bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron status in pregnant women: which measurements are valid?
Authors: Van Den Broek  , Letsky  , White  , Shenkin
Published: March 2003

Anaemia in pregnancy in developing countries continues to be a public health problem of significant proportion. At least 50% of the anaemia has been blamed on iron deficiency. In populations where chronic inflammation and iron deficiency anaemia coexist, the criteria to accurately define iron status are not always clear. Similarly, in pregnancy, with marked physiological changes, cut‐off points for biochemical parameters need to be re‐examined. In this study we examined the diagnostic accuracy of iron parameters including mean cellular volume (MCV), serum iron, transferrin, total iron binding capacity (TIBC) and its saturation, zinc protoporphyrin (ZPP), ferritin and serum transferrin receptor (TfR) for the assessment of iron status in a population of anaemic pregnant women in Malawi. Stained bone marrow aspirates were used as the standard for comparison.

Results show that for the purpose of screening, serum ferritin is the best single indicator of storage iron provided a cut‐off point of 30 μg/l is used. A number of other commonly used parameters of iron status were shown to have limited diagnostic accuracy. Logistic regression was used to obtain mathematical models for the prediction of bone marrow iron status using a combination of available parameters.
Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes
Authors: Tamura  , Ogata  , Luo  , Nakamura  , Yokose  , Dan  , Tohyama  , Yoshida  , Hamaguchi  , Sakamaki  , Kuwaki  , Tahara  , Kato  , Nomura
Published: March 2003

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO‐R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I‐TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects (P < 0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB‐t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB‐t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB‐t. The number of TPO‐R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB‐t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB‐t. Thus, in addition to thrombocytopenia, a reduced platelet TPO‐R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO‐R and marrow megakaryocytes, such as blasts expressing TPO‐R, may operate in RAEB/RAEB‐t.
The P2Y1 receptor, necessary but not sufficient to support full ADP‐induced platelet aggregation, is not the target of the drug clopidogrel
Authors: Hechler Béatrice , Eckly Anita , Ohlmann Philippe , Cazenave Jean‐Pierre , Gachet Christian
Published: March 2003

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP‐induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP‐induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP‐induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP‐induced shape change and aggregation in platelets from both control and clopidogrel‐treated rats. A2P5P and A3P5P (100 μM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 μM) in control and clopidogrel‐treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM). A3P5P (1 m
M) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m
M). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.
The haemochromatosis mutations do not modify the clinical picture of thalassaemia major in patients regularly transfused and chelated
Authors: Borgna‐Pignatti Caterina , Solinas Agostina , Bombieri Cristina , Micciolo Rocco , Gamberini Maria Rita , De Stefano Piero , De Menis Ernesto , Pignatti Pier Franco
Published: March 2003

Iron overload is the main cause of morbidity and mortality in patients with thalassaemia major. In order to establish if the presence of the mutations recently described in the haemochromatosis gene affects the severity of iron overload in thalassaemia patients, we compared the prevalence of mutations C282Y and H63D in 216 young adults regularly transfused and chelated in North‐Eastern Italy with the frequency found in a group of blood donors from the same area. For each patient, mean serum ferritin over the last 3 years, liver iron concentration, and the presence of diabetes, hypogonadism and heart disease, were considered. The frequency of the C282Y allele was 1.9% in patients with thalassaemia major and 2.3% in blood donors (P = ns). The frequency of the H63D allele was 16.2% in patients with thalassaemia major and 15.3% in blood donors (P = ns). When age, liver iron concentration and mean yearly serum ferritin levels were compared in patients with and without mutations C282Y and H63D, no significant differences were found. Also, the prevalence of iron‐induced complications was not significantly different between patients carrying or not carrying the mutations. The presence of the HH mutations does not seem to influence the degree of iron overload and its consequences in regularly transfused and chelated patients with thalassaemia major.
Symptoms of inherited factor V deficiency in 35 Iranian patients
Authors: Lak  , Sharifian  , Peyvandi  , Mannucci
Published: March 2003

The type of bleeding symptom has been evaluated in 35 Iranian patients with an inherited deficiency of factor V, with plasma levels between 1% and 10%. The most frequent symptoms included epistaxis and excessive bleeding after surgery. Haemarthroses and muscle haematomas were less common, even in severely deficient patients. More severe symptoms such as gastrointestinal and central nervous system bleeding were rare. The severity of bleeding symptoms was only partially related to the degree of factor V deficiency in plasma. On the whole, human factor V deficiency is characterized by a moderately severe bleeding phenotype.
Acute and chronic arsenic poisoning associated with treatment of acute promyelocytic leukaemia
Authors: Huang Shang‐Yi , Chang Cheng‐Shyong , Tang Jih‐Luh , Tien Hwei‐Fang , Kuo Tsung‐Li , Huang Shiu‐Feng , Yao Yu‐Tung , Chou Wen‐Chien , Chung Chih‐Yuan , Wang Chiu‐Hwa , Shen Ming‐Ching , Chen Yao‐Chang
Published: March 2003

