% were compound heterozygotes for C282Y and H63D. The general population allele frequencies were high (8% and 15.7% for C282Y and H63D respectively). Although it is likely that HH is under diagnosed, these figures suggest that disease expression is variable, and many of those with the genetic predisposition HH will never develop the clinical consequences of iron overload. This has implications for diagnosis and predictive testing.
Detection of a Hodgkin/Reed‐Sternberg cell specific immunoglobulin gene rearrangement in the serum DNA of a patient with Hodgkin's disease
Authors: Kornacker M. , Jox A. , Vockerodt M. , Tesch H. , Bohlen H. , Diehl V. , Wolf J.
Published: March 2003

We analysed multiple serum samples from a patient with mixed cellularity Hodgkin's disease for the Hodgkin/Reed‐Sternberg cell clone‐specific rearranged Ig gene sequence. The clone‐specific sequence could be detected in DNA extracted from a serum sample obtained during clinical relapse but not in serum samples obtained during or after treatment following relapse.
Response to cladribine in previously treated patients with chronic lymphocytic leukaemia identified by ex vivo assessment of drug sensitivity by DiSC assay
Authors: Bosanquet Andrew G. , Copplestone J. Adrian , Johnson Stephen A. N. , Smith Alastair G. , Povey Sara J. , Orchard Jennifer A. , Oscier David G.
Published: March 2003

The ability to identify non‐responders to cytotoxic chemotherapy has significant clinical and economic benefits. Differential staining cytotoxicity (DiSC) assays were performed in 34 previously treated patients with chronic lymphocytic leukaemia prior to treatment with cladribine. Of the 28 identified as ex vivo sensitive, 26 achieved a complete (CR) or partial response (PR) (median length of response 1.5 years, median survival 3.37 years) and two had a >70% fall in lymphocytes: six identified as ex vivo resistant failed to respond. The DiSC assay can accurately identify a subgroup of patients resistant to cladribine.
Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis
Authors: Cazzola Mario , Beguin Yves , Bergamaschi Gaetano , Guarnone Roberta , Cerani Paola , Barella Susanna , Cao Antonio , Galanello Renzo
Published: March 2003

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X‐linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601 μg/l, range 105–2855 μg/l). Multiple regression analysis showed that 62% (P < 0.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.
Phase III efficacy study of interleukin‐3 after autologous bone marrow transplantation in patients with malignant lymphoma
Authors: Brouwer Rolf E. , Vellenga Edo , Zwinderman Koos H. , Bezwoda W. R. , Durrant Simon T. S. , Herrmann Richard P. , Kiese Beate , Maraninchi Dominique , Milligan Donald W. , Sklenar Ivo , Tabilio A. , Volonte José‐Luis Pico , Winfield David A. , Fibbe Willem E.
Published: March 2003

We evaluated the efficacy of recombinant human interleukin‐3 (rhIL‐3) in reducing the number of platelet transfusions and major infections after autologous bone marrow transplantation (ABMT) in patients with malignant lymphoma. 198 patients with non‐Hodgkin's lymphoma (NHL, n = 111) and Hodgkin's disease (HD, n = 87) were randomized to receive rhIL‐3 10 μg/kg/d (n = 130) or placebo (n = 68) for a maximum of 28 d after ABMT. Several well‐known conditioning regimens were used. From day 1 after ABMT patients were treated with placebo or rhIL‐3 at a dose of 10 μg/kg/d by continuous i.v. infusion for 7 d and then by s.c. administration for 21 d or until platelet (50 × 109/l) and neutrophil (0.5 × 109/l) recovery had occurred. Treatment was completed in 54% of the patients in the rhIL‐3 group versus 75% in the placebo group (P < 0.004). Adverse events were the main reason for premature discontinuation in the IL‐3 group (23% IL‐3 v 5% placebo). The median number of platelet transfusions was not significantly different between the IL‐3 group and the placebo group (8.0 IL‐3 v 6.0 placebo, P = 0.09). Platelet engraftment ( 20 × 109/l) was not significantly faster in the IL‐3 group (28 d in the IL‐3 and 27 d in the placebo group, P = 0.06) and the incidence of haemorrhagic complications was similar in both groups. In patients receiving the full intended dose of rhIL‐3, platelet engraftment to  20 × 109/l was delayed (P = 0.007). The median time to neutrophil engraftment was 23 d in the IL‐3 and 25 d for the placebo group (P = 0.39). There was no difference in the incidence of major infections. We conclude that treatment with IL‐3 has no clinical benefit in patients receiving ABMT for malignant lymphoma.