Seven relapsed and/or refractory acute promyelocytic leukaemia patients were treated by arsenic trioxide (As2O3). Four patients (4/7, 57%) achieved complete remission after one to three cycles of treatment and the most common acute side‐effect was fluid retention (in six patients, 86%), including weight gains and pleuro‐pericardial effusions. Evident polyneuropathy compatible with chronic arsenic toxicity was noted in two of the three patients who received As2O3 maintenance therapy and one of them had marked distal muscular atrophy. We suggest that As2O3 may be a useful salvage therapy for relapsed and refractory APL patients, but the acute or chronic arsenic toxicity should be carefully monitored.
The Fyx phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein
Authors: Olsson Martin L. , Smythe Jonathan S. , Hansson Carola , Poole Joyce , Mallinson Gary , Jones Jeff , Avent Neil D. , Daniels Geoff
Published: March 2003

The molecular basis of the three major alleles (Fya/Fyb/Fy) of the Duffy (FY) blood group system has recently been established but the Fyx phenotype associated with weak expression of the Fyb and other FY antigens is poorly understood.

In the Fyx genes of five unrelated British and Swedish donors with the Fy(a+b+weak) phenotype we found two missense mutations predicting amino acid changes Arg89Cys and Ala100Thr in the FY glycoprotein.

The same mutations were found in two Fy(a−b+weak) samples from individuals of Swedish and Algerian origin. Their red blood cells showed a marked decrease in Fyb, Fy3 and Fy6 expression measured by routine serology and flow cytometry. The rare FY genotypes FyxFyx and FyxFy were confirmed by family studies and DNA sequencing.

Screening by allele‐specific primer PCR (ASP‐PCR) for these mutations among 100 Caucasian and 100 Black random blood donors indicated allele frequencies of 2.5% and 0%, respectively. Ala100Thr alone was present in 33% of the Caucasians (but none of the Blacks) with no weakening of FY expression.

A novel allele at the FY locus associated with the Fyx phenotype was studied. Mistyping of this weak Fyb antigen in clinical transfusion medicine may lead to delayed haemolytic transfusion reactions in immunized patients. A potential role for genomic typing is proposed.
A new variant of Bernard‐Soulier syndrome characterized by dysfunctional glycoprotein (GP) Ib and severely reduced amounts of GPIX and GPV
Authors: Noris Patrizia , Arbustini Eloisa , Spedini Pierangelo , Belletti Simona , Balduini Carlo Luigi
Published: March 2003

We describe a new variant of Bernard‐Soulier syndrome characterized by almost normal amounts of GPIb and severely reduced GPIX and GPV. Despite surface expression, GPIbα failed to support ristocetin‐induced platelet agglutination and to bind two conformation‐dependent monoclonal antibodies, suggesting a qualitative defect. Sequence analysis of the gene coding for GPIX revealed a T‐to‐C substitution at base 1811, leading to a Leu40Pro conversion, whereas no defects were found in the coding region of the GPIbα gene. Allele‐specific restriction enzyme analysis showed that the propositus and one of his sisters, both with severe bleeding diathesis, were homozygous for the GPIX mutation; the members of the family with mild bleeding diathesis and/or giant platelets in the peripheral blood were heterozygous, whereas the healthy ones were homozygous for the normal allele.

Infusion of 1‐desamino‐8‐ d‐arginine vasopressin normalized bleeding time in the two severely affected patients, although it did not modify ristocetin‐induced platelet agglutination or membrane expression of GPIbα, GPIX, GPIIb–IIIa and GMP‐140. Moreover, in one patient, normalization of bleeding time and rise of von Willebrand factor plasma concentration did not seem to be directly related.
Leukapheresis cells of patients with multiple myeloma collected after mobilization with chemotherapy and G‐CSF do not bear Kaposi's sarcoma associated herpesvirus DNA
Authors: Bellos Frauke , Cremer Friedrich W. , Ehrbrecht Edith , Goldschmidt Hartmut , Moos Marion
Published: March 2003

The presence of Kaposi's sarcoma associated herpesvirus (KSHV) in bone marrow dendritic cells, in bone marrow biopsies and in dendritic cells of peripheral blood from patients with multiple myeloma (MM) has been reported. These data suggested an association between infection with KSHV and the development of MM. The mobilization of infected cells into leukapheresis products (LP) has also been described. We assessed the LP of 35 patients with MM for the presence of KSHV using a sensitive and specific nested PCR assay, capable of detecting one copy of the virus genome. None of the samples tested revealed positivity for KSHV after amplification and subsequent hybridization with a KSHV‐specific probe. Amplification products of approximately the same size as the positive control seen in eight samples did not hybridize with the specific oligonucleotide. No homologies of these products to the KSHV genome could be discovered after sequencing. Therefore we have no evidence that LP of patients with MM bear KSHV, and they can therefore be used as a source for dendritic cells for immunotherapy.
Thymus‐independent T‐cell differentiation in vitro
Authors: Sanchez  , Alfani  , Visconti  , Passarelli  , Migliaccio  , Migliaccio
Published: March 2003

The generatation of large quantities of novel human T‐cell clones ex vivo would make a wide range of gene‐ and immuno‐therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal‐neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co‐cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum‐deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRαβ+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T‐cell receptor β‐chain rearrangments were generated over time. These results pave the way for the development of very simple culture conditions for ex‐vivo production of naive helper or cytotoxic T cells which could be very useful for gene‐ and immuno‐therapy of human diseases.
Characterization of anti‐CD138 monoclonal antibodies as tools for investigating the molecular polymorphism of syndecan‐1 in human lymphoma cells
Authors: Gattei Valter , Godeas Cristiana , Degan Massimo , Rossi Francesca Maria , Aldinucci Donatella , Pinto Antonio
Published: March 2003

Syndecan‐1 (CD138) is a surface proteoglycan consisting of long unbranched glycosaminoglycan (GAG) chains covalently attached to a protein backbone. High levels of a putatively syndecan‐1 isoform have recently been found on neoplastic cells of primary effusion lymphoma (PEL). As opposed to murine systems, studies on syndecan‐1 isoforms in humans have been hampered by the lack of a precise characterization of anti‐CD138 monoclonal antibodies (mAbs). We have therefore investigated the reactivity of anti‐CD138 mAbs (B‐B4, B‐B2, 1D4, MI15 and 104‐9) with either intact native proteoglycans or a recombinant unglycosylated form of syndecan‐1 core protein, and utilized these reagents to dissect the molecular heterogeneity of syndecan‐1 in human lymphoma cells. Our results indicated that: (a) mAb B‐B2 recognized only non‐denatured syndecan‐1, being poorly reactive by immunoblotting with both intact and recombinant syndecan‐1 protein; (b) mAb 104‐9 was unable to recognize native syndecan‐1, but showed a significant reactivity with intact and unglycosylated syndecan‐1 protein upon immunoblotting; (c) mAbs B‐B4, 1D4 and MI15 recognized both the intact molecule and the core protein of syndecan‐1, and showed a comparable reactivity in flow cytometry and immunoblotting. Cross‐blocking experiments indicated these latter mAbs recognizing the same or closely related epitopes of syndecan‐1. Using these mAbs, we have demonstrated that: (a) tumour cells from PEL expressed a syndecan‐1 isoform with a higher molecular weight than that present on malignant plasma cells; (b) syndecan‐1 expressed by PEL cells had a core protein identical in size to that expressed by plasma cells, suggesting that differences in syndecan‐1 size were due to different GAG chains attached to an identical protein backbone; (c) the PEL‐specific isoform of syndecan‐1, which probably represented the major proteoglycan expressed by these cells, was effective in mediating cell adhesion to type I collagen substrates. This data represents the first evidence describing the existence of a molecular polymorphism, of syndecan‐1 in human lymphomas.
CD34+‐selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: tumour cell contamination and outcome
Authors: Gupta D. , Bybee A. , Cooke F. , Giles C. , Davis J. G. , McDonald C. , Armitage S. E. , McGuigan D. , Lyttelton M. P. A. , Kanfer E. J. , Apperley J. F. , Samson Diana
Published: March 2003

Thirty‐six patients with multiple myeloma (23 PR1, nine PR2, four stable disease) were entered into a pilot study evaluating the use of CD34+‐selected peripheral blood progenitor cell transplantation (PBPCT) following high‐dose melphalan alone or high‐dose melphalan and total body irradiation. Peripheral blood progenitor cells (PBPCs) were mobilized with cyclophosphamide and granulocyte colony stimulating factor (G‐CSF). CD34+ selection using the Cellpro Ceprate‐SC system was performed in 22 cases with an adequate yield in 20. 10 patients failed to mobilize sufficient cells to permit selection and in four cases selection was not performed for other reasons. 16 patients therefore received unselected PBPC. Tumour cell contamination was evaluated by IgH gene fingerprinting (fpPCR). Harvested PBPC were fpPCR positive in 13/20 CD34+‐selected cases and remained positive after selection in seven. Harvested PBPC were studied in 9/16 patients receiving unselected cells; fpPCR was positive in five and negative in four. There was no difference in event‐free survival (EFS) between the CD34+‐selected group and the unselected group (median 21 and 26 months, respectively, P=ns). The CD34+‐selection process therefore reduced contamination but did not eliminate it completely, and in this small non‐randomized study there was no apparent clinical benefit of CD34+selection.
Identification and characterization of two missense mutations causing factor XIIIA deficiency
Authors: Kangsadalampai Sasichai , Chelvanayagam Gareth , Baker Rohan , Tiedemann Karin , Kuperan Ponnudurai , Board Philip G.
Published: March 2003

In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patients of Syrian and Indian descent, respectively. To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast. Determination of the transglutaminase activity and immuno detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yeast.

The mutations Arg260His and Val414Phe occur within the ‘core’ domain of the enzyme. Computer modelling of the mutant enzymes reveals that the substitution of the Arg260 by His results in the loss of a conserved electrostatic interaction whereas the effect of the Val414Phe substitution is a consequence of the large increase in side‐chain volume. Although both mutations do not effect the active site directly, they are predicted to reduce the stability of the enzyme.

The effects of these two amino acid substitutions on enzyme expression and three‐dimensional structure strongly confirm that residues which are located outside of the active site can have a significant effect on protein stability and function.
Late graft rejection and second infusion of bone marrow in children with aplastic anaemia
Authors: Eapen Mary , Davies Stella M. , Ramsay Norma K. C.
Published: March 2003

Late graft rejection following allogeneic bone marrow transplantation (BMT) for aplastic anaemia is a significant clinical problem and is associated with a high risk of mortality. We report two children with severe aplastic anaemia (SAA) who developed very late graft rejection 2 years and 4 months and 10 years respectively after allogeneic BMT from HLA‐identical siblings. Following a second BMT from their initial donors, engraftment has been sustained in both cases. The patients are alive with full donor chimaerism, 18 and 19 years from initial transplant. These cases illustrate that graft failure can be an extremely late event after allogeneic BMT for SAA, and that long‐term sustained engraftment can be achieved in these patients with second BMT from the original donors.
Enhanced retention of in vitro functional activity of platelets from recombinant human thrombopoietin‐treated patients following long‐term cryopreservation with a platelet‐preserving solution (ThromboSol) and 2% DMSO
Authors: Vadhan‐Raj Saroj , Currie Laura M. , Bueso‐Ramos Carlos , Livesey Stephen A. , Connor Jerome
Published: March 2003

Chemotherapy‐induced thrombocytopenia represents a significant clinical problem in the management of patients with malignancy. Recombinant human thrombopoietin (rhTPO) is a potent stimulator of platelet production in vivo. The ability to cryopreserve rhTPO‐derived platelets would enable the use of autologous platelets during the period of thrombocytopenia. ThromboSolTM is a platelet‐stabilizing formulation consisting of second messenger effectors that inhibit specific activation pathways endogenous to platelets. To investigate the effect of ThromboSol cryopreservation, platelets from rhTPO‐treated patients (n = 23) and normal donors were treated with ThromboSol and 2% DMSO and cryopreserved for up to 6 months. The platelets were thawed at different intervals and tested for retention of platelet functional activity in vitro. Following a short‐term storage (1 week), the cryopreserved platelets from patients treated with rhTPO exhibited significantly higher retention of functional activities including discoid morphology (70% v 57%), extent of shape change (19% v 13%) stirring shape change (15% v 11%) and hypotonic shock response (56% v 25%), as compared to the cryopreserved platelets from controls. Furthermore, there was no further significant loss of functional activity following cryopreservation for up to 6 months. These findings suggest that cryopreservation of platelets from rhTPO‐treated donors may provide a useful novel strategy for autologous or allogeneic donation for subsequent transfusions to manage treatment‐related thrombocytopenia.
A reverse dot‐blot method for rapid detection of non‐deletion α thalassaemia
Authors: CHAN VIVIAN , YAM IRENE , CHEN F. E. , CHAN T. K.
Published: March 2003

A reverse dot blot method based on membrane‐bound allele‐specific oligonucleotides as hybridization targets for amplified α‐gene fragments has been developed for the rapid detection of four non‐deletion α thalassaemia defects found in the Chinese. Since these non‐deletion defects account for 22.8% of haemoglobin H disease, a sensitive, specific and rapid screening method should be of value.
The Italian Consensus Conference on Diagnostic Criteria for Myelofibrosis with Myeloid Metaplasia
Authors: Barosi Giovanni , Ambrosetti Achille , Finelli Carlo , Grossi Alberto , Leoni Pietro , Liberato Nicola L. , Petti Maria C. , Pogliani Enrico , Ricetti Marilena , Rupoli Serena , Visani Giuseppe , Tura Sante ,   for the ICSG on MMM (Italian Cooperative Study Group on Myelofibrosis with Myeloid Metaplasia)
Published: March 2003

The purpose of this work was to develop a definition of myelofibrosis with myeloid metaplasia (MMM) using diagnostic criteria that would remain valid within the set of patients with chronic myeloproliferative disorders or myelodysplastic syndromes. A list of 12 names for the disease and 37 diagnostic criteria were proposed to a Consensus Panel of 12 Italian experts who ranked them in order so as to identify a core set of criteria. The Panel was then asked to score the diagnosis of 46 patient profiles as appropriate or not appropriate for MMM. Using the experts' consensus as the gold standard, the performance of 90 possible definitions of the disease obtained through the core set was evaluated. ‘Myelofibrosis with myeloid metaplasia’ ranked as the preferred name of the disease. Necessary criteria consisted of ‘diffuse bone marrow fibrosis’ and ‘absence of Philadelphia chromosome or BCR‐ABL rearrangement in peripheral blood cells’. The six optional criteria in the core set consisted of: splenomegaly of any grade; anisopoikilocytosis with tear‐drop erythrocytes; the presence of circulating immature myeloid cells; the presence of circulating erythroblasts; the presence of clusters of megakaryoblasts and anomalous megakaryocytes in bone marrow sections; myeloid metaplasia. The definition of the disease with the highest final score was as follows: necessary criteria plus any other two criteria when splenomegaly is present or any four when splenomegaly is absent. The use of this definition will help to standardize the conduct and reporting of clinical studies and should help practitioners in clinical practice.
Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin‐2 receptor α chain
Authors: Shikami Masato , Miwa Hiroshi , Nishii Kazuhiro , Takahashi Takahiro , Shiku Hiroshi , Tsutani Hiroshi , Oka Kouji , Hamaguchi Hiroyuki , Kyo Taiichi , Tanaka Kimio , Kamada Nanao , Kita Kenkichi
Published: March 2003

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH‐1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The immunophenotypes of the AML cells, and YNH‐1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA‐DR−/+. Cells from all samples strongly expressed c‐kit, granulocyte colony‐stimulating factor receptor (G‐CSFR), c‐fms (macrophage colony‐stimulating factor receptor), interleukin‐3 receptor α chain (IL‐3Rα), and granulocyte macrophage colony‐stimulating factor receptor α chain (GM‐CSFRα), and these data corresponded well to the growth responsiveness to the cytokines. IL‐2Rα expression was also found in all t(16;21) samples, but IL‐2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G‐CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c‐kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL‐2Rα expression) and FUS/ERG protein remains undetermined.
Inhibition of p34cdc2 dephosphorylation in DNA damage‐ and topoisomerase II inactivation‐induced G2 arrests in HL‐60 cells
Authors: Sugimoto Koichi , Sasaki Makoto , Tamayose Kenji , Oshimi Kazuo
Published: March 2003

Doxorubicin induces DNA breakage by stabilizing a cleavable topoisomerase II–DNA complex. In contrast, topoisomerase II catalytic inhibitor ICRF‐193 and uncoupling inhibitor aclarubicin interfere with the cleavable complex formation. We analysed combination effects of these drugs using two‐dimensional flow cytometry of DNA content and the terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labelling assay. Both ICRF‐193 and aclarubicin attenuated the cytotoxic effect of doxorubicin on HL‐60 cells (85% and 46% maximum reduction, respectively), which suggested that doxorubicin exerts its cytotoxic effect at least partially through the topoisomerase II‐dependent DNA cleavage. Doxorubicin and ICRF‐193 both induced G2 arrest in HL‐60 cells, by which they may have reduced the cytotoxic effect of vincristine. Indeed, although ICRF‐193 inhibited doxorubicin‐induced apoptosis, ICRF‐193 and doxorubicin cooperated in arresting HL‐60 cells at G2 phase. These results indicated that G2 arrest was caused not only by DNA damage but also through a DNA damage‐free, topoisomerase II inactivation‐induced pathway. Western blot analysis showed that both types of G2 arrest were mediated by the inhibition of p34cdc2 dephosphorylation.
An improved micro‐method for obtaining chromosome preparations from individual haemopoietic colonies
Authors: Kadhim M. A. , Pocock D. A. , Lorimore S. A. , Wright E. G.
Published: March 2003

A robust method for obtaining chromosome preparations from individual haemopoietic colonies in semi‐solid media is described. The accumulation of metaphases and the hypotonic treatment of cells were carried out in the culture dish and individual colonies were transferred onto poly‐l‐lysine‐treated slides and fixed stepwise prior to staining. The technique produced high yields of well‐spread metaphases facilitating clonal cytogenetic analysis.
HHV‐6‐related secondary graft failure following allogeneic bone marrow transplantation
Authors: Johnston Rosalynd E. , Geretti Anna‐Maria , Prentice H. Grant , Clark A. Duncan , Wheeler Anne C. , Potter Michael , Griffiths Paul D.
Published: March 2003

We report the case of an 11‐year‐old boy who underwent allogeneic bone marrow transplantation (BMT) for relapsed acute lymphoblastic leukaemia. Despite adequate engraftment, on day 45 he developed marrow aplasia with haemophagocytosis. HHV‐6 was detected in blood and bone marrow by nested PCR. Retrospective testing showed that viraemia had started on day 24. Following therapy with foscarnet and ganciclovir, viral load declined to undetectable levels and his donor marrow recovered contemporaneously. This case suggests that HHV‐6 may be a treatable cause of graft failure following BMT and provides clinical and virological evidence for the anti‐HHV‐6 activity of ganciclovir and foscarnet.
Expression of CD41 and c‐mpl does not indicate commitment to the megakaryocyte lineage during haemopoietic development
Authors: Basch Ross S. , Zhang Xin‐Min , Dolzhanskiy Alexandr , Karpatkin Simon
Published: March 2003

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+CD41+) were isolated from normal human bone marrow or induced in culture from CD34+CD41− bone marrow cells by treatment with thrombopoietin (TPO) or IL‐3. We found that although this population included the majority of cells that can form CFU‐MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+CD41+‐induced cells was greater than that of isolated CD34+CD41+ cells in that the isolated cells only formed CFU‐MK and BFU‐e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU‐e. A major portion of the cells that expressed CD34+CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c‐mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+CD38− cells), expressed c‐mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c‐mpl was predictive of commitment to the MK lineage.
Diagnostic importance of the two‐stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954 → Leu substitution in the factor VIII A3 domain
Authors: Keeling D. M. , Sukhu K. , Kemball‐Cook G. , Waseem N. , Bagnall R. , Lloyd J. V.
Published: March 2003

In some families with mild haemophilia higher results are obtained for factor VIII activity (FVIII:C) determined by one‐stage assay than by two‐stage or chromogenic assays. Amino‐acid substitutions in the A1, A2 and A3 domains of factor VIII have been described in affected individuals with this phenotype. We describe a case of mild haemophilia A in which FVIII:C measured by one‐stage assay was normal at 106%. However, FVIII:C levels measured by two‐stage and chromogenic assays were 18% and 35% respectively. DNA analysis revealed a novel mutation in the A3 domain of factor VIII, His1954 → Leu. In a molecular model of the FVIII A domains, His1954 is placed in close proximity to two other mutations that have previously been shown also to be associated with one‐stage/two‐stage discrepancies. In this patient the diagnosis of haemophilia A would be missed if only the one‐stage assay was used.
Differentiation of autoimmune thrombocytopenia from thrombocytopenia associated with immune complex disease: systemic lupus erythematosus, hepatitis‐cirrhosis, and HIV‐1 infection by platelet and serum immunological measurements
Authors: Samuel Hala , Nardi Michael , Karpatkin Margaret , Hart David , Belmont Michael , Karpatkin Simon
Published: March 2003

A method and approach are described to differentiate classic autoimmune thrombocytopenia (ATP) from immune complex‐associated thrombocytopenia in systemic lupus (SLE), hepatitis/chronic liver disease (LIV‐ITP) and HIV‐1 related thrombocytopenia (HIV‐1‐ITP). The platelet immunologic profile of IgG, C3C4 and IgM was measured with a solid‐phase ELISA, employing 125I‐staphylococcal protein A to detect indicator antibody binding. Polyethylene glycol was employed to precipitate immune complexes (PEG‐IC). Platelet‐associated IgG (PAIgG) was 2.8‐, 5.6‐ and 5.8‐fold higher in SLE, LIV‐ITP and HIV‐1‐ITP patients respectively compared to ATP patients; platelet C3C4 was 3.2‐, 4.8‐ and 4.5‐fold higher respectively; platelet IgM was 2.2‐, 3.7‐ and 3.8‐fold higher respectively; serum PEG‐IC levels were 4.2‐, 4.8‐ and 2.1‐fold higher respectively. With all parameters measured, there was no overlap between the 75th percentile for ATP patients and the 25th percentile for all three cohorts. The likelihood of having a platelet C3C4 level higher than the highest ATP level was 69% for SLE, 90% for LIV‐ITP and 94% for HIV‐1‐ITP respectively; with PEG‐IC measurements the likelihood was 83%, 100% and 100% respectively. Serum IgG, C3, C4, IgM and PEG‐IC were examined for a possible relationship with platelet measurements. Except for a positive correlation between serum and platelet IgM in ATP, r = 0.5, P < 0.04, there was no positive correlation with any of the parameters measured. An inverse correlation was noted between PEG‐IC level and platelet C3C4 in SLE, r = 0.7, P < 0.04. Thus platelet immunologic profile and serum PEG‐IC level measurements differentiated classic ATP from immune complex‐associated thrombocytopenias (SLE, LIV‐ITP, HIV‐1‐ITP). Except for IgM measurements in ATP, platelet measurements could not be attributed to their respective serum concentration.
Platelet activation responses in vitro to human mast cell activation
Authors: Gardiner  , Harrison  , Chavda  , Mackie  , Machin
Published: March 2003

Mast cells are thought to play an important role in atherogenesis and plaque rupture, but their role in the subsequent platelet activation and thrombus formation is unclear. Tryptase positive cells (KU812T+) were established from the KU812 cell line as an in vitro model of human mast cells and used to study the effect of mast cell activation on human platelets. Overnight incubation of KU812T+ with IgE and subsequent challenge with anti‐IgE caused the release of heparinoid substances which inhibited 1 μg/ml collagen‐induced platelet aggregation. KU812T+ challenged with compound 48/80 produced a releasate that had no apparent heparinoid content but caused full platelet aggregation. These findings showed that, although activation of KU812T+ via FcɛR1 partially abrogated collagen‐induced platelet aggregation, activation of the C5a receptor signalling pathway, by compound 48/80, caused the release of potent platelet‐activating substances. This cell culture model offers a unique insight into the role of platelet–mast cell interactions in arterial thrombogenesis.
Cyclin D2 promoter disrupted by t(12;22)(p13;q11.2) during transformation of chronic lymphocytic leukaemia to non‐Hodgkin's lymphoma
Authors: Qian L. , Gong J. , Liu J. , Broome J. D. , Koduru P. R. K.
Published: March 2003

In a unique case of chronic lymphocytic leukaemia (CLL) we performed a longitudinal cytogenetic and molecular genetic study of tumour cells from diagnosis through progression and transformation to non‐Hodgkin's lymphoma (NHL) and lymphomatous meningitis. CLL cells at diagnosis had trisomy 12 and a t(14;19)(q32;q13.3). At relapse, the leukaemic cells had a subclone carrying a t(12;22)(p13;q11.2) in addition to the initial changes.

We cloned reciprocal translocation junctions at the 22q11.2− chromosome and the 12p13+ chromosome and the corresponding germline DNA fragments. Restriction map analysis and nucleotide sequence analysis of the cloned DNA fragment from the 22q11.2− chromosome mapped the translocation break within the immunoglobulin (Ig)‐λ‐C complex at the nt3889; nts 3890, 3891 were lost from the translocation site. A probe from the 3′‐end of the clone derived from the 22q11.2− chromosome showed single copy hybridization which was different from the Ig‐λ probe. Nucleotide sequence analysis of the exact junction region and the corresponding germline DNA showed that the translocation at 12p13 occurred in the negative regulatory region of the cyclin D2 gene at the nt −1602, and a pentamer consisting of nts −1603 to −1599 was lost at the break site. We sequenced another 227 bp upstream of the known 5′‐end of the promoter and did not find any open reading frame. From these results we hypothesize that, in this patient, the t(12;22) disrupted the negative regulator in the promoter of cyclin D2 which in turn might have deregulated cyclin D2.
α4β1 integrin‐mediated adhesion of CD34+ cells from patients with chronic myeloid leukaemia: influence of IL‐3
Authors: Schofield K. P. , Duerig J. , Rushton G. , Chang J.
Published: March 2003

Interactions between integrins on haemopoietic progenitor cells and their stromal ligands have an important role in the control of haemopoiesis. Growth factors can modulate these interactions (so‐called ‘inside‐out’ signalling) resulting in changes in ligand binding activity. We have studied α4β1 integrin‐mediated adhesion to the H120 fragment of fibronectin (which contains the strongest α4β1 binding site) in CD34+ cells from patients with chronic myeloid leukaemia (CML) and have determined the effect of IL‐3 on the level of adhesion. Compared to normal CD34+ cells isolated from cord blood and peripheral blood progenitor harvests (mean of 61.4 ± 14.9% of cells attached) the CML CD34+ cells showed reduced levels of adhesion (mean of 41.9 ± 14.7%, P < 0.05). The effect of 10 ng/ml of IL‐3 resulting in reduced adhesion of normal CD34+ cells at 30 min was absent in 6/7 patients with CML. Abnormalities of adhesion to fibronectin may thus be related to IL‐3 pathways affected by BCR‐ABL. These findings will have implications for understanding the dysregulation of growth and adhesion in CML.
Incompatibility for CD31 and human platelet antigens and acute graft‐versus‐host disease after bone marrow transplantation
Authors: Balduini C. L. , Noris P. , Giorgiani G. , Martinetti M. , Klersy C. , Spedini P. , Belletti S. , Maccario R. , Gusberti L. , Locatelli F.
Published: March 2003

Bone marrow transplantation (BMT) is often complicated by acute graft‐versus‐host disease (aGVHD). In patients transplanted with an HLA‐matched donor the occurrence of this complication is believed to be favoured by disparities at the minor histocompatibility antigens (mHA). However, few of these polymorphic molecules have been identified. We sought to determine whether donor/recipient incompatibility for HPA‐1, HPA‐2, HPA‐3, HPA‐5 or CD31 (codon 125) antigens represented a risk factor for aGVHD and genotyped these antigens in 70 bone marrow donors and their HLA‐identical recipients. All patients were children who received BMT for haematological malignancies at a single institution according to well‐defined therapy protocols. Statistical analysis showed that incompatibility for CD31 (codon 125) was a risk factor for grade II–IV aGVHD in the overall patient population, whereas HPA‐3 incompatibility predicted aGVHD occurrence in HLA‐A2 patients only. The magnitude of the aGVHD risk was directly related to the number of HPA/CD31 incompatibilities. No correlation was found between non‐identity for HPA/CD31 and aGVHD. Since incompatibility but not non‐identity for CD31 or HPA‐3 was a risk factor for aGVHD, we suggest that allelic variants of these molecules can serve as mHA in BMT recipients from HLA‐identical donors.
Effect of factor VIII concentrate on leucocyte cytokine production: characterization of TGF‐beta as an immunomodulatory component in plasma‐derived factor VIII concentrate
Authors: Hodge G. , Flower R. , Han P.
Published: March 2003

Clinical and subclinical immunological abnormalities have been reported in HIV‐seronegative haemophiliacs. The mechanisms by which these abnormalities arise remain unclear. As cytokines are important biological response modifiers, the effect of a FVIII concentrate on production of a range of cytokines, by a variety of cells, was investigated. A whole blood technique was used and the in vitro modulation of cytokine synthesis by an intermediate‐purity plasma‐derived factor VIII (pdFVIII) concentrate was analysed using multiparameter flow cytometry. In cell cultures exposed to pdFVIII, T cells showed reduced production of TNF‐α, IL‐2 and IFN‐γ; monocytes showed reduced production of TNF‐α, IL‐1α, IL‐1β, IL‐6, IL‐8 and IL‐12 but an increase in IL‐10 synthesis; IFN‐γ synthesis by NK cells was reduced. All changes in cytokine synthesis and the reduction in cell surface expression of CD69, a signal transduction molecule contributing to both cytokine and cytokine receptor synthesis, were in a dose‐dependent manner in cultures exposed to FVIII concentrate. These changes were characteristic of TGF‐β. Addition of anti‐TGF‐β to FVIII reduced these changes in T‐cell cytokine production, suggesting TGF‐β may be an important immunomodulatory agent in the pdFVIII concentrate. The Th2 cytokine bias shown in the presence of pdFVIII concentrate, in vitro, may explain the increase in rates of certain types of infections reported in these patients, which require Th1 cytokine production for an effective response.
Quantification of minimal residual disease in patients with BCR‐ABL‐positive acute lymphoblastic leukaemia using quantitative competitive polymerase chain reaction
Authors: Mitterbauer Gerlinde , Nemeth Peter , Wacha Stefan , Cross Nicholas C. P. , Schwarzinger Ilse , Jaeger Ulrich , Geissler Klaus , Greinix Hildegard T. , Kalhs Peter , Lechner Klaus , Mannhalter Christine
Published: March 2003

We analysed 20 patients with BCR‐ABL‐positive acute lymphoblastic leukaemia (ALL) by quantitative competitive polymerase chain reaction (QC‐PCR) to study the kinetics of the leukaemic clone. Consecutive samples of 16 patients (minor‐bcr, n = 10; major‐bcr, n = 6) were analysed after conventional chemotherapy and/or bone marrow transplantation (BMT). DNA competitor templates co‐amplifying with either p210 or p190 BCR‐ABL cDNA were used for quantification of leukaemia‐specific BCR‐ABL mRNA. In all samples, total ABL transcripts were measured as internal control, and the percentage of BCR‐ABL/ABL molecules was calculated. Following induction chemotherapy the number of BCR‐ABL transcripts was reduced by a maximum of 2–3 logs. In most patients, additional chemotherapy did not lead to further reduction of BCR‐ABL mRNA. In two patients, conventional chemotherapy plus autologous BMT in complete haematological remission resulted in a total reduction of the transcript level of more than 3 logs. In two other patients, allogeneic BMT caused a transient reduction of the BCR‐ABL transcripts below the detection level of our method (<1 blast cell in 105 normal cells) for a period of 7 and 11 months, respectively. The achievement of PCR negativity did not guarantee sustained remission. Both patients relapsed and BCR‐ABL transcript levels rose by more than 1 log prior to frank relapse. Our data demonstrate that quantification of BCR‐ABL mRNA allows the evaluation of the dynamics of the leukaemic clone and thus is valuable for the evaluation of minimal residual leukaemia following various therapies and the early detection of increasing BCR‐ABL transcripts prior to relapse.
Suppression of CDA II expression in a homozygote
Authors: Beauchamp‐Nicoud  , Schischmanoff  , Alloisio  , Boivin  , Parsons  , Mielot  , Tchernia  , Legrand  , Miraglia del Giudice  , Gasparini  , Iolascon  , Wickramasinghe  , Delaunay
Published: March 2003

The CDAN2 gene, responsible for congenital dyserythropoietic anaemia, type II (CDA II), was recently mapped to 20q11.2. We report data on an additional member of a previously studied CDA II family. This member had always been regarded as haematologically normal. Unexpectedly, she had the same microsatellite assortments around the CDAN2 alleles as her three sisters with CDA II. In particular, she was a homozygote for microsatellites D20S863 and D20S841. This prompted an analysis of all facets of her phenotype. The Ham test was negative. The bone marrow smears contained a normal proportion of binucleate erythroblasts. Electron microscopy revealed the absence of extensive stretches of cisternae beneath and parallel to the inner surface of the erythroblast plasma membrane. Proteins of the endoplasmic reticulum, which contaminate the reticulocyte plasma membrane in CDA II patients, were missing. Only the shape of the band 3 peak appeared slightly altered. This case exemplifies that homozygosity (or compound heterozygosity) for a deleterious gene may be silenced, or almost completely silenced. In recessively inherited diseases, suppressed phenotypes tend to be overlooked in siblings where both patients and unaffected individuals are expected